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Sample records for indirect immunofluorescence microscopy

  1. Indirect immunofluorescent antibody test in chicken leucocytozoonosis.

    PubMed

    Isobe, T; Akiba, K

    1982-01-01

    The indirect immunofluorescent antibody technique was applied to detection of antigens of different developmental stages of Leucocytozoon caulleryi and antibodies in the sera of chickens infected with L. caulleryi by using second generation merozoite and gametocyte antigens. Zygotes, ookinetes and sporozoites in midges, and second generation merozoites and gametocytes in chickens indicated specific fluorescence. Indirect immunofluorescent antibody titers were higher than agar gel precipitation antibody titers. So the detection of the antibody by the indirect immunofluorescent antibody technique was possible in the sera in which the antibody could not be detected by the agal gel precipitation test. Therefore, the indirect immunofluorescent antibody technique was applicable to chicken leucocytozoonosis as a highly sensitive serological diagnostic method.

  2. Establishment of indirect immunofluorescence assay for rotavirus.

    PubMed

    Tao, J; Zhang, J; Liu, X; Jin, H; Jiang, C; Yin, Y

    2016-03-01

    Rotavirus infection is the most frequent cause of infantile gastroenteritis worldwide and a significant cause of death in infants and young children, following severe diarrhea and dehydration. Rotavirus vaccines are considered the most effective way to prevent rotavirus infections. In the process of developing rotavirus vaccines, it is crucial to establish a reliable and standardized method to determine vaccine titer. In this study, we developed an indirect immunofluorescence assay (IFA) to determine the infectious titer of Lanzhou lamb rotavirus (LLR) vaccine grown in MA104 cells. The activating concentration of trypsin was 1 µg/ml for healthy monolayers of MA104 cells at 100% confluence. After incubation for 18 hr, a rabbit anti-SA11 polyclonal antibody, diluted at 1:800 in PBS, was added to all wells, followed by an Alexa-488-conjugated secondary antibody diluted at 1:500 in PBS. Cells were examined with a fluorescence microscope. Our results show that IFA was more reproducible, more sensitive, simpler, and more rapid than the log 50% cell culture infectious dose-ELISA (lgCCID50-ELISA) in measuring the rotavirus vaccines. IFA provided a reliable basis for the qualitative and quantitative analysis of rotavirus, and the certification of rotavirus vaccine production.

  3. Epidermolysis bullosa acquisita and anti-p200 pemphigoid as major subepidermal autoimmune bullous diseases diagnosed by floor binding on indirect immunofluorescence microscopy using human salt-split skin.

    PubMed

    Goyal, Nupur; Rao, Raghavendra; Shenoi, Shrutakirthi D; Pai, Sathish; Kumar, Pramod; Bhogal, Balbir S; Schmidt, Enno; Zillikens, Detlef

    2017-01-01

    Subepidermal autoimmune bullous diseases are a diverse group of diseases with overlapping clinical and immunopathological features. Indirect immunofluorescence microscopy on artificially split skin helps to classify these conditions into those with staining on the epidermal side of the split ("roof-binding") and those with staining on the dermal side ("floor-binding"). Epidermolysis bullosa acquisita is the prototype of "floor-binding" subepidermal autoimmune bullous diseases. However, not all floor-binding sera are associated with epidermolysis bullosa acquisita. The aim of this study was to evaluate the clinical and immunological profile of patients with floor-binding subepidermal autoimmune bullous disease by indirect immunofluorescence microscopy and to identify the target antigens in them. Ten patients who showed a floor-binding pattern were studied with regard to their clinical and immunopathological characteristics. Target antigens were identified by modified indirect immunofluorescence microscopy using recessive dystrophic epidermolysis bullosa skin, enzyme linked immunosorbent assay, and immunoblotting. Diagnosis of epidermolysis bullosa acquisita was confirmed in six patients. Three patients with an inflammatory subepidermal autoimmune bullous disease mimicking bullous pemphigoid reacted with a 200 kDa protein on immunoblotting with dermal extract, as is characteristic of anti-p200 pemphigoid. One serum showed both roof and floor binding, and reacted with the BP180 antigen. We could not perform serration pattern analysis in our patients. In this study, we report three cases of anti-p200 pemphigoid from India. These cases, though indistinguishable clinically from bullous pemphigoid, revealed a floor-binding pattern on indirect immunofluorescence using salt-split skin.

  4. A novel automated indirect immunofluorescence autoantibody evaluation.

    PubMed

    Kivity, Shaye; Gilburd, Boris; Agmon-Levin, Nancy; Carrasco, Marina Garcia; Tzafrir, Yaron; Sofer, Yael; Mandel, Matilda; Buttner, Thomas; Roggenbuck, Dirk; Matucci-Cerinic, Marco; Danko, Katalin; Hoyos, Marcos López; Shoenfeld, Yehuda

    2012-03-01

    Autoantibodies (AAb), especially antinuclear (ANAs) and anticytoplasmatic antibodies (ACyA), are essential diagnosing markers for several autoimmune diseases. The current gold standard method for ANA detection is manual indirect immunofluorescence (IIF) on human epithelial-2 (HEp-2) cells. However, this technique is cost and time consuming, and characterized by considerable intra- and interlaboratory variability. Thus, an automated IIF-HEp-2 reader has been developed recently. In the current study, we compared the performance of the automated AAb IIF-HEp-2 interpretation to conventional detection methods. Autoantibody detection by IIF on HEp-2 cells was performed in a total of 260 sera of patients, including 34 with systemic lupus erythematosus, 111 with dermatomyositis or polymyositis, 74 with systemic sclerosis, 41 with rare AAb patterns, and 137 healthy individuals. Visual interpretation and routine immunoassays were compared with a novel automated IIF-HEp-2 system using Aklides pattern recognition algorithms. Positive AAbs were detected in 95-100% of rheumatic patients by automated interpretation, in 74-100% with manual reading, and in 64-100% by immunodot assay. Receiver operating characteristic curve analysis of fluorescent intensity revealed a high sensitivity and specificity for automated reading of AAb with an agreement ranging from 90% to 95% between manual and automated interpretation (kappa 0.554-0.69) for systemic sclerosis and myositis, respectively. This study demonstrates a good correlation between manual and automated interpretation of AAb including ANA and ACyA in patients with autoimmune diseases. Full automation of HEp-2 cell assay reading may minimize errors in ANA pattern interpretation and thus help in the standardization of ANA assessment.

  5. [Autoantibody detection by indirect immunofluorescence on HEp-2 cells].

    PubMed

    Sack, U; Conrad, K; Csernok, E; Frank, I; Hiepe, F; Krieger, T; Kromminga, A; Landenberg, P von; Messer, G; Witte, T; Mierau, R

    2009-06-01

    Systemic autoimmune diseases are characterized by the presence of antinuclear autoantibodies (ANA). Diluted patient sera are typically used to screen for the presence of ANA by immunfluorescence microscopy with fixed HEp-2 cells. Despite high-quality test kits, reports of different laboratories frequently present controversial results. This article recommends unified processing and interpretation of HEp-2 based screening for autoantibodies. Suggestions are made for the selection of high-quality test kits, optimized processing and diagnostic procedures. In addition to a relevant clinical diagnosis and an experienced laboratory specialist, the following procedure is highly recommended to achieve good laboratory practice: Initial HEp-2 based screening by indirect immunofluorescence, starting with a 1:80 serum dilution, and evaluation in a bright fluorescence microscope, pathological values from a titer of 1:160 upwards, internal quality checks and unified interpretation. We aim to improve diagnosis and care of patients with autoimmune diseases as a central focus of the European Autoimmunity Standardization Initiative (EASI).

  6. Indirect Immunofluorescence for the Detection of Autoimmune Urticaria.

    PubMed

    Bahrani, Bahar; Gattey, Natasha T; Hull, Peter R

    2016-01-01

    An autoimmune basis is believed to be responsible for about half of chronic spontaneous urticaria (CSU) cases. The autologous serum skin test is used as a possible indicator, but there is currently no test that directly indicates an autoimmune etiology. In this study, an indirect immunofluorescence was used to identify patients with autoantibodies directed at mast cells. Two substrates were used including paraffin embedded sections of skin biopsies from an infant with bullous mastocytosis and cord blood-derived mast cells (CBMC). Sera from 76 patients with CSU were incubated with substrates and conjugated with human IgG. Using the bullous mastocytosis preparations, positive indirect immunofluorescence was found in 46% (n = 76), while the CBMC substrate was positive in 39% (n = 70). The IgG autoantibodies directed at mast cells could be detected in about half the patients with CSU. Indirect immunofluorescence should be considered as an indicator of the autoimmune form of CSU. © The Author(s) 2015.

  7. Autoantibody detection using indirect immunofluorescence on HEp-2 cells.

    PubMed

    Sack, Ulrich; Conrad, Karsten; Csernok, Elena; Frank, Ingrid; Hiepe, Falk; Krieger, Thorsten; Kromminga, Arno; von Landenberg, Philipp; Messer, Gerald; Witte, Torsten; Mierau, Rudolf

    2009-09-01

    The detection of autoantibodies is an important element in the diagnosis and monitoring of disease progression in patients with autoimmune diseases. In laboratory diagnostic tests for connective tissue and autoimmune liver diseases, indirect immunofluorescence on HEp-2 cells plays a central role in a multistage diagnostic process. Despite the high quality of diagnostics, findings at different laboratories can differ considerably due to a lack of standardization, as well as subjective factors. The present paper formulates recommendations for the standardized processing and interpretation of the HEp-2 cell test for the detection of non-organ-specific (especially antinuclear) antibodies. It provides requirements regarding the diagnostic tests used, instructions for laboratory procedure and evaluation, and recommendations for interpretation. For an optimal laboratory diagnostic process, it is useful to have an informative, tentative clinical diagnosis and an experienced laboratory diagnostician. In addition, the following key elements are recommended: initial screening using indirect immunofluorescence on carefully chosen HEp-2 cells beginning with a serum dilution of 1:80 and evaluation under a microscope with powerful illumination; results from a titer of 1:160 upwards being considered positive; internal laboratory quality control; and standardized interpretation. The aim is to improve diagnostic tests and care of patients with autoimmune diseases as a central concern of the European Autoimmunity Standardization Initiative (EASI).

  8. [Standardized indirect immunofluorescence. Differentiation of mitochondrial, microsomal and ribosomal antibodies].

    PubMed

    Storch, W

    1977-02-15

    By an extensive standardisation of the indirect immunofluorescence for the demonstration espeially of mitochondrial antibodies we succeeded in recognizing atypical fluorescence patterns and in describing their exact localisation. On the basis of absorption studies with mitochondrias, microsomas and ribosomas by comparative observation of sections of liver, stomach and kidneys of rats the preferred sort of reaction and the intensity of fluorescence of antibodies against mitochondria, microsomas and ribosomas were empirically established. Antimitochondrial antibodies react above all with the parietal cells of the stomach and the distal epithelia of the tubulus of the kidney. Antibodies against microsomas of liver and kidney are characterized by a brilliant diffuse cytoplasmatic fluorescence of the hepatocytes and by a comparatively weaker fluorescence of exclusively proximal tubuli of the kidneys of rats. Antibodies against ribosomas lead to a fluorescence especially of the main cells of the stomach. The differentiation of several cytoplasmatic antibodies is among others of interest for the diagnosis of certain autoimmune diseases. Although there are numerous still unclear findings and "overlap" phenomena the existence of high titre antibodies against mitochondrias speaks for a primarily biliary cirrhosis or a pseudo-LE-syndrome, the existence of antibodies against microsomas of kidney and liver of rats for a special form of a chronically active hepatitis and the existence of the very rare antibodies against ribosomas for an active lupus erythematodes disseminatus.

  9. Photoelectron microscopy and immunofluorescence microscopy of cytoskeletal elements in the same cells.

    PubMed Central

    Nadakavukaren, K K; Chen, L B; Habliston, D L; Griffith, O H

    1983-01-01

    Pt K2 rat kangaroo epithelial cells and Rat-1 fibroblasts were grown on conductive glass discs, fixed, and permeabilized, and the cytoskeletal elements actin, keratin, and vimentin were visualized by indirect immunofluorescence. After the fluorescence microscopy, the cells were postfixed and dehydrated for photoelectron microscopy. The contrast in these photoelectron micrographs is primarily topographical in origin, and the presence of fluorescent dyes at low density does not contribute significantly to the material contrast. By comparison with fluorescence micrographs obtained on the same individual cells, actin-containing stress fibers, keratin filaments, and vimentin filaments were identified in the photoelectron micrographs. The apparent volume occupied by the cytoskeletal network in the cells as judged from the photoelectron micrographs is much less than it appears to be from the fluorescence micrographs because the higher resolution of photoelectron microscopy shows the fibers closer to their true dimensions. Photoelectron microscopy is a surface technique, and the images highlight the exposed cytoskeletal structures and suppress those extending along the substrate below the nuclei. The results reported here show marked improvement in image quality of photoelectron micrographs and that this technique has the potential of contributing to higher resolution studies of cytoskeletal structures. Images PMID:6191327

  10. Monitoring Chemokine Receptor Trafficking by Confocal Immunofluorescence Microscopy

    PubMed Central

    Marchese, Adriano

    2016-01-01

    Here, we describe a protocol to detect chemokine receptor CXCR4 by confocal immunofluorescence microscopy in HeLa cells treated with its chemokine ligand CXCL12. Typically, ligand-activated chemokine receptors undergo a multistep process of desensitization and/or internalization from the plasma membrane in order to terminate signaling. Once internalized to endosomes, chemokine receptors readily enter the recycling pathway and return to the cell surface, giving rise to resensitization of signaling. The chemokine receptor CXCR4, when activated by CXCL12 is also internalized to endosomes, but in contrast to many chemokine receptors it is mainly sorted to the degradative pathway, contributing to a loss in the cellular complement of CXCR4 and long-term downregulation of signaling. The trafficking of CXCR4 from early endosomes to lysosomes can be easily detected by confocal immunofluorescence microscopy by immunostaining fixed cells for the receptor and with markers of these vesicular compartments. This approach is advantageous because it can be used to identify factors that regulate the trafficking of CXCR4 from early endosomes to lysosomes. The protocol described here focuses on CXCR4, but it can be easily adapted to other chemokine receptors. PMID:26921951

  11. Detection of antinuclear antibodies by indirect immunofluorescence and by solid phase assay.

    PubMed

    Op De Beeck, Katrijn; Vermeersch, Pieter; Verschueren, Patrick; Westhovens, René; Mariën, Godelieve; Blockmans, Daniel; Bossuyt, Xavier

    2011-10-01

    Testing for antinuclear antibodies is useful for the diagnosis of systemic rheumatic diseases. Solid phase assays are increasingly replacing indirect immunofluorescence for detection of antinuclear antibodies. In the most recent generation of solid phase assays, manufacturers attempt to improve the performance of the assays by adding extra antigens. Solid phase assay (EliA CTD Screen, Phadia, in which antibodies to 17 antigens are detected) was compared to indirect immunofluorescence for the detection of antinuclear antibodies in diagnostic samples of 236 patients with autoimmune connective tissue diseases, in 149 healthy blood donors, 139 patients with chronic fatigue syndrome, and 134 diseased controls. The sensitivity of EliA CTD Screen for systemic lupus erythematosus, systemic sclerosis, primary Sjögren's syndrome, mixed connective tissue disease, and inflammatory myopathy was 74%, 72%, 89%, 100%, and 39%, respectively. The reactivity in blood donors, in patients with chronic fatigue syndrome, and in diseased controls was <4%. Likelihood ratios increased with increasing antibody concentrations. Generally, a positive test result by EliA CTD Screen had a higher likelihood ratio for systemic rheumatic disease than a positive test result by indirect immunofluorescence. A negative test result by indirect immunofluorescence, however, had a lower likelihood ratio than a negative test result by EliA CTD Screen, indicating that the negative predictive value was higher for indirect immunofluorescence than for EliA CTD screen.

  12. Indirect Immunofluorescence, Serum Neutralization, and Viremia Responses of Thesus Monkeys (Macaca Mulatta) to Machupo Virus,

    DTIC Science & Technology

    1985-08-22

    immunofluorescent antibody tests (iFAT) have been developed V: for several arenaviruses , none has been applied to the rhesus monkey model for Bolivian...though indirect immunofluorescent antibody tests (IUAT) have been developed for several arenaviruses , none has been applied to the rhesus monkey model for...specific for individual arenaviruses than the SN procedure. Substantial cross- reactivity by IFAT has been described between New World arenaviruses

  13. EUROPattern Suite technology for computer-aided immunofluorescence microscopy in autoantibody diagnostics.

    PubMed

    Krause, C; Ens, K; Fechner, K; Voigt, J; Fraune, J; Rohwäder, E; Hahn, M; Danckwardt, M; Feirer, C; Barth, E; Martinetz, T; Stöcker, W

    2015-04-01

    Antinuclear autoantibodies (ANA) are highly informative biomarkers in autoimmune diagnostics. The increasing demand for effective test systems, however, has led to the development of a confusingly large variety of different platforms. One of them, the indirect immunofluorescence (IIF), is regarded as the common gold standard for ANA screening, as described in a position statement by the American College of Rheumatology in 2009. Technological solutions have been developed aimed at standardization and automation of IIF to overcome methodological limitations and subjective bias in IIF interpretation. In this review, we present the EUROPattern Suite, a system for computer-aided immunofluorescence microscopy (CAIFM) including automated acquisition of digital images and evaluation of IIF results. The system was originally designed for ANA diagnostics on human epithelial cells, but its applications have been extended with the latest system update version 1.5 to the analysis of antineutrophil cytoplasmic antibodies (ANCA) and anti-dsDNA antibodies.

  14. Indirect immunofluorescence assay for intra vitam diagnosis of avian bornavirus infection in psittacine birds.

    PubMed

    Herzog, Sibylle; Enderlein, Dirk; Heffels-Redmann, Ursula; Piepenbring, Anne; Neumann, Daniel; Kaleta, Erhard F; Müller, Hermann; Lierz, Michael; Herden, Christiane

    2010-06-01

    Different avian bornavirus (ABV) genotypes have recently been detected in psittacine birds with proventricular dilatation disease (PDD), an inflammatory fatal central and peripheral nervous system disorder. An indirect immunofluorescence assay (IIFA) for intra vitam demonstration of ABV-specific serum antibodies was established since reverse transcription-PCR (RT-PCR) assays may not detect all ABV variants.

  15. Indirect Immunofluorescence Assay for Intra Vitam Diagnosis of Avian Bornavirus Infection in Psittacine Birds ▿

    PubMed Central

    Herzog, Sibylle; Enderlein, Dirk; Heffels-Redmann, Ursula; Piepenbring, Anne; Neumann, Daniel; Kaleta, Erhard F.; Müller, Hermann; Lierz, Michael; Herden, Christiane

    2010-01-01

    Different avian bornavirus (ABV) genotypes have recently been detected in psittacine birds with proventricular dilatation disease (PDD), an inflammatory fatal central and peripheral nervous system disorder. An indirect immunofluorescence assay (IIFA) for intra vitam demonstration of ABV-specific serum antibodies was established since reverse transcription-PCR (RT-PCR) assays may not detect all ABV variants. PMID:20392921

  16. The use of Biochip immunofluorescence microscopy for the serological diagnosis of epidermolysis bullosa acquisita.

    PubMed

    Marzano, Angelo Valerio; Cozzani, Emanuele; Biasin, Matteo; Russo, Irene; Alaibac, Mauro

    2016-05-01

    Epidermolysis bullosa acquisita is a rare autoimmune bullous disease characterized by the presence of circulating antibodies directed against the collagen type VII. Diagnosis is generally based on clinical history, clinical features, histology, direct and indirect immunofluorescence, immunoblotting and ELISA. Our study aims to determine the validity of the Biochip immunofluorescence microscopy for the serological diagnosis of epidermolysis bullosa acquisita. Six patients with epidermolysis bullosa acquisita and presence of antibodies against type VII collagen confirmed by ELISA were included in the study. Subsequently, all sera of patients were analyzed using Biochip. Antibodies anti-collagen type VII were detected in all sera by means of the Biochip technology. Thus, Biochip shows a good correlation with ELISA and seems to be an appropriate method for the diagnosis of epidermolysis bullosa acquisita. It is an easy, fast and standardized method which could facilitate the diagnosis of this autoimmune bullous disease. We suggest that it could be used as an initial screening test to identify patients with epidermolysis bullosa acquisita.

  17. Automated Evaluation of Crithidia luciliae Based Indirect Immunofluorescence Tests: A Novel Application of the EUROPattern-Suite Technology.

    PubMed

    Gerlach, Stefan; Affeldt, Kai; Pototzki, Lena; Krause, Christopher; Voigt, Jörn; Fraune, Johanna; Fechner, Kai

    2015-01-01

    Systemic lupus erythematosus (SLE) is a severe rheumatic autoimmune disease with various clinical manifestations. Anti-dsDNA antibodies are an important immunological hallmark of SLE and their occurrence represents a major criterion for the diagnosis. Among the commonly applied test systems for determination of anti-dsDNA antibodies, the indirect immunofluorescence test (IIFT) using the flagellated kinetoplastida Crithidia luciliae is considered to be highly disease specific at moderate sensitivity. Since IIFT, however, is claimed to be affected by subjective interpretation and a lack of standardization, there has been an increasing demand for automated pattern interpretation of immunofluorescence reactions in recent years. Corresponding platforms are already available for evaluation of anti-nuclear antibody (ANA) IIFT on HEp-2 cells, the recommended "gold standard" for ANA screening in the diagnosis of various systemic rheumatic autoimmune diseases. For one of these systems, the "EUROPattern-Suite" computer-aided immunofluorescence microscopy (CAIFM), automated interpretation of microscopic fluorescence patterns was extended to the Crithidia luciliae based anti-dsDNA IIFT.

  18. An automated approach to the segmentation of HEp-2 cells for the indirect immunofluorescence ANA test.

    PubMed

    Tonti, Simone; Di Cataldo, Santa; Bottino, Andrea; Ficarra, Elisa

    2015-03-01

    The automatization of the analysis of Indirect Immunofluorescence (IIF) images is of paramount importance for the diagnosis of autoimmune diseases. This paper proposes a solution to one of the most challenging steps of this process, the segmentation of HEp-2 cells, through an adaptive marker-controlled watershed approach. Our algorithm automatically conforms the marker selection pipeline to the peculiar characteristics of the input image, hence it is able to cope with different fluorescent intensities and staining patterns without any a priori knowledge. Furthermore, it shows a reduced sensitivity to over-segmentation errors and uneven illumination, that are typical issues of IIF imaging.

  19. Reconstructing a 3-dimensional image of the results of antinuclear antibody testing by indirect immunofluorescence.

    PubMed

    Murai, Ryosei; Yamada, Koji; Tanaka, Maki; Kuribayashi, Kageaki; Kobayashi, Daisuke; Tsuji, Naoki; Watanabe, Naoki

    2013-01-31

    Indirect immunofluorescence anti-nuclear antibody testing (IIF-ANAT) is an essential screening tool in the diagnosis of various autoimmune disorders. ANA titer quantification and interpretation of immunofluorescence patterns are determined subjectively, which is problematic. First, we determined the examination conditions under which IIF-ANAT fluorescence intensities are quantified. Next, IIF-ANAT was performed using homogeneous, discrete speckled, and mixed serum samples. Images were obtained using Bio Zero BZ-8000, and 3-dimensional images were reconstructed using the BZ analyzer software. In the 2-dimensional analysis, homogeneous ANAs hid the discrete speckled pattern, resulting in a diagnosis of homogeneous immunofluorescence. However, 3-dimensional analysis of the same sample showed discrete speckled-type ANA in the homogeneous background. This study strengthened the current IIF-ANAT method by providing a new approach to quantify the fluorescence intensity and enhance the resolution of IIF-ANAT fluorescence patterns. Reconstructed 3-dimensional imaging of IIF-ANAT can be a powerful tool for routine laboratory examination.

  20. Simple method for the detoxification of wastewater ultrafiltration concentrates for rotavirus assay by indirect immunofluorescence.

    PubMed Central

    Oragui, J I; Mara, D D

    1989-01-01

    A simple method for the detoxification of ultrafiltration concentrates of wastewaters for rotavirus assay by the indirect immunofluorescence technique has been developed. Polyacrylamide (Bio-Gel) or dextran (Sephadex G50) beads were mixed with concentrates (0.5 g/10 ml, wt/vol) of wastewaters seeded with simian rotavirus SA11 and allowed to stand for 2 h. The supernatant was decontaminated with antibiotics and then assayed for rotaviruses. Concentrates from raw sewage and treated effluents seeded with SA11 were used to infect MA104 or LLC MK2 cell lines. The concentrates, particularly those from raw sewage and anaerobic waste stabilization ponds, were very toxic to the tissue culture cells. These toxic effects were determined by the detachment and subsequent loss of cells after incubation with concentrates and assay medium for 24 h. They were either completely eliminated or were reduced by greater than 80% after treatment with beads. PMID:2541662

  1. Staining Pattern Classification of Antinuclear Autoantibodies Based on Block Segmentation in Indirect Immunofluorescence Images

    PubMed Central

    Li, Jiaqian; Tseng, Kuo-Kun; Hsieh, Zu Yi; Yang, Ching Wen; Huang, Huang-Nan

    2014-01-01

    Indirect immunofluorescence based on HEp-2 cell substrate is the most commonly used staining method for antinuclear autoantibodies associated with different types of autoimmune pathologies. The aim of this paper is to design an automatic system to identify the staining patterns based on block segmentation compared to the cell segmentation most used in previous research. Various feature descriptors and classifiers are tested and compared in the classification of the staining pattern of blocks and it is found that the technique of the combination of the local binary pattern and the k-nearest neighbor algorithm achieve the best performance. Relying on the results of block pattern classification, experiments on the whole images show that classifier fusion rules are able to identify the staining patterns of the whole well (specimen image) with a total accuracy of about 94.62%. PMID:25474260

  2. Comparison of the indirect immunobead, radiolabeled, and immunofluorescence assays for immunoglobulin G serum antibodies to human sperm

    SciTech Connect

    Haas, G.G. Jr.; D'Cruz, O.J.; DeBault, L.E. )

    1991-02-01

    The relative sensitivities of the indirect immunobead test, the indirect flo cytometric immunofluorescence assay, and an indirect radiolabeled antiglobulin assay were compared. Eighteen immunobead test positive sera and 18 negative sera were used as the standard for the other two assays. Of the 18 positive sera, 14 (77%) and 5 (27%) were positive in the immunofluorescence assay and the radiolabeled antiglobulin assay, respectively. Four (22%) of the low titer immunobead test positive sera were negative by both the immunofluorescence assay and the radiolabeled antiglobulin assay. However, there was a significant positive correlation between the results of the immunofluorescence assay and the radiolabeled antiglobulin assay (r = 0.73) and between the results of the radiolabeled antiglobulin assay and the titer of the immunobead test (r = 0.82). The use of an unselected sperm population in the radiolabeled antiglobulin assay and the classical indirect immunofluorescence method using methanol-fixed sperm gave false-positive results in the radiolabeled antiglobulin assay and the immunofluorescence assay. These results suggested that immunoglobulin G antisperm antibody positive sera may be reactive both to sperm surface and internalized sperm antigens.

  3. The incidence of antinuclear antibodies (ANA) detected by indirect immunofluorescence assay (IFA) method.

    PubMed

    Karamehic, Jasenko; Subasic, Djemo; Gavrankapetanovic, Faris; Zecevic, Lamija; Eminovic, Izet; Memic, Senaid; Seric, Natasa; Drace, Zahida

    2007-01-01

    Human anti-nuclear antibodies (ANA) in Systemic Lupus Erythematosus (SLE) react specificaly with DNA, RNA, several proteins and ribonucleoproteins. Systemic Lupus Erythematosus is the classic type of polysystemic autoimmune disease. The high frequency of ANA is determined in these patients. Actually, all SLE patients are ANA positive. ANA testing by IFA (Indirect Immunofluorescence Assay) is an excellent screening tool for SLE cases, but it is not so highly specific test. Patients with connective tissue diseases, such as rheumatoid arthritis, scleroderma and dermatomyositis are also frequently positive. Results of IFA ANA have relative low specific degree, and for this reason the titration of these specimens to the end point is usually recommended. Indirect immunoflourescence is reference method for ANA testing. Common substrates are thin sections of rodent organs or various types of cell lines. The cell line substrates are preferable to organ sections, because these rapidly dividing cells have higher level for detection of certain clinically relevant antigens such as (e.g., centromere, SSA (Ro) and Scl-70). In this paper we present the results evaluation of ANA incidence, detected by IFA in serum specimens of corresponding clinical patients, during 2005 and 2006.

  4. Indirect immunofluorescence test and enzyme-linked immunosorbent assay for detection of Campylobacter pylori.

    PubMed Central

    Schaber, E; Umlauft, F; Stöffler, G; Aigner, F; Paulweber, B; Sandhofer, F

    1989-01-01

    An indirect immunofluorescence test (IIF) has been developed for detecting Campylobacter pylori in gastroduodenal biopsies. This test was compared with standard methods of C. pylori diagnosis, namely Gram staining and urease test, in a study population of 226 patients; 121 of the biopsy specimens were cultured for C. pylori as well. C. pylori colonization was detected in 154 of 226 patients (68%) by at least one of these methods (IIF, 96%; Gram staining, 78%; urease test, 60%; cultivation, 55%). Serum samples from 191 patients of the study population were screened for circulating antibodies to C. pylori by an indirect enzyme-linked immunosorbent assay with whole, untreated bacteria as antigen. Of these serum specimens, 140 (73%) revealed absorbance readings above the limit of positivity, which was determined as an optical density of greater than 0.35 at 405/620 nm. Of 132 serum specimens, 128 (97%) from patients with C. pylori detected in biopsies, but only 12 (20%) of 59 specimens from those without C. pylori detection showed elevated specific antibody levels. Our data revealed that IIF proved to be the superior rapid, sensitive, and specific diagnostic method. The correlation between microbiological findings and the immune response favors our enzyme-linked immunosorbent assay as an additional tool in C. pylori diagnosis. PMID:2644295

  5. Automated indirect immunofluorescence evaluation of antinuclear autoantibodies on HEp-2 cells.

    PubMed

    Voigt, Jörn; Krause, Christopher; Rohwäder, Edda; Saschenbrecker, Sandra; Hahn, Melanie; Danckwardt, Maick; Feirer, Christian; Ens, Konstantin; Fechner, Kai; Barth, Erhardt; Martinetz, Thomas; Stöcker, Winfried

    2012-01-01

    Indirect immunofluorescence (IIF) on human epithelial (HEp-2) cells is considered as the gold standard screening method for the detection of antinuclear autoantibodies (ANA). However, in terms of automation and standardization, it has not been able to keep pace with most other analytical techniques used in diagnostic laboratories. Although there are already some automation solutions for IIF incubation in the market, the automation of result evaluation is still in its infancy. Therefore, the EUROPattern Suite has been developed as a comprehensive automated processing and interpretation system for standardized and efficient ANA detection by HEp-2 cell-based IIF. In this study, the automated pattern recognition was compared to conventional visual interpretation in a total of 351 sera. In the discrimination of positive from negative samples, concordant results between visual and automated evaluation were obtained for 349 sera (99.4%, kappa = 0.984). The system missed out none of the 272 antibody-positive samples and identified 77 out of 79 visually negative samples (analytical sensitivity/specificity: 100%/97.5%). Moreover, 94.0% of all main antibody patterns were recognized correctly by the software. Owing to its performance characteristics, EUROPattern enables fast, objective, and economic IIF ANA analysis and has the potential to reduce intra- and interlaboratory variability.

  6. [Comparative study of indirect immunofluorescence (IIF) and ELISA techniques in the detection of parvovirus B19].

    PubMed

    González, M; Hassanhi, M; Rivera, S; Bracho, M P

    2000-03-01

    To determine the seroprevalence of IgG and IgM antibodies against Parvovirus B19 (P. B19), we studied the sera of 53 patients with different hematologic disorders and the sera of 15 controls using indirect immunofluorescence (IFI) and the ELISA method. The prevalence of IgG in the control group was 46.6%, in patients with aplastic crisis was 83.3% (IFI) and 66.7% (ELISA) and, in patients without crisis was 68.9% (IFI) and 72.4% (ELISA). IgM was negative except for patients with crisis: 8.3% (IFI) and 29.1% (ELISA). The higher seroprevalence (IgG) found in patients in comparison with controls might be due to a greater exposure of of patients to the virus. The agreement for both techniques was 81%(IgG) and 93% (IgM) however ELISA technique was more sensitive for detecting IgM of P. B19. In spite of serologic evidence and evaluating a simple serum sample per patient, we could establish an association between aplastic crisis and viral infection for IgM ELISA but not for IgG between hematologic disorders and infection for the P. B19.

  7. Effect of substrate selection on indirect immunofluorescence testing of canine autoimmune subepidermal blistering diseases.

    PubMed

    Favrot, C; Dunston, S; Deslandes, J; Paradis, M; Olivry, T

    2002-01-01

    The detection by indirect immunofluorescence (IIF) of circulating antibodies in the serum of dogs with autoimmune subepidermal blistering diseases (AISBD) was regarded for a long time as an unrewarding tool. It was, however, demonstrated in humans that the sensitivity of IIF assays depended on the selection of the substrates used. The effects of substrate selection on IIF tests was thus studied by examining sera from 12 dogs with AISBD tested against 8 different substrates from 3 different normal dogs. Patients with AISBD suffered from bullous pemphigoid (n = 4 sera), mucous membrane pemphigoid (n = 4 sera), and epidermolysis bullosa acquisita (n = 4 sera). Substrates included canine tongue, canine lip, canine dorsal haired skin, and ventral haired skin. The same 4 substrates were also split with salt splitting technique (using 1 M sodium chloride), in order to cleave the basement membrane within the lamina lucida and to expose the targeted antigens. The strength of the specific fluorescence of each slide was scored after processing for IIF testing with anti-canine IgG polyclonal antibody. Other criteria, such as background fluorescence, easiness of the interpretation, and variations within a same substrate, were also assessed. Intact canine lip and canine salt-split lip demonstrated consistently stronger intensity of fluorescence and a better ease of interpretation. We concluded that the performance of IIF tests with such substrates was a reliable tool for the detection of circulating IgG autoantibodies of canine patients with AISBD.

  8. Automated Image Analysis for Determination of Antibody Titers Against Occupational Bacterial Antigens Using Indirect Immunofluorescence.

    PubMed

    Brauner, Paul; Jäckel, Udo

    2016-06-01

    Employees who are exposed to high concentrations of microorganisms in bioaerosols frequently suffer from respiratory disorders. However, etiology and in particular potential roles of microorganisms in pathogenesis still need to be elucidated. Thus, determination of employees' antibody titers against specific occupational microbial antigens may lead to identification of potentially harmful species. Since indirect immunofluorescence (IIF) is easy to implement, we used this technique to analyze immunoreactions in human sera. In order to address disadvantageous inter-observer variations as well as the absence of quantifiable fluorescence data in conventional titer determination by eye, we specifically developed a software tool for automated image analysis. The 'Fluorolyzer' software is able to reliably quantify fluorescence intensities of antibody-bound bacterial cells on digital images. Subsequently, fluorescence values of single cells have been used to calculate non-discrete IgG titers. We tested this approach on multiple bacterial workplace isolates and determined titers in sera from 20 volunteers. Furthermore, we compared image-based results with the conventional manual readout and found significant correlation as well as statistically confirmed reproducibility. In conclusion, we successfully employed 'Fluorolyzer' for determination of titers against various bacterial species and demonstrated its applicability as a useful tool for reliable and efficient analysis of immune response toward occupational exposure to bioaerosols. © The Author 2016. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.

  9. Automation in indirect immunofluorescence testing: a new step in the evolution of the autoimmunology laboratory.

    PubMed

    Tozzoli, Renato; Antico, Antonio; Porcelli, Brunetta; Bassetti, Danila

    2012-08-01

    Indirect immunofluorescence (IIF) plays an important role in immunological and immunometric assays for detecting and measuring autoantibodies. This technology was the first multiplex method used to detect cardinal autoantibodies for the diagnosis of autoimmune diseases. Over the last 20 years, research has enabled the progressive identification of cell and tissue autoantigens which are the target of autoantibodies originally detected by IIF. Accordingly, newer immunometric methods, capable of measuring concentrations of specific autoantibodies directed against these autoantigens, allowed for a gradual replacement of the IIF method in the autoimmunology laboratory. Currently, IIF remains the method of choice only in selected fields of autoimmune diagnostics. Following the recent statement by the American College of Rheumatology that the IIF technique should be considered as the standard screening method for the detection of ANA, the biomedical industry has developed technological solutions which significantly improve automation of the procedure, not only in the preparation of substrates and slides, but also in microscope reading. This review summarizes the general and specific features of new available commercial systems (Aklides, Medipan; Nova View, Inova; Zenit G Sight, A. Menarini Diagnostics; Europattern, Euroimmun; Helios, Aesku.Diagnostics; Image Navigator, Immuno Concepts; Cytospot, Autoimmun Diagnostika) for automation of the IIF method. The expected advantages of automated IIF are the reduction in frequency of false negative and false positive results, the reduction of intra- and inter-laboratory variability, the improvement of correlation of staining patterns with corresponding autoantibody reactivities, and higher throughput in the laboratory workflow.

  10. Comparison of techniques and immunoreagents used for indirect immunofluorescence and immunoperoxidase identification of enteroviruses.

    PubMed

    Herrmann, J E; Morse, S A; Collins, M F

    1974-07-01

    Peroxidase-conjugated antibodies were found to be as sensitive as those conjugated to fluorescein-isothiocyanate (FITC) for identification of selected enterovirus types by the indirect technique. Peroxidase conjugates, however, were found to give fewer nonspecific reactions. The reasons for the higher specificity of the immunoperoxidase technique appear to be related to the relative size, charge, and uniformity of the preparations. Monomers of peroxidase-conjugated globulin are larger than those of FITC conjugates, but the latter readily form aggregates. This was shown by chromatography on Sepharose 6B columns: various FITC-conjugated globulins eluted before those conjugated to peroxidase and before (125)I-labeled immunoglobulin G. The net charge of the conjugates was determined by adsorption to ion-exchange columns. FITC-labeled globulins had a negative net charge, eluting at pH 5.1 from diethylamino-ethyl-Sephadex A-50 columns. Peroxidase conjugates were not retained by either cationic or anionic exchangers at pH values ranging from 4.0 to 10.5. Further, the fluorescein/protein ratios of FITC conjugates from different commercial sources and of those prepared in the laboratory were found to be variable; higher fluorescein/protein ratios (>2:1) give a higher degree of nonspecific reactions, whereas the peroxidase/protein ratio does not appear to affect specificity. These characteristics of peroxidase conjugates make the immunoperoxidase technique easier to standardize and more reliable for enterovirus identification than the immunofluorescence technique.

  11. Evaluation of antinuclear antibodies by indirect immunofluorescence and line immunoassay methods': four years' data from Turkey.

    PubMed

    Sener, Asli Gamze; Afsar, Ilhan; Demirci, Mustafa

    2014-12-01

    The presence of antinuclear antibodies (ANAs), directed against intracellular antigens, is a hallmark of systemic autoimmune rheumatic diseases. The indirect immunofluorescence (IIF) assay is among the most commonly used routine methods for ANA detection as the screening test. The objective of the study was to evaluate ANA patterns in a 4-year period retrospectively. All 19 996 serum samples that were sent to the Laboratory of Medical Microbiology of the tertiary Hospital by any hospital department between 1 January 2009 and 1 January 2013 with a request to test for ANA, anti-ENA or both were included in the study. Of these samples, 4375 (21.9%) were ANA-IIF-positive and 15621 (78.1%) were ANA-IIF-negative. The presented ANA-positive samples consisted of 2392 (54.67%) homogenous, 818 (18.70%) speckled, 396 (9.05%) centromere, 242 (5.53%) nucleolar, 213 (4.87%) nuclear dots, 178 (4.07%) cytoplasmic (except for actin and golgi), 24 (0.55%) actin, 9 (0.21%) golgi, 53 (1.21%) nuclear membrane and 50 (1.14%) mixed pattern. Totally 7800 samples were examined by LIA. Of these samples, 3440 were positive and 4307 were negative with IIF and LIA. In addition, 22 samples were detected as IIF-positive but LIA-negative, whereas the rest 31 samples were IIF-negative but LIA-positive. ANA patterns in 22 IIF-positive samples were homogenous (9), speckled (5), golgi (4), cytoplasmic (3) and nucleolar (1). SSA/Ro-52, SSB/La and Scl-70 positivity were detected in 31 IIF-negative/LIA-positive samples by LIA. The present study comes forward with its overall scope, which covers 4-year data obtained in tertiary hospital located in the western part of Turkey.

  12. Autoantibody detection with indirect immunofluorescence on HEp-2 cells: starting serum dilutions for systemic rheumatic diseases.

    PubMed

    González, Delia Almeida; León, Antonio Cabrera de; Varela, Alfredo Roces; García, Miriam García; Rahola, Marta de Sequera; Pérez, María Del Cristo Rodríguez; Hernández, Ana González; Falcón, María José Falcón; Díaz, Buenaventura Brito

    2011-10-30

    Antinuclear antibodies (ANA) are determined, among other reasons, to identify samples which need a second test to detect the associated specificities. Our aim was to evaluate the clinical and economic impact generated by using an initial dilution for ANA of 1:160. We analyzed all samples for which ANA, anti-ENA and anti-dsDNA were requested over a 1-year period. ANA were detected by indirect immunofluorescence. Anti-ENA were analyzed with a combination of techniques. Anti-dsDNA were detected by radioimmunoassay. Cost analysis was performed by calculating the difference between two cut-offs (ANA 1:40 and 1:160). A total of 13,233 samples were processed for ANA, of which 59.9% were positive with the 1:40 cut-off and 39.2% with the 1:160 cut-off. At ANA titer 1:40, 0.2% of the samples were anti-ENA-positive and 2.2% were anti-dsDNA positive. Only ANA dilutions of 1:160 and higher showed significantly increased positive predictive value for anti-ENA (1.5 versus 0.2, p=0.029) and anti-dsDNA (8.3 versus 2.2, p<0.001) compared to the 1:40 titer. With the 1:160 cut-off, 16.6% fewer ANA tests, 41.8% fewer anti-ENA determinations and 36.4% fewer anti-dsDNA tests would have been needed. The average saving was 0.87 cost-units per sample (1 unit=2.06euro). We conclude that setting the starting dilution for ANA at 1:160 avoids unnecessary studies, increases the positive predictive values of ANA for anti-ENA and anti-dsDNA, and generates clinical and economic benefits.

  13. Dosimetry of Exposure to Sulfur Mustard of Human Skin: Immunofluorescence Microscopy of DNA-Adducts.

    DTIC Science & Technology

    used to develop methods for dosimetry by immunofluorescence microscopy of adducts to DNA after exposure to HD. This technique might be of great help...distinguished by cell-type specific immunochemical staining. Such techniques will facilitate investigations on the healing of skin injuries....recent years, monoclonal antibodies have been raised against a major HD-DNA adduct , i.e. N7-(2’-hydroxyethylthioethyl)-guanine. These antibodies have been

  14. New automated indirect immunofluorescent antinuclear antibody testing compares well with established manual immunofluorescent screening and titration for antinuclear antibody on HEp-2 cells.

    PubMed

    Daves, M; Blecken, J; Matthias, T; Frey, A; Perkmann, V; Dall Acqua, A; Joos, A; Platzgummer, S

    2017-02-01

    The IIF using the HEp-2 cell substrate should be still considered the "gold standard" techniques for determination of antinuclear antibody (ANA). Standardization and automation can be considered to be still in progress. Aim of this study was to evaluate the performance of the commercially automated indirect immunofluorescent antinuclear HEp-2 antibody assay. The study was designed to compare two commercially available HEp-2 ANA by indirect immunofluorescent antibody assays using a sensitivity panel (120 clinically determined patients) and a specificity panel consisting of 78 clinically confirmed negative patients. We compared the NOVA View(®) system [INOVA Diagnostics San Diego, USA] with the new HELIOS Processor from AESKU Systems/AESKU.Diagnostics (Wendelsheim, Germany) to assess their capability for screening, pattern recognition and titration of the samples. These automated methods were directly compared to manual reading of the same processed slides on respective microscopes and also compared with the known clinical information. The results of the two automated methods were in very good agreement with recognizing negative and positive samples. The HELIOS system detected 188 samples correctly as negative or positive versus 187 detected by the NOVA View(®) system. The diagnostic sensitivity of the systems was 95.8 versus 96.7 % for HELIOS and NOVA View(®), respectively. The systems exhibited a diagnostic specificity of 93.5 % for the HELIOS system and 91.0 % for the NOVA View(®). Both systems are suitable for fast and reliable detection of positivity/negativity due to their high sensitivity and will lead to a further increase of standardization in autoimmunity.

  15. The use of biochip immunofluorescence microscopy for the diagnosis of Pemphigus vulgaris.

    PubMed

    Russo, Irene; Saponeri, Andrea; Peserico, Andrea; Alaibac, Mauro

    2014-06-01

    Pemphigus vulgaris is an autoimmune intraepithelial blistering skin disease characterized by the presence of circulating autoantibodies directed against surfaces of keratinocytes. Diagnosis is generally based on clinical features, histology, direct and indirect immunofluorescence and ELISA. This study describes a new BIOCHIP mosaic-based indirect immunofluorescence technique based on recombinant antigenic substrates and transfected cells. We investigated the diagnostic use of BIOCHIP for the serological diagnosis of Pemphigus vulgaris. Autoantibodies against desmoglein 3 were detected in 97.62% of patients (41/42) with P. vulgaris. There were no positive results in the negative control group. Our study revealed that BIOCHIP has high sensitivity and specificity comparable to that of the ELISA assays. Therefore the BIOCHIP technique seems to be an appropriate method for the diagnosis of P. vulgaris as it has been shown to be a simple, standardized and readily available novel tool, which could facilitate the diagnosis of this autoimmune bullous disease. We suggest that it could be used as an initial screening test to identify patients with P. vulgaris before using the ELISA approach.

  16. [Extramembranous glomerulonephritis of acquired syphilis in a patient recently infected by the hepatitis B virus. Demonstration of the treponemal antigen in the kidney by indirect immunofluorescence].

    PubMed

    Schillinger, F; Montagnac, R; Goclowski, C; Dine, G; Alessandri, E; Hopfner, C; Birembaut, P

    1983-01-22

    A 38 years old male homosexual with active secondary syphilis presented with pure nephrotic syndrome while HBs and HBe tests were positive without clinical hepatitis. He had circulating immune complexes, IgG--IgM cryoglobulinemia and high IgA, IgM and IgE levels; the C3 and C4 complement constituents were normal. Examination of renal biopsy sections under light, fluorescent and electronic microscopy showed stage I membranous glomerulonephritis the syphilitic origin of which was confirmed by indirect immunofluorescence and by rapid cure under penicillin treatment. This case calls for the following comments: (1) glomerular deposits are extramembranous rather than subendothelial in syphilitic nephrosis, a disease now classified among circulating immune complexes diseases; (2) in the kidney, the treponema antigen can be demonstrated by indirect immunofluorescence and the anti-treponema antibody, by elution; (3) the outcome of the nephrotic syndrome is always favourable, either spontaneously or after penicillin treatment; (4) syphilis and HBs antigens are frequently associated, particularly in homosexual patients; one should be looked for when the other is discovered.

  17. Application of photostable quantum dots for indirect immunofluorescent detection of specific bacterial serotypes on small marine animals

    NASA Astrophysics Data System (ADS)

    Decho, Alan W.; Beckman, Erin M.; Chandler, G. Thomas; Kawaguchi, Tomohiro

    2008-06-01

    An indirect immunofluorescence approach was developed using semiconductor quantum dot nanocrystals to label and detect a specific bacterial serotype of the bacterial human pathogen Vibrio parahaemolyticus, attached to small marine animals (i.e. benthic harpacticoid copepods), which are suspected pathogen carriers. This photostable labeling method using nanotechnology will potentially allow specific serotypes of other bacterial pathogens to be detected with high sensitivity in a range of systems, and can be easily applied for sensitive detection to other Vibrio species such as Vibrio cholerae.

  18. Short report: improved diagnosis and follow-up of canine leishmaniasis using amastigote-based indirect immunofluorescence.

    PubMed

    Fernández-Pérez, F J; Méndez, S; de la Fuente, C; Gómez-Muñoz, M T; Cuquerella, M; Alunda, J M

    1999-10-01

    A comparison of an indirect immunofluorescence test using promastigotes (IFATp) or cultured amastigotes (IFATa) in the diagnosis and follow-up of canine leishmaniasis caused by Leishmania infantum was carried out. Results obtained with both diagnostic methods were in good agreement although the IFATa titration was more sensitive than the currently used IFATp without losing specificity. The higher sensitivity of the amastigote-based IFAT resulted in an earlier diagnosis in the absence of clinical signs. Both methods showed comparable results for monitoring the clinical evolution of naturally infected and treated (meglumine antimoniate plus allopurinol) dogs.

  19. Reversible optical control of cyanine fluorescence in fixed and living cells: optical lock-in detection immunofluorescence imaging microscopy

    PubMed Central

    Yan, Yuling; Petchprayoon, Chutima; Mao, Shu; Marriott, Gerard

    2013-01-01

    Optical switch probes undergo rapid and reversible transitions between two distinct states, one of which may fluoresce. This class of probe is used in various super-resolution imaging techniques and in the high-contrast imaging technique of optical lock-in detection (OLID) microscopy. Here, we introduce optimized optical switches for studies in living cells under standard conditions of cell culture. In particular, a highly fluorescent cyanine probe (Cy or Cy3) is directly or indirectly linked to naphthoxazine (NISO), a highly efficient optical switch that undergoes robust, 405/532 nm-driven transitions between a colourless spiro (SP) state and a colourful merocyanine (MC) state. The intensity of Cy fluorescence in these Cy/Cy3-NISO probes is reversibly modulated between a low and high value in SP and MC states, respectively, as a result of Förster resonance energy transfer. Cy/Cy3-NISO probes are targeted to specific proteins in living cells where defined waveforms of Cy3 fluorescence are generated by optical switching of the SP and MC states. Finally, we introduce a new imaging technique (called OLID-immunofluorescence microscopy) that combines optical modulation of Cy3 fluorescence from Cy3/NISO co-labelled antibodies within fixed cells and OLID analysis to significantly improve image contrast in samples having high background or rare antigens. PMID:23267183

  20. Reversible optical control of cyanine fluorescence in fixed and living cells: optical lock-in detection immunofluorescence imaging microscopy.

    PubMed

    Yan, Yuling; Petchprayoon, Chutima; Mao, Shu; Marriott, Gerard

    2013-02-05

    Optical switch probes undergo rapid and reversible transitions between two distinct states, one of which may fluoresce. This class of probe is used in various super-resolution imaging techniques and in the high-contrast imaging technique of optical lock-in detection (OLID) microscopy. Here, we introduce optimized optical switches for studies in living cells under standard conditions of cell culture. In particular, a highly fluorescent cyanine probe (Cy or Cy3) is directly or indirectly linked to naphthoxazine (NISO), a highly efficient optical switch that undergoes robust, 405/532 nm-driven transitions between a colourless spiro (SP) state and a colourful merocyanine (MC) state. The intensity of Cy fluorescence in these Cy/Cy3-NISO probes is reversibly modulated between a low and high value in SP and MC states, respectively, as a result of Förster resonance energy transfer. Cy/Cy3-NISO probes are targeted to specific proteins in living cells where defined waveforms of Cy3 fluorescence are generated by optical switching of the SP and MC states. Finally, we introduce a new imaging technique (called OLID-immunofluorescence microscopy) that combines optical modulation of Cy3 fluorescence from Cy3/NISO co-labelled antibodies within fixed cells and OLID analysis to significantly improve image contrast in samples having high background or rare antigens.

  1. Multiparametric serological testing in autoimmune encephalitis using computer-aided immunofluorescence microscopy (CAIFM).

    PubMed

    Fraune, Johanna; Gerlach, Stefan; Rentzsch, Kristin; Teegen, Bianca; Lederer, Sabine; Affeldt, Kai; Fechner, Kai; Danckwardt, Maick; Voigt, Jörn; Probst, Christian; Komorowski, Lars; Stöcker, Winfried

    2016-10-01

    Autoantibodies against neuronal cell surface antigens are tightly associated with immunotherapy-responsive autoimmune encephalitis, and a considerable number of corresponding autoantigens has been identified in recent years. Most patients initially present with overlapping symptoms, and a broad range of autoantibodies has to be considered to establish the correct diagnosis and initiate treatment as soon as possible to prevent irreversible and sometimes even life-threatening damage to the brain. Recombinant cell-based immunofluorescence allows to authentically present fragile membrane-associated surface antigens and, in combination with multiparametric analysis in the form of biochip mosaics, has turned out to be highly beneficial for parallel and prompt determination of anti-neuronal autoantibodies and comprehensive differential diagnostics. For the evaluation of recombinant cell-based IIFT, a semi-automated novel function was introduced into an established platform for computer-aided immunofluorescence microscopy. The system facilitates the microscopic analysis of the tests and supports the laboratory personnel in the rapid issuance of diagnostic findings, which is of major importance for autoimmune encephalitis patients since timely initiation of treatment may lead to their full recovery. Copyright © 2016. Published by Elsevier B.V.

  2. Microtubules in Plant Cells: Strategies and Methods for Immunofluorescence, Transmission Electron Microscopy and Live Cell Imaging

    PubMed Central

    Celler, Katherine; Fujita, Miki; Kawamura, Eiko; Ambrose, Chris; Herburger, Klaus; Wasteneys, Geoffrey O.

    2016-01-01

    Microtubules are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labelling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography. PMID:26498784

  3. Localization of vicilin peptidohydrolase in the cotyledons of mung bean seedlings by immunofluorescence microscopy

    PubMed Central

    1978-01-01

    Vicilin peptidohydrolase, the protease that hydrolyzes the reserve proteins in the cotyledons of mung bean (Vigna radiata) seedlings, has been localized intracellularly by immunofluorescence microscopy using monospecific antibodies against the enzyme and rhodamine-coupled goat- anti-rabbit immunoglobulin G's. The enzyme can first be visualized after 3 days of seedling growth and is associated with small foci within the cytoplasm of the storage parenchyma cells farthest from the vascular bundles. On the 4th day of growth, the protease is also present in the numerous large protein bodies within these cells. Vicilin peptidohydrolase is known to be synthesized de novo starting on the 3rd day of growth. Our observations are therefore consistent with the interpretation that the enzyme is synthesized in the cytoplasm and subsequently transported to the protein bodies. PMID:359572

  4. Parietal cell surface reactive autoantibody in pernicious anaemia demonstrated by indirect membrane immunofluorescence.

    PubMed Central

    de Aizpurua, H J; Toh, B H; Ungar, B

    1983-01-01

    We examined, in a 'double blind' study, 60 sera from patients with pernicious anaemia for immunofluorescence reactivity with the surface membranes of viable parietal cells isolated from dog stomachs. Fifty-three sera (88%) gave an IgG autoantibody reaction with the surface membranes of parietal cells. Surface staining was also seen with parietal cells from monkey, pig, rat and mouse. The parietal cell surface reactive autoantibody was not found in any of 14 sera from patients with chronic active hepatitis, 10 from patients with systemic lupus erythematosus and 50 from healthy persons. The surface reactivity autoantibody was present in 13 of 14 sera without parietal cell microsomal antibody, 28 of 31 sera without intrinsic factor antibody and in four of four sera without microsomal and intrinsic factor antibodies. Absorption with parietal cell enriched gastric mucosal cells neutralized the activity of the surface reactive but not the microsomal antibody and cross absorption with gastric microsomes neutralized the activity of the microsomal but not the surface reactive antibody. Surface staining of parietal cells was not abolished by absorption with dog or rat hepatocytes, dog or rat kidney cells, human fibroblasts or human AB red blood cells. The results suggest that the parietal cell surface reactive antibody is probably different from the microsomal antibody. Immune reactions of the cell surface reactive antibody with parietal cell surface antigens may play a role in the pathogenesis of the gastric lesion in pernicious anaemia. Images Fig. 1 Fig. 2 PMID:6345039

  5. In-vivo immunofluorescence confocal microscopy of herpes simplex virus type 1 keratitis

    NASA Astrophysics Data System (ADS)

    Kaufman, Stephen C.; Laird, Jeffery A.; Beuerman, Roger W.

    1996-05-01

    The white-light confocal microscope offers an in vivo, cellular-level resolution view of the cornea. This instrument has proven to be a valuable research and diagnostic tool for the study of infectious keratitis. In this study, we investigate the direct visualization of herpes simplex virus type 1 (HSV-1)-infected corneal epithelium, with in vivo confocal microscopy, using HSV-1 immunofluorescent antibodies. New Zealand white rabbits were infected with McKrae strain of HSV-1 in one eye; the other eye of each rabbit was used as an uninfected control. Four days later, the rabbits were anesthetized and a cellulose sponge was applied to each cornea, and a drop of direct HSV fluorescein-tagged antibody was placed on each sponge every 3 to 5 minutes for 1 hour. Fluorescence confocal microscopy was then performed. The HSV-infected corneas showed broad regions of hyperfluorescent epithelial cells. The uninfected corneas revealed no background fluorescence. Thus, using the confocal microscope with a fluorescent cube, we were able to visualize HSV-infected corneal epithelial cells tagged with a direct fluorescent antibody. This process may prove to be a useful clinical tool for the in vivo diagnosis of HSV keratitis.

  6. Demonstration of common antigens on cell surface of Clostridium chauvoei and C. septicum by indirect-immunofluorescence assay.

    PubMed

    Hamaoka, T; Terakado, N

    1994-04-01

    The common antigens between Clostridium chauvoei and C. septicum were examined by indirect-immunofluorescence assay (IFA). Antisera to formalized cells of C. chauvoei and C. septicum strains and to EDTA-soluble antigens of these strains were used. The antisera to formalized cells, which have reacted only with homologous antigens in agglutination tests, reacted not only with homologous antigens but also with heterologous antigens in IFA. The antisera to EDTA-soluble antigens, which have shown no reactivities in somatic agglutination tests, reached with both homologous and heterologous antigens in IFA. These results indicate that these species possess common antigens, which are undetected by agglutination tests, on the cell surface and a some of them are solubilized by EDTA-treatment.

  7. Detection of Schistosoma mansoni Antibodies in a Low-Endemicity Area Using Indirect Immunofluorescence and Circumoval Precipitin Test

    PubMed Central

    Carvalho do Espírito-Santo, Maria Cristina; Pinto, Pedro Luiz; Gargioni, Cybele; Viviana Alvarado-Mora, Monica; Pagliusi Castilho, Vera Lúcia; Pinho, João Ranato Rebello; de Albuquerque Luna, Expedito José; Borges Gryschek, Ronaldo Cesar

    2014-01-01

    Parasitological diagnostic methods for schistosomiasis lack sensitivity, especially in regions of low endemicity. The objective of this study was to determine the prevalence of Schistosoma mansoni infections by antibody detection using the indirect immunofluorescence assay (IFA-IgM) and circumoval precipitin test (COPT). Serum samples of 572 individuals were randomly selected. The IFA-IgM and COPT were used to detect anti-S. mansoni antibodies. Of the patients studied, 15.9% (N = 91) were IFA-IgM positive and 5.1% (N = 29) had COPT reactions (P < 0.001 by McNemar's test). Immunodiagnostic techniques showed higher infection prevalence than had been previously estimated. This study suggests that combined use of these diagnostic tools could be useful for the diagnosis of schistosomiasis in epidemiological studies in areas of low endemicity. PMID:24639303

  8. Expression of Calcitonin Gene-related Peptide in Rat Pulp and Periodontal Tissues by Indirect Immunofluorescence Method

    PubMed Central

    Tao, Rui; Zhang, Mengjie; Cao, Xue; Wang, Hong; Xue, Lufeng; Wu, Meng

    2013-01-01

    The aim of this study was to investigate the expression of nerve fibers immunoreactive to calcitonin gene-related peptide (CGRP) in pulp and periodontal tissues of rats. Male Sprague-Dawley rats, aged 6 weeks, were sacrificed, and the jaws were excised, demineralized, and processed for indirect immunofluorescence staining. A considerably higher density of nerve fibers immunoreactive to CGRP was found in the dental pulp and gingiva than in periodontal ligament. The majority of pulpal CGRP immunopositive fibers that were located followed blood vessels parallel to the long axis of the root. A subodontoblastic network of fibers IR to CGRP was found in the coronal pulp in rat molars. In the periodontium, CGRP immunopositive fibers were mainly located in the periapical area and close to the alveolar bone. Gingiva was also well supplied with CGRP-IR nerves. PMID:24328744

  9. [Evaluation of a mouse anti-IgG-fluorescein conjugate using indirect immunofluorescence and flow cytometry techniques].

    PubMed

    Vilaseca, J C; Pérez, L; Savón, C; Chacón, D; Fernández, S O; Rodríguez, H; Goyenechea, A; Otero, A

    1997-01-01

    An immunoglobulin G of mouse was purified from sera by affinity chromatography in protein A. The rabbits whose sera were able to recognize the antigen injected by double immunodiffusion were immunized with this preparation. The antibodies were precipitated from the rabbit's serum and purified by ion exchange chromatography. This preparation was conjugated to fluorescin isothiocyanate according to the conventional technique. The conjugated obtained was evaluated with the reference strains of Parainfluenza virus 1, 2, 3; Adenovirus; respiratory syncytial virus; and influenza virus A and B, by an indirect immunofluorescence technique and HIV positive samples by flow citometry. Specific monoclonal antibodies were used in both cases. Clinical specimens of patients with acute respiratory infection were evaluated.

  10. [Diagnostics of ataxia-telangiectasia by the express-test found on the method of indirect immunofluorescence].

    PubMed

    Kuranova, M L; Ledashcheva, T A; Tulush, E K; Beliaev, D L; Zherebtsov, S V; Pleskach, N M; Prokof'eva, V V; Mikhel'son, V M; Spivak, I M

    2013-01-01

    Ataxia-telangiectasia (AT) is a hereditary severe neurodegenerative disease developing, when mutations take place in both alleles of the atm gene, which encodes the key protein of the cellular response to DNA damage (DDR)--ATM proteinkinase. In response to the occurrence of double-strand DNA breaks, the ATM proteinkinase pass the autophosphorylation, and its active form--the phospho-ATM (P-ATM) appears in cells. In the nuclei of cells having the atm gene, P-ATM is revealed, being absent in cells with mutated forms of this gene, by means of the application of the modified method of indirect immunofluorescence. This peculiarity may be applied in the clinic, in order to confirm the diagnosis of AT.

  11. Immunofluorescence of spirochetes with serum from swine recovered from swine dysentery using an indirect fluorescent antibody test.

    PubMed Central

    Lee, C H; Olson, L D

    1976-01-01

    Using an indirect fluorescent antibody test, immunofluorescence of large spirochetes was observed with serum from swine that had recovered from swine dysentery. The spirochetes were obtained from scrapings of the colonic mucosa on the first day of diarrhea which was the time when the spirochete population was observed to be the highest. Of 29 exposed nonmedicated swine which developed and recovered from a diarrhea characteristic of swine dysentery 27 had antispirochete serum titers which ranged from 1:2 to 1:16. None of the 50 nonexposes swine developed a titer. Of 19 swine with a serum titer and reexposed with infective swine dysentery inoculum, 18 did not develop a diarrhea and were presumed to be immune. Considering these findings it is possible that this test could be used to detect antispirochete antibody in unknown swine serum. Images Fig. 1. PMID:793698

  12. Comparison between Indirect Immunofluorescence Assay and Shell Vial Culture for Detection of Mumps Virus from Clinical Samples

    PubMed Central

    Reina, Jordi; Ballesteros, Francisca; Ruiz de Gopegui, Enrique; Munar, Maria; Mari, Margarita

    2003-01-01

    We report a prospective comparison of the efficacies of an indirect immunofluorescence assay (IFA) and shell vial culture (SVC) of throat swab and urine samples from patients with mumps. Throat swab samples were used for the IFA; the urine samples and throat swabs were inoculated into vials of Vero cells. We studied 62 patients by using 62 throat swabs and 50 urine samples (50 patients with both samples). Sixty (96.7%) throat samples were positive in the SVC, and 61 (98.3%) were positive in the IFA. For the 50 patients from whom both samples were available, the IFA was positive in 50 (100%) cases, the urine sample was positive in 49 (98%) cases, and the throat swab was positive in 48 (96%) cases (P > 0.05). This comparison of throat swabs and urine samples has shown that the two clinical samples are similar in efficacy. PMID:14605158

  13. Enzyme-linked immunosorbent assay screening then indirect immunofluorescence confirmation of antinuclear antibodies: a statistical analysis.

    PubMed

    Copple, Susan S; Sawitzke, Allen D; Wilson, Andrew M; Tebo, Anne E; Hill, Harry R

    2011-05-01

    The purpose of this study was to analyze antinuclear antibody (ANA) screening by enzyme-linked immunosorbent assay (ELISA) followed by indirect fluorescent antibody (IFA) testing to confirm and characterize the pattern and titer of the antibody. We evaluated 4 ANA ELISAs and 1 HEp-2 IFA substrate in 224 clinically defined serum samples consisting of 30 from systemic lupus erythematosus (SLE) cases, 94 from rheumatoid arthritis cases, and 100 from healthy donors plus 495 serum samples submitted for routine ANA testing and 12 reference serum samples distributed by the Centers for Disease Control and Prevention. IFA tests were read independently by 2 certified medical technologists. ELISA sensitivities ranged from 90% to 97% compared with 80% by IFA in the SLE serum samples. The ELISAs had specificities of 36% to 94%, whereas the IFA had 99% specificity. Overall, ELISAs for ANA assays demonstrated better sensitivity and good specificity, suggesting ELISA is a more cost-effective alternative to IFA testing for initial ANA screening. Samples positive by ANA ELISA should be tested on HEp-2 to determine the titer and pattern.

  14. Furcation Perforation: Periradicular Tissue Response to Biodentine as a Repair Material by Histopathologic and Indirect Immunofluorescence Analyses.

    PubMed

    Silva, Lea Assed Bezerra; Pieroni, Karina Alessandra Michelão Grecca; Nelson-Filho, Paulo; Silva, Raquel Assed Bezerra; Hernandéz-Gatón, Patrícia; Lucisano, Marília Pacífico; Paula-Silva, Francisco Wanderley Garcia; de Queiroz, Alexandra Mussolino

    2017-07-01

    The purpose of this study was to evaluate the in vivo response of periradicular tissues after sealing of furcation perforations with Biodentine, mineral trioxide aggregate (MTA), and gutta-percha by means of histopathologic and indirect immunofluorescence analyses. Thirty teeth of 3 dogs were divided into 3 groups: Biodentine (n = 14 teeth), MTA (negative control, n = 10 teeth), and gutta-percha (positive control, n = 6 teeth). After endodontic treatment, perforations were made on the center of the pulp chamber floor and filled with the materials. After 120 days, the animals were killed, and blocks containing the teeth and periradicular tissues were processed histotechnically for histopathologic semiquantitative (new mineralized tissue formation and bone resorption at the perforation site) and quantitative (thickness and area of newly formed mineralized tissue and number of inflammatory cells) analyses and RUNX2 immunofluorescence assay. Data were analyzed by χ(2), Fisher exact test, Mann-Whitney test, one-way analysis of variance, Kruskal-Wallis test, and Dunn posttest (α = 0.05). MTA and Biodentine induced the formation of significantly more new mineralized tissue (P < .0001) than gutta-percha, which did not induce the formation of mineralized tissue in any case. Complete sealing of the perforations was more frequent with MTA, which formed mineralized tissue with greater thickness and area. Biodentine and MTA exhibited no bone resorption in the furcation region, fewer inflammatory cells, and greater RUNX2 immunostaining intensity than gutta-percha. Although MTA presented higher frequency of complete sealing and greater thickness and area of newly formed mineralized tissue, Biodentine also had good histopathologic results and can be considered as an adequate furcation perforation repair material. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  15. Comparison of complement-dependent cytotoxicity and indirect immunofluorescence for enumeration of T-cell subpopulations in human peripheral blood.

    PubMed

    Gratama, J W; Schuurman, R K; Van Leeuwen, A; Jansen, J; Oljans, P; Tanke, H J; Van Rood, J J

    1983-11-11

    The monitoring of T-lymphocyte subsets of recipients of organ grafts enables studies on immune reconstitution (after bone-marrow transplantation) and may predict graft rejection (after kidney transplantation). Quantitation of human peripheral T-lymphocyte subsets from healthy volunteers and from recipients of a bone-marrow graft by a complement dependent cytotoxicity (CDC) assay, based on the use of propidium iodide, and by an indirect immunofluorescence (IIF) technique has been compared using the monoclonal antibodies OKT3, OKT4 and OKT8. Except for OKT3 in healthy individuals--for which no significant difference was found between CDC and IIF--CDC detected significantly more cells of each subset than IIF. Furthermore, the CDC results indicated the presence of low numbers of OKT4+8+ cells in the peripheral blood of healthy individuals and--with higher numbers--following marrow transplantation. Results of depletion experiments, obtained by fluorescence activated cell sorting (FACS) for either OKT4 or OKT8, supported this conclusion. OKT4/OKT8 ratios were calculated from enumerations by the CDC assay and by the IIF assay and found to be linearly related, both in healthy persons and in marrow-graft recipients. Thus, the CDC assay is a reliable method for monitoring T-cell subsets, allowing detection of lymphocytes carrying low densities of membrane determinants.

  16. Sexing murine embryos with an indirect immunofluorescence assay using phage antibody B9-Fab against SDM antigen

    PubMed Central

    WANG, Naidong; YUAN, Anwen; MA, Jun; DENG, Zhibang; XUE, Liqun

    2015-01-01

    The use of serologically detectable male (SDM; also called H-Y) antigens to identify male embryos may be limited by the source of anti-SDM antibody. In the present study, novel anti-SDM B9-Fab recombinant clones (obtained by chain shuffling of an A8 original clone) were used to detect SDM antigens on murine embryos. Murine morulae and blastocysts (n=138) were flushed from the oviducts of Kunming mice and incubated with anti-SDM B9-Fab for 30 min at 37°C. With an indirect immunofluorescence assay, the membrane and inner cell mass had bright green fluorescence (presumptive males). Overall, 43.5% (60/138) were classified as presumptive males and 56.5% (78/138) as presumptive females, with 85.0 and 88.5% of these, respectively, confirmed as correct predictions (based on PCR analysis of a male-specific [Sry] sequence). We concluded that the anti-SDM B9-Fab molecule had potential for non-invasive, technically simple immunological sexing of mammalian embryos. PMID:25715803

  17. The nuclear pore complex protein Tpr is a common autoantigen in sera that demonstrate nuclear envelope staining by indirect immunofluorescence.

    PubMed

    Ou, Y; Enarson, P; Rattner, J B; Barr, S G; Fritzler, M J

    2004-05-01

    We studied the autoantigen targets of 75 human sera that had antibodies to the nuclear envelope (NE) as identified by indirect immunofluorescence (IIF) on HEp-2 cells. Several different IIF staining patterns could be identified when antibodies to different components of the nuclear membrane (NM) and nuclear pore complexes (NuPC) were identified: a smooth membrane pattern characteristic of antibodies to nuclear lamins, a punctate pattern typical of antibodies to the nuclear pore complex and more complex patterns that included antibodies to nuclear and cytoplasmic organelles. Western immunoblotting of isolated nuclear and NE proteins and immunoprecipitation of radiolabelled recombinant proteins prepared by using the full-length cDNAs of the Translocated promoter region (Tpr), gp210 and p62 were used to identify specific autoantibody targets. Fifty-two of the 75 (70%) sera bound to Tpr, 25 (33%) bound to lamins A, B or C, 15 (20%) reacted with gp210 and none reacted with p62. Sixteen (21%) did not react with any of the NE components tested in our assays. The clinical features of 37 patients with anti-NE showed that there were 34 females and three males with an age range of 16-88 years (mean 59 years). The most frequent clinical diagnosis (9/37 = 24%) was autoimmune liver disease (ALD; two with primary biliary cirrhosis), followed by seven (19%) with systemic lupus erythematosus (SLE), four (11%) with a motor and/or sensory neuropathy, three (8%) with anti-phospholipid syndrome (APS), two with systemic sclerosis (SSc), two with Sjögren's syndrome (SjS), and others with a variety of diagnoses. This report indicates that Tpr, a component of the NuPC, is a common target of human autoantibodies that react with the NE.

  18. [Evaluation of a Computer-Aided Microscope System and Its Anti-Nuclear Antibody Test Kit for Indirect Immunofluorescence Assay].

    PubMed

    Hayashi, Nobuhide; Saegusa, Jun; Uto, Kenichi; Oyabu, Chinami; Saito, Toshiharu; Sato, Itsuko; Kawano, Seiji; Kumagai, Shunichi

    2016-02-01

    Antinuclear antibody (ANA) testing is indispensable for diagnosing and understanding clinical conditions of autoimmune diseases. The indirect immunofluorescence assay (IFA) is the gold standard for ANA screening, and it can detect more than 100 different antibodies, such as anti-PCNA as well as anti-cytoplasmic antibodies. However, complicated procedures of conventional IFA and visual interpretation require highly skilled laboratory staff. This study evaluates the capability, characteristics, and applicability of the recently developed ANA detection system (EUROPattern Cosmic IFA System, EPA) using HEp20-10 cells and the automated pattern recognition microscope. Findings using EPA and conventional methods were compared in 282 sera obtained from connective tissue disease patients and 250 sera from healthy individuals. The concordance of the positivity rate, antibody titer (within +/- 1 tube difference), and the accurate recognition rate of ANA patterns between the automated EPA method and the microscopic judgement of the EPA image by eye was 98.9, 97.4, and 55.3%, respectively. The EPA method showed concordance of the positivity rate as high as 93.3% and concordance of the antibody titer as high as 94.0% (within +/- 1 titer) compared with the conventional method. Regarding the four typical patterns of ANA (homogeneous, speckled, nucleolar, and centromere), large differences between the EPA and conventional methods were not observed, and the rate of concordance between the final EPA result and the conventional method was from 94.1 to 100%. The positivity rate of ANA using the EPA and conventional methods showed marked agreement among the six connective tissue diseases (SLE, MCTD, SSc, PM/DM, and SS) and healthy individuals. Although the EPA system is not considered a complete system and laboratory staff should verify the results, it is a useful system for routine ANA analysis because it contributes to ANA standardization and an efficient workflow.

  19. Multiplex RT-PCR and indirect immunofluorescence assays for detection and subtyping of human influenza virus in Tunisia.

    PubMed

    Ben M'hadheb, Manel; Harrabi, Myriam; Souii, Amira; Jrad-Battikh, Nadia; Gharbi, Jawhar

    2015-03-01

    Influenza viruses are negative stranded segmented RNA viruses belonging to Orthomyxoviridae family. They are classified into three types A, B, and C. Type A influenza viruses are classified into subtypes according to the antigenic characters of the surface glycoproteins: hemagglutinin (H) and neuraminidase (N). The aim of the present study is to develop a fast and reliable multiplex RT-PCR technique for detecting simultaneously the subtypes A/H1N1 and A/H3N2 of influenza virus. Our study included 398 patients (mean age 30.33 ± 19.92 years) with flu or flu-like syndromes, consulting physicians affiliated with collaborating teams. A multiplex RT-PCR detecting A/H1N1 and A/H3N2 influenza viruses and an examination by indirect immunofluorescence (IFI) were performed. In the optimized conditions, we diagnosed by IFI a viral infection in 90 patients (22.6 %): 85 cases of influenza type A, four cases of influenza type B, and only one case of coinfection with types A and B. An evaluation of the technique was performed on 19 clinical specimens positive in IFI, and we detected eight cases of A/H3N2, five cases of A/H1N1, one case of influenza virus type A which is not an H1N1 nor H3N2, and five negative cases. Multiplex RT-PCR is a sensitive technique allowing an effective and fast diagnosis of respiratory infections caused by influenza viruses in which the optimization often collides with problems of sensibility.

  20. Indirect immunofluorescence assay for the simultaneous detection of antibodies against clinically important old and new world hantaviruses.

    PubMed

    Lederer, Sabine; Lattwein, Erik; Hanke, Merle; Sonnenberg, Karen; Stoecker, Winfried; Lundkvist, Åke; Vaheri, Antti; Vapalahti, Olli; Chan, Paul K S; Feldmann, Heinz; Dick, Daryl; Schmidt-Chanasit, Jonas; Padula, Paula; Vial, Pablo A; Panculescu-Gatej, Raluca; Ceianu, Cornelia; Heyman, Paul; Avšič-Županc, Tatjana; Niedrig, Matthias

    2013-01-01

    In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n=97); SEOV (China, n=5); DOBV (Romania, n=7); SNV (Canada, n=23); ANDV (Argentina and Chile, n=52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n=177; 96%) and/or IgM (n=131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV).

  1. Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses

    PubMed Central

    Lederer, Sabine; Lattwein, Erik; Hanke, Merle; Sonnenberg, Karen; Stoecker, Winfried; Lundkvist, Åke; Vaheri, Antti; Vapalahti, Olli; Chan, Paul K. S.; Feldmann, Heinz; Dick, Daryl; Schmidt-Chanasit, Jonas; Padula, Paula; Vial, Pablo A.; Panculescu-Gatej, Raluca; Ceianu, Cornelia; Heyman, Paul; Avšič-Županc, Tatjana; Niedrig, Matthias

    2013-01-01

    In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n = 97); SEOV (China, n = 5); DOBV (Romania, n = 7); SNV (Canada, n = 23); ANDV (Argentina and Chile, n = 52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n = 177; 96%) and/or IgM (n = 131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV). PMID:23593524

  2. Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy.

    PubMed

    Sun, Yulong; Ip, Philbert; Chakrabartty, Avijit

    2017-09-03

    Immunofluorescence is a common method used to visualize subcellular compartments and to determine the localization of specific proteins within a tissue sample. A great hindrance to the acquisition of high quality immunofluorescence images is endogenous autofluorescence of the tissue caused by aging pigments such as lipofuscin or by common sample preparation processes such as aldehyde fixation. This protocol describes how background fluorescence can be greatly reduced through photobleaching using white phosphor light emitting diode (LED) arrays prior to treatment with fluorescent probes. The broad-spectrum emission of white phosphor LEDs allow for bleaching of fluorophores across a range of emission peaks. The photobleaching apparatus can be constructed from off-the-shelf components at very low cost and offers an accessible alternative to commercially available chemical quenchers. A photobleaching pre-treatment of the tissue followed by conventional immunofluorescence staining generates images free of background autofluorescence. Compared to established chemical quenchers which reduced probe as well as background signals, photobleaching treatment had no effect on probe fluorescence intensity while it effectively reduced background and lipofuscin fluorescence. Although photobleaching requires more time for pre-treatment, higher intensity LED arrays may be used to reduce photobleaching time. This simple method can potentially be applied to a variety of tissues, particularly postmitotic tissues that accumulate lipofuscin such as the brain and cardiac or skeletal muscles.

  3. Differentiation of antineutrophil nuclear antibodies in inflammatory bowel and autoimmune liver diseases from antineutrophil cytoplasmic antibodies (p-ANCA) using immunofluorescence microscopy.

    PubMed

    Terjung, B; Worman, H J; Herzog, V; Sauerbruch, T; Spengler, U

    2001-10-01

    Perinuclear antineutrophil cytoplasmic antibodies (p-ANCA) directed against cytoplasmic proteins of neutrophils have been studied extensively in patients with systemic vasculitides. Recent data indicate that antineutrophil antibodies in sera from patients with chronic inflammatory bowel diseases (IBD) or autoimmune liver disorders, currently called 'atypical p-ANCA', recognize a nuclear target antigen, rendering the term 'ANCA' inaccurate. Specific microscopic criteria to distinguish atypical p-ANCA from p-ANCA are lacking. We used planar and confocal laser scanning indirect immunofluorescence microscopy to examine the labelling characteristics of ethanol-, methanol- and formaldehyde-fixed neutrophils by antineutrophil antibodies in 153 serum samples from patients with IBD, autoimmune liver disorders, systemic vasculitides or healthy blood donors. On ethanol- or methanol-fixed neutrophils, multiple intranuclear fluorescent foci together with either a rim-like peripheral nuclear staining ('type A') or a combined cytoplasmic and peripheral nuclear staining ('type B') was noted exclusively with atypical p-ANCA in sera from patients with IBD or autoimmune liver disorders. Intranuclear foci, which probably corresponded to invaginations of the nuclear envelope, were not labelled by p-ANCA from patients with microscopic polyangiitis or cytoplasmic ANCA (c-ANCA) from patients with Wegener's granulomatosis. On formaldehyde-fixed neutrophils, atypical p-ANCA gave a fine rim-like staining of the nuclear periphery, whereas ANCA diffusely labelled the cytoplasm. To distinguish reliably between the patterns produced by atypical p-ANCA or p-ANCA, particularly p-ANCA, careful indirect immunofluorescence microscopy on ethanol- as well as on formaldehyde-fixed neutrophils is necessary, with particular emphasis on the presence of multiple intranuclear fluorescent foci.

  4. Evaluation of Vircell Enzyme-Linked Immunosorbent Assay and Indirect Immunofluorescence Assay for Detection of Antibodies against Legionella pneumophila

    PubMed Central

    Diederen, Bram M. W.; Kluytmans, Jan A. J. W.; Peeters, Marcel F.

    2006-01-01

    We evaluated the abilities of the Vircell immunoglobulin G (IgG) and IgM indirect immunofluorescence assay (IFA) for Legionella pneumophila serogroup 1, the IgM and IgG enzyme-linked immunosorbent assay (ELISA) for Legionella pneumophila serogroup 1, and the IgM-plus-IgG ELISA for Legionella pneumophila serogroups 1 to 6 to diagnose Legionnaires' disease (LD) in a well-described sample of patients with and without LD. Also, we determined the agreements, sensitivities, and specificities of the different Vircell assays in comparison to a validated ELISA (Serion classic ELISA). Clinical sensitivity and specificity were 74.6% and 96.6%, respectively, for the IgM IFA, 65.1% and 88.0% for the IgG IFA, 92.3% and 100% for the IgM ELISA, 43.3% and 96.6% for the IgG ELISA, and 90.8% and 100% for the IgM-plus-IgG ELISA. Compared to Serion classic ELISA, agreement, sensitivity, and specificity were 80.0%, 83.1%, and 78.4%, respectively, for the IgM IFA, 75.2%, 66.0%, and 79.5% for the IgG IFA, 89.5%, 82.0%, and 97.6% for the IgM ELISA, 81.9%, 88.9%, and 78.0% for the IgG ELISA, and 93.5%, 90.0%, and 96.6% for the IgM-plus-IgG ELISA. The value of a positive diagnostic result obtained by the Vircell IgM IFA, the Vircell IgG IFA, and the Vircell IgG ELISA might not be acceptable for a diagnostic assay. Both the high specificities and sensitivities of the Vircell IgM ELISA and the IgM-plus-IgG ELISA and the high correlation with the Serion classic ELISA indicate that they are useful in the diagnosis of LD. PMID:16522778

  5. Specific chemoluminescence and immunoasdorption tests for anti-DFS70 antibodies avoid false positive results by indirect immunofluorescence.

    PubMed

    Bizzaro, Nicola; Tonutti, Elio; Tampoia, Marilina; Infantino, Maria; Cucchiaro, Francesco; Pesente, Fiorenza; Morozzi, Gabriella; Fabris, Martina; Villalta, Danilo

    2015-12-07

    To evaluate two new diagnostic methods for the identification of anti-DFS70 antibodies in samples showing a DFS70-staining pattern by indirect immunofluorescence (IIF). We studied 731 patients: 576 were collected consecutively among those that in the ANA test on HEp-2 cells had produced a DFS70 fluorescence pattern and 155 were a consecutive series of patients sent by referring physicians for routine ANA testing. As controls we studied 50 patients with autoimmune diseases and 120 patients with active infectious disease. All 731 sera were assayed for anti-DFS70 antibodies by a specific chemoluminescence assay (CLIA); 70 randomly selected IIF-positive sera and 35 samples from patients with autoimmune diseases were studied by inhibition tests using the HEp-2 Select method. Assays performed with the CLIA-DFS70 method were positive in 30.4% of the samples presenting a DFS70 pattern by IIF, in 1.3% of the routine ANA sera, in 1.6% of the infectious sera and in none of the 50 autoimmune controls. However, as the IIF-DFS70 positive group included 106 patients with systemic autoimmune rheumatic diseases (SARD), 11 of which were DFS70 positive by CLIA, the prevalence of DFS70 antibodies in SARD was 7.5%. The ANA test performed after the use of HEp-2 Select showed an inhibition in 95.7% of the sera. No change in fluorescence intensity and pattern morphology between the native sera and the same sera tested with the solution containing the DFS70 antigen was observed in the 35 samples from patients with autoimmune diseases. To avoid misinterpretation of ANA pattern and consequent diagnostic errors, confirmation of the DFS70-IIF pattern by CLIA or other specific methods is mandatory before reporting the presence of anti-DFS70 antibodies. The HEp-2 Select test in most cases eliminates the interference by anti-DFS70 antibodies and avoids the possible reporting of false positive results. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. [The possibilities for using anti-A, anti-B and anti-H immunoreagents obtained by new technology in direct and indirect immunofluorescence reactions].

    PubMed

    Iudina, G S; Zaretskaia, E F

    1999-01-01

    The authors propose to prepare group-specific immunoreagents anti-A, anti-B, and anti-H for direct and indirect immunofluorescence from non-precipitated antisera of the appropriate specificities by a new technology making use of routine bioadsorbents and synthetic oligosaccharides. The method has been developed at Research Center of Forensic Medical Expert Evaluations of the Russian Ministry of Health. The immunoreagents were tried on a vast scope of experimental and expert specimens at several institutions.

  7. Development of confocal immunofluorescence FRET microscopy to Investigate eNOS and GSNOR localization and interaction in pulmonary endothelial cells

    NASA Astrophysics Data System (ADS)

    Rehman, Shagufta; Brown-Steinke, Kathleen; Palmer, Lisa; Periasamy, Ammasi

    2015-03-01

    Confocal FRET microscopy is a widely used technique for studying protein-protein interactions in live or fixed cells. Endothelial nitric oxide synthase (eNOS) and S-nitrosoglutathione reductase (GSNOR) are enzymes involved in regulating the bioavailability of S-nitrosothiols (SNOs) in the pulmonary endothelium and have roles in the development of pulmonary arterial hypertension. Labeling of endogenous proteins to better understand a disease process can be challenging. We have used immunofluorescence to detect endogenous eNOS and GSNOR in primary pulmonary endothelial cells to co-localize these proteins as well as to study their interaction by FRET. The challenge has been in selecting the right immunofluorescence labeling condition, right antibody, the right blocking reagent, the right FRET pair and eliminating cross-reactivity of secondary antibodies. We have used Alexa488 and Alexa568 as a FRET pair. After a series of optimizations, the data from Confocal Laser Scanning Microscopy (CLSM) demonstrate co-localization of eNOS and GSNOR in the perinuclear region of the pulmonary endothelial cell primarily within the cis-Golgi with lower levels of co-localization seen within the trans-Golgi. FRET studies demonstrate, for the first time, interaction between eNOS and GSNOR in both murine and bovine pulmonary endothelial cells. Further characterization of eNOSGSNOR interaction and the subcellular location of this interaction will provide mechanistic insight into the importance of S-nitrosothiol signaling in pulmonary biology, physiology and pathology.

  8. Diagnostic immunofluorescence*

    PubMed Central

    Cherry, William B.; Reimer, Charles B.

    1973-01-01

    The standardization of diagnostic immunofluorescence is a complex problem because diagnostic results are greatly influenced by interacting factors, such as the equipment, materials, and techniques for expressing and recording fluorescence. Furthermore, the characteristics of immunofluorescence reagents depend on how they are manufactured and used. The adoption of stable reference preparations of such reagents appears to be the only practicable way of standardizing laboratory test results. Several professional and regulatory organizations are actively promoting this objective. Consensus evaluation may be the best method of introducing proposed standards. Basic and applied research must provide the information needed to improve reagents and tests. Material fluorescent standards are proving helpful in standardizing fluorescence emission, but the most promising development is the use of insolubilized antigens to provide standards for more relevant immunological-fluorescence comparisons. Several important direct and indirect diagnostic immunofluorescence tests and reagents currently used in microbiological, histological, and pathological examinations require standardization. The medical profession should insist that commercial reagents be adequately characterized and that manufacturers supply the data necessary for their safe and informed use. PMID:4594319

  9. Monoclonal Antibody-Based Immunofluorescence Microscopy for the Rapid Identification of Burkholderia pseudomallei in Clinical Specimens

    PubMed Central

    Tandhavanant, Sarunporn; Wongsuvan, Gumphol; Wuthiekanun, Vanaporn; Teerawattanasook, Nittaya; Day, Nicholas P. J.; Limmathurotsakul, Direk; Peacock, Sharon J.; Chantratita, Narisara

    2013-01-01

    The diagnosis of melioidosis depends on the culture of Burkholderia pseudomallei, which takes at least 48 hours. We used a polyclonal-FITC-based immunofluorescence microscopic assay (Pab-IFA) on clinical samples to provide a rapid presumptive diagnosis. This has limitations including photobleaching and batch-to-batch variability. This study evaluated an IFA based on a monoclonal antibody specific to B. pseudomallei (Mab-IFA) and Alexa Fluor 488. A diagnostic evaluation was performed on a prospective cohort of 951 consecutive patients with suspected melioidosis. A total of 1,407 samples were tested. Test accuracy was defined against culture as the gold standard, and was also compared against Pab-IFA. A total of 88 samples from 64 patients were culture positive for B. pseudomallei. The diagnostic sensitivity and specificity of the Mab-IFA was comparable to the Pab-IFA (48.4% versus 45.3% for sensitivity, and 99.8% versus 98.8% for specificity). We have incorporated the Mab-IFA into our routine practice. PMID:23716405

  10. Dual channel confocal laser scanning microscopy of lucifer yellow-microinjected human brain cells combined with Texas red immunofluorescence.

    PubMed

    Belichenko, P V; Dahlström, A

    1994-06-01

    A method for visualization of individual human brain cells and their dendritic extensions in combination with immunofluorescence is described. Microinjection of Lucifer Yellow was used to reveal the dendritic morphology of cortical brain cells. Indirect immunofluorescence with Texas Red as label was used to investigate the distribution of 3 different groups of immunogens: enzymes (monoamine oxidase A and B), receptors (beta-adrenoceptor protein), and synaptic vesicle proteins (synapsin I and synaptophysin) in each cortical slice. A dual-channel confocal laser scanning microscope with an argon/krypton laser was used for imaging these double-stained fluorescent specimens. Lucifer Yellow and Texas Red were recorded simultaneously or separately, taking advantage of the different activating lines (488 lambda and 568 lambda) of the laser and using the two filter blocks (K1 and K2) supplied with the instrument (BioRad MRC-600) for recording the emission of either fluorophore. Using this technique we have demonstrated the localization of immunoreactive material in relation to the dendritic morphology of cortical cells.

  11. Detection of platelet antibodies by enzyme-linked immunosorbent assay (ELISA). Comparative studies with the indirect immunofluorescence assay.

    PubMed

    Horai, S; Claas, F H; van Rood, J J

    1981-08-01

    A newly developed enzyme-linked immunosorbent assay (ELISA) for the detection of platelet antibodies was compared with the platelet immunofluorescence test (PIF). A good correlation was found between both assays. However, ELISA seems to be more sensitive than PIF. Some sera reacted only in ELISA whereas no sera were found that were negative in ELISA and positive in PIF. When comparing the antibody titres, ELISA is at least 8 times more sensitive than PIF.

  12. Detecting seasonal variation of antifreeze protein distribution in Rhagium mordax using immunofluorescence and high resolution microscopy.

    PubMed

    Buch, J L; Ramløv, H

    2017-02-01

    Larvae of the blackspotted pliers support beetle, Rhagium mordax, were collected monthly, for the duration of 2012 and fixed. The larvae were embedded in paraffin wax and sectioned. Using fluorophore-coupled antibodies specific to the R. mordax antifreeze protein, RmAFP1, sections were visualised with UV reflected light microscopy. An automated software analysis method was developed in order to discard autofluorescence, and quantify fluorescence from bound antibodies. The results show that R. mordax cuticle and gut exhibit a higher degree of fluorophore-bound fluorescence during summer, than in the cold months. It is hypothesised that R. mordax stores RmAFP1 in, or near, the fat body during times when freeze avoidance is not needed.

  13. Automated Analysis of siRNA Screens of Virus Infected Cells Based on Immunofluorescence Microscopy

    NASA Astrophysics Data System (ADS)

    Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

    We present an image analysis approach as part of a high-throughput microscopy screening system based on cell arrays for the identification of genes involved in Hepatitis C and Dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in cells, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behavior of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

  14. [The automated analysis of anti-nuclear antibodies using technique of indirect reaction of immunofluorescence with application of HEP-2-cells].

    PubMed

    Aleksandrova, E N; Verijnikova, J G; Novikov, A A; Baranov, A A; Abaitova, N E; Lapkina, N A; Roggenbuk, D; Nasonov, E L

    2015-03-01

    The identification of antinuclear antibodies in blood serum based on indirect reaction of immunofluorescence using cells of line HEp-2 (IRIF HEp-2)--a "golden standard" and key screening technique of laboratory diagnostic of autoimmune rheumatic diseases. The automated systems of interpretation of samples offluorescence promote standardization and increase effectiveness of detection of content of antinuclear antibodies with IRIF HEp-2 technique. The study was organized to comparatively analyze automated and visual interpretation of results of IRIF HEp-2 in detection of content antinuclear antibodies in patients with rheumatic diseases. The level of antinuclear antibodies in blood serums of 1178 patients with rheumatic diseases was detected using IRIF HEp-2 technique. The results of IRIF HEp-2 were evaluated by visual microscopy and using automated platform "AKLIDES". The degree of consistency of positive/negative results of detection (k = 0.5), types (k = 0.7) and titers/intensity of fluorescence (k = 0.45) of antinuclear antibodies under automated and traditional interpretation of IRIF HEp-2 was "good". The discordance of positive/negative results of analysis of content of IRIF HEp-2 was established in 18.5% of patients. The automated technique more often detected homogeneous (37.6%) and speckled (32.3%) fluorescence of nucleus. At the same time, there were no differentiation of type of fluorescence in 21.4% of patients. The visual technique detected mixed type of fluorescence in blood serums of most of the patients (72.8%). The mixed fluorescence was identified by system "AKLIDES" as homogeneous (40.5%), speckled (32.7%), nucleolar (2.4%), centromeric (0.9%), undifferentiated (23.5%). Under visual analysis of samples of fluorescence with undifferentiated type of fluorescence was identified as mixed (79.8%), homogeneous (5.9%) and speckled (14.3%). The titers of antinuclear antibodies less than 1:160 associated with intensity of fluorescence 0/B±; 1:160-0, B

  15. A case of nonscarring inflammatory epidermolysis bullosa acquisita: characterization of IgG autoantibodies by immunofluorescence, immunoblotting and immunogold electron microscopy.

    PubMed

    Honoki, K; Muramatsu, T; Nakatani, C; Iida, T; Shirai, T

    1998-10-01

    We report a case of nonscarring inflammatory epidermolysis bullosa acquisita in a 59-year-old Japanese woman. She developed blisters and erosions on her lip, trunk and extremities. Sodium aurothiomalate was effective for the skin lesions. The patient had been free from bullous skin lesions for the last 13 years and had shown no scarring. Indirect immunofluorescence (IF) study on 1 M NaCl-split skin revealed IgG autoantibodies against the dermal side of the split skin. Immunoblotting using normal human dermal extracts disclosed IgG autoantibodies reactive with the 290 and 145 kD antigens. Circulating IgG autoantibodies were deposited on the lamina densa by immunoelectron microscopy. IF mapping using several antibodies for the components of the basement membrane zone revealed blister formation at the lamina densa. These results suggest that the cleavage at the lamina lucida does not necessarily exclude the diagnosis of EBA and that the definite diagnosis of EBA should be confirmed by immunoblotting or immunoelectron microscopic study.

  16. Immunofluorescence and confocal microscopy for ex-vivo diagnosis of melanocytic and non-melanocytic skin tumors: a pilot study.

    PubMed

    Hartmann, Daniela; Krammer, Sebastian; Vural, Secil; Bachmann, Mario Raphael; Ruini, Cristel; Sárdy, Miklós; Ruzicka, Thomas; Berking, Carola; von Braunmühl, Tanja

    2017-09-26

    Ex-vivo confocal laser scanning microscopy (ex-vivo CLSM) offers rapid examination of freshly excised tissue. During the conventional examination immunohistochemistry enables to distinguish various cell types. The possibility of immunofluorescent techniques could enhance the accuracy of the diagnosis performed by ex-vivo CLSM. The tissue probes from various skin tumors were stained with FITC labelled S-100A10, Melan-A and anti-Ber-EP4 antibodies before examination with ex-vivo CLSM in the fluorescence (FM) and reflectance modes (RM). Results were compared to negative controls and conventional histopathology. The staining protocols were evaluated by establishing a scoring system according to the signal intensity found in ex-vivo CLSM. S100 immunostaining was successful in 55.6%. Dilution of 1:200 resulted in the best possible evaluation of the tumor. The best suitable protocol was protocol B (phosphate buffered saline /PBS/, without blocking agent). Melan A immunostaining was positive in 66.7%; 1:500 was the best dilution and protocol B (PBS, without blocking agent) the most suitable. Ber-EP4 immunostaining presented a signal in 85.7%, the best dilutions were 1:200 and 1:500 and protocol A (PBS, with blocking agent) showed most optimal results. The use of fluorescent-labeled antibodies in ex-vivo CLSM is possible and could improve intraoperative diagnostics of skin tumors. This article is protected by copyright. All rights reserved.

  17. Quantitative immunofluorescence microscopy of subcellular GLUT4 distribution in human skeletal muscle: effects of endurance and sprint interval training.

    PubMed

    Bradley, Helen; Shaw, Christopher S; Worthington, Philip L; Shepherd, Sam O; Cocks, Matthew; Wagenmakers, Anton J M

    2014-07-01

    Increases in insulin-mediated glucose uptake following endurance training (ET) and sprint interval training (SIT) have in part been attributed to concomitant increases in glucose transporter 4 (GLUT4) protein content in skeletal muscle. This study used an immunofluorescence microscopy method to investigate changes in subcellular GLUT4 distribution and content following ET and SIT. Percutaneous muscle biopsy samples were taken from the m. vastus lateralis of 16 sedentary males in the overnight fasted state before and after 6 weeks of ET and SIT. An antibody was fully validated and used to show large (> 1 μm) and smaller (<1 μm) GLUT4-containing clusters. The large clusters likely represent trans-Golgi network stores and the smaller clusters endosomal stores and GLUT4 storage vesicles (GSVs). Density of GLUT4 clusters was higher at the fibre periphery especially in perinuclear regions. A less dense punctate distribution was seen in the rest of the muscle fibre. Total GLUT4 fluorescence intensity increased in type I and type II fibres following both ET and SIT. Large GLUT4 clusters increased in number and size in both type I and type II fibres, while the smaller clusters increased in size. The greatest increases in GLUT4 fluorescence intensity occurred within the 1 μm layer immediately adjacent to the PM. The increase in peripheral localisation and protein content of GLUT4 following ET and SIT is likely to contribute to the improvements in glucose homeostasis observed after both training modes.

  18. Combined analysis of in situ hybridization, cell cycle and structural markers using reflectance and immunofluorescence confocal microscopy.

    PubMed

    Linares-Cruz, G; Millot, G; De Cremoux, P; Vassy, J; Olofsson, B; Rigaut, J P; Calvo, F

    1995-01-01

    A method for the simultaneous detection of mRNA by reflectance in situ hybridization (RISH), cell cycle and structural markers by immunofluorescence using confocal laser scanning microscopy is presented. The mRNA expression of two ras-related genes rhoB and rhoC was analysed in human breast cancer cell lines and human histological specimens (breast cancer tissues and skin biopsies). In breast cancer cell lines, the conditions were optimized to detect RNA-RNA hybrids and DNA synthesis after pulse-labelling with bromodeoxyuridine. Endonuclease-exonuclease digestion, which allows the accessibility to specific antibodies of halogenated pyrimidine molecules, was carried out following ISH. Finally, cytokeratin or vimentin staining was performed. The detection of signals, arising from 1-nm colloidal gold particles without silver enhancement, by reflectance confocal laser scanning microscopy is described. Bromodeoxybiridine DNA markers and cytokeratin/vimentin staining were detected concomitantly using different fluorochromes. To allow comparative expression of two related genes, the mRNA of rhoB and rhoC were detected using digoxigenin- or biotin-labelled riboprobes and, after 3-D imaging, a detailed analysis by optical horizontal (x, y) and vertical (x, z) sectioning was undertaken. The subsequent bromodeoxyuridine detection procedure permitted to us explore the specific transcription of these two genes during S and non-S phases. This method allows the identification and localization of several subcellular components in cells within a complex tissue structure and makes it possible to analyse further transcript localization in relation to the function of the encoded protein and to the cell cycle.

  19. Indirect immunofluorescence on HEp-2000 substrate is a sensitive method for detecting anti-Ro/SSA antibodies in patients with lupus erythematosus and/or photosensitivity.

    PubMed

    Cozzani, Emanuele; Drosera, Massimo; Siccardi, Marina; Babbini, Giovanni; Parodi, Aurora

    2008-07-01

    To evaluate the sensitivity of indirect immunofluorescence (IIF) utilizing HEp-2000 cells to detect antinuclear and anti-Ro/SSA antibodies. We examined 5949 sera samples from 2316 patients with connective tissue diseases. All sera were tested by IIF on HEp-2 cells as substrate and by counterimmunoelectrophoresis (CIE). The positive sera for anti-Ro/SSA antibodies by CIE were retested by IIF using HEp-2000 cells. The sera that were anti-Ro/SSA-positive in CIE but negative on HEp-2000 cells were tested by ELISA for the 60 and 52 subunits of Ro/SSA antigen. Our results confirm the high sensitivity (81%) of HEp-2000 cells in detecting anti Ro/SSA antibodies. Using the HEp-2000 substrate is a reliable, simple alternative method for detecting anti-Ro/SSA antibodies in a first-level screening test such as IIF, especially in patients with lupus erythematosus and/or photosensitivity.

  20. Accuracy of molecular diagnostics in pemphigus and bullous pemphigoid: comparison of commercial and modified mosaic indirect immunofluorescence tests as well as enzyme-linked immunosorbent assays

    PubMed Central

    Seraszek-Jaros, Agnieszka; Bowszyc-Dmochowska, Monika; Kaczmarek, Elżbieta; Pietkiewicz, Paweł; Bartkiewicz, Paweł; Dmochowski, Marian

    2017-01-01

    Introduction Pemphigus and bullous pemphigoid (BP) are identified by autoantibodies (abs) against desmoglein 1, 3 (DSG1/3) and BP180/BP230, respectively. A novel mosaic to indirect immunofluorescence (IIF) using purified BP180 recombinant proteins spotted on slide and transfected cells expressing BP230, DSG1, DSG3 is available. The commercial (IgG detection) and modified (IgG4 detection) mosaic for indirect immunofluorescence (IIFc – IIF commercial, IIFm – IIF modified) and IgG ELISAs were evaluated in pemphigus and bullous pemphigoid (BP) molecular diagnostics. Aim To compare diagnostic accuracy of commercial (IgG detection) and modified (IgG4 detection) mosaic IIF assay and to examine the diagnostic value of ELISAs in relation to mosaic IIF in routine laboratory diagnostics of pemphigus and BP. Material and methods Sera from 37 BP and 19 pemphigus patients were studied. Associations between tests were assessed using Fisher’s exact test. Results There are associations between the positive/negative samples detected by IIFc with desmoglein1 (DSG1)/desmoglein3 (DSG3)/BP230 transfected cells and ELISAs and no association between anti-BP180 IgG detection by IIFc and ELISA. IIFm with DSG1 and DSG3 showed both 100% sensitivity and 100% and 78% specificity, respectively, and 100% and 83% positive predictive value in relation to IIFc. IIFm with BP230 had 87% specificity, 55% sensitivity, whereas IIFm with BP180 had a 100% sensitivity and 13% specificity in relation to IIFc. Conclusions The IIFc with DSG1/DSG3/BP230 transfected cells, excluding BP180 spots, is an alternative method to ELISA in pemphigus/BP diagnostics. IgG4 antibodies, both pathogenically and diagnostically important, are inconsistently detectable with IIFm. PMID:28261028

  1. An indirect immunofluorescence assay using a cell culture-derived antigen for detection of antibodies to the agent of human granulocytic ehrlichiosis.

    PubMed Central

    Nicholson, W L; Comer, J A; Sumner, J W; Gingrich-Baker, C; Coughlin, R T; Magnarelli, L A; Olson, J G; Childs, J E

    1997-01-01

    An indirect immunofluorescence assay for the detection of human antibodies to the agent of human granulocytic ehrlichiosis (HGE) was developed and standardized. Antigen was prepared from a human promyelocytic leukemia cell line (HL-60) infected with a tick-derived isolate of the HGE agent (USG3). Suitable antigen presentation and preservation of cellular morphology were obtained when infected cells were applied and cultured on the slide, excess medium was removed, and cells were fixed with acetone. Use of a buffer containing bovine serum albumin and goat serum reduced background fluorescence, and use of an immunoglobulin G (gamma-specific) conjugate reduced nonspecific binding. The assay readily detected specific antibody from HGE patients and did not detect antibody from healthy individuals. No significant reactivity was noted in sera from patients with high titers of antibodies to other rickettsial species. We were able to identify antibodies reactive to USG3 antigen in samples from areas where HGE is endemic that had tested negative to other rickettsial agents. Animal sera reactive against Ehrlichia equi or Ehrlichia phagocytophila bound to the HGE antigen, indicating that the assay may be useful for veterinary use. Comparability between two different laboratories was assessed by using coded human sera exchanged between laboratories. Results from the two laboratories were similar, indicating that the assay can be easily integrated into use for routine testing for HGE. The assay was then compared to an assay using horse neutrophils infected with ehrlichiae. The two assays gave comparable results, indicating that the cell culture-derived antigen can be used for testing samples that have been previously tested with E. equi as an antigen. The new assay offers several advantages over other immunofluorescence methods that use animal-derived antigen and is suitable for use in testing for human antibodies to the HGE agent. PMID:9163471

  2. Serological diagnosis of autoimmune bullous skin diseases: prospective comparison of the BIOCHIP mosaic-based indirect immunofluorescence technique with the conventional multi-step single test strategy.

    PubMed

    van Beek, Nina; Rentzsch, Kristin; Probst, Christian; Komorowski, Lars; Kasperkiewicz, Michael; Fechner, Kai; Bloecker, Inga M; Zillikens, Detlef; Stöcker, Winfried; Schmidt, Enno

    2012-08-09

    Various antigen-specific immunoassays are available for the serological diagnosis of autoimmune bullous diseases. However, a spectrum of different tissue-based and monovalent antigen-specific assays is required to establish the diagnosis. BIOCHIP mosaics consisting of different antigen substrates allow polyvalent immunofluorescence (IF) tests and provide antibody profiles in a single incubation. Slides for indirect IF were prepared, containing BIOCHIPS with the following test substrates in each reaction field: monkey esophagus, primate salt-split skin, antigen dots of tetrameric BP180-NC16A as well as desmoglein 1-, desmoglein 3-, and BP230gC-expressing human HEK293 cells. This BIOCHIP mosaic was probed using a large panel of sera from patients with pemphigus vulgaris (PV, n=65), pemphigus foliaceus (PF, n=50), bullous pemphigoid (BP, n=42), and non-inflammatory skin diseases (n=97) as well as from healthy blood donors (n=100). Furthermore, to evaluate the usability in routine diagnostics, 454 consecutive sera from patients with suspected immunobullous disorders were prospectively analyzed in parallel using a) the IF BIOCHIP mosaic and b) a panel of single antibody assays as commonly used by specialized centers. Using the BIOCHIP mosaic, sensitivities of the desmoglein 1-, desmoglein 3-, and NC16A-specific substrates were 90%, 98.5% and 100%, respectively. BP230 was recognized by 54% of the BP sera. Specificities ranged from 98.2% to 100% for all substrates. In the prospective study, a high agreement was found between the results obtained by the BIOCHIP mosaic and the single test panel for the diagnosis of BP, PV, PF, and sera without serum autoantibodies (Cohen's κ between 0.88 and 0.97). The BIOCHIP mosaic contains sensitive and specific substrates for the indirect IF diagnosis of BP, PF, and PV. Its diagnostic accuracy is comparable with the conventional multi-step approach. The highly standardized and practical BIOCHIP mosaic will facilitate the serological

  3. Comparison of indirect immunofluorescence assays for diagnosis of scrub typhus and murine typhus using venous blood and finger prick filter paper blood spots.

    PubMed

    Phetsouvanh, Rattanaphone; Blacksell, Stuart D; Jenjaroen, Kemajittra; Day, Nicholas P J; Newton, Paul N

    2009-05-01

    We performed indirect immunofluorescence assays (IFAs) to compare levels of IgM and IgG antibodies to Orientia tsutsugamushi and Rickettsia typhi in admission-phase serum samples and filter paper blood spots (assayed immediately and stored at 5.4 degrees C and 29 degrees C for 30 days) collected on the same day from 53 adults with suspected scrub typhus and murine typhus admitted to Mahosot Hospital Vientiane, Lao People's Democratic Republic. The sensitivities and specificities of admission-phase filter paper blood spots in comparison to paired sera were between 91% and 95% and 87% and 100%, respectively, for the diagnosis of scrub typhus and murine typhus. The classification of patients as having or not having typhus did not significantly differ after storage of the blood spots for 30 days (P > 0.4) at 5.4 degrees C and 29 degrees C. Because filter paper blood samples do not require sophisticated and expensive storage and transport, they may be an appropriate specimen collection technique for the diagnosis of rickettsial disease in the rural tropics.

  4. Development of a Recombinant Cell-Based Indirect Immunofluorescence Assay for the Determination of Autoantibodies against Soluble Liver Antigen in Autoimmune Hepatitis

    PubMed Central

    Radzimski, Christiane; Probst, Christian; Teegen, Bianca; Rentzsch, Kristin; Blöcker, Inga Madeleine; Dähnrich, Cornelia; Schlumberger, Wolfgang; Stöcker, Winfried; Bogdanos, Dimitrios P.; Komorowski, Lars

    2013-01-01

    Autoantibodies against soluble liver antigen (SLA) are specific markers for autoimmune hepatitis (AIH) type 1. In contrast to the determination of other AIH-associated autoantibodies by indirect immunofluorescence assay (IFA), detection of anti-SLA relied up to now on ELISA or immunoblot based on bacterially expressed recombinant protein. In order to develop a complementary IFA substrate, SLA isoform 1 was recombinantly produced in the human cell line HEK293 and controlled by a rabbit hyperimmune serum against SLA. The recombinant cells were used in IFA (RC-IFA) to analyze sera from 20 AIH patients with anti-SLA positivity predetermined by ELISA together with 80 controls (20 anti-SLA negative AIH, 15 primary biliary cirrhosis, 15 HCV, and 30 healthy blood donors). Using RC-IFA, anti-SLA was detected in all ELISA positive AIH sera but in none of the controls. Furthermore, a cytosolic fraction of HEK293 containing SLA was able to neutralize the autoantibodies in all positive sera in a dose-dependent manner. HEK293 cells expressing SLA are a valid substrate for the serodiagnosis of AIH relevant autoantibodies by IFA. In concert with cryosections of primate liver, rat kidney, rat liver, rat stomach, and HEp-2 cells, they enable the parallel determination of all autoantibodies associated with autoimmune liver diseases. PMID:23401700

  5. Development of a recombinant cell-based indirect immunofluorescence assay for the determination of autoantibodies against soluble liver antigen in autoimmune hepatitis.

    PubMed

    Radzimski, Christiane; Probst, Christian; Teegen, Bianca; Rentzsch, Kristin; Blöcker, Inga Madeleine; Dähnrich, Cornelia; Schlumberger, Wolfgang; Stöcker, Winfried; Bogdanos, Dimitrios P; Komorowski, Lars

    2013-01-01

    Autoantibodies against soluble liver antigen (SLA) are specific markers for autoimmune hepatitis (AIH) type 1. In contrast to the determination of other AIH-associated autoantibodies by indirect immunofluorescence assay (IFA), detection of anti-SLA relied up to now on ELISA or immunoblot based on bacterially expressed recombinant protein. In order to develop a complementary IFA substrate, SLA isoform 1 was recombinantly produced in the human cell line HEK293 and controlled by a rabbit hyperimmune serum against SLA. The recombinant cells were used in IFA (RC-IFA) to analyze sera from 20 AIH patients with anti-SLA positivity predetermined by ELISA together with 80 controls (20 anti-SLA negative AIH, 15 primary biliary cirrhosis, 15 HCV, and 30 healthy blood donors). Using RC-IFA, anti-SLA was detected in all ELISA positive AIH sera but in none of the controls. Furthermore, a cytosolic fraction of HEK293 containing SLA was able to neutralize the autoantibodies in all positive sera in a dose-dependent manner. HEK293 cells expressing SLA are a valid substrate for the serodiagnosis of AIH relevant autoantibodies by IFA. In concert with cryosections of primate liver, rat kidney, rat liver, rat stomach, and HEp-2 cells, they enable the parallel determination of all autoantibodies associated with autoimmune liver diseases.

  6. [Comparison of the indirect immunofluorescence assay performance of Bartonella henselae antigens obtained by co-cultivation in Vero and HeLa cells].

    PubMed

    Ergin, Cağrı; Akkaya, Yüksel; Kiriş Satılmış, Ozgün; Yılmaz, Cansev

    2011-07-01

    The laboratory diagnosis of Bartonella henselae infection is mainly based on serological testing by indirect immunofluorescence assay (IFA). Cell line co-cultivation with B.henselae and agar derivated antigens are the two major procedures used for evaluation of anti-Bartonella antibodies. Vero and Hep-2 cell lines are the most commonly used media for co-cultivation both in-house and commercial diagnostic kits production. However, HeLa cells which are easily supplied and grown, also can easily be infected by B.henselae. The aim of this study was to compare the performances of antigens obtained by co-cultivation of B.henselae ATCC 49882 (Houston-1) in Vero and HeLa Cells in IFA serology. Out of 381 sera samples, 127 (33.3%) were found positive and 195 (51.2%) were found negative by IFA performed by both cell line co-cultivations. The total agreement between the methods were found as 84.5% (322/381), and Cohen kappa value was calculated as 0.68 (strong, coherent). As a result, He-La cells were found to be useful for the preparation of B.henselae antigens to be used in IFA for the serologic diagnosis of B.henselae infections. However different genotype strains and cross reactions with other infectious agents should be investigated by further studies before routine applications of HeLa cell co-cultivations procedure is established.

  7. Hep-2 cell based indirect immunofluorescence assay for antinuclear antibodies as a potential diagnosis of drug-induced autoimmunity in nonclinical toxicity testing.

    PubMed

    Hong, Min; Ma, Ben; Lin, Zhi; Zhou, Xiaobing; Geng, Xingchao; Shen, Lianzhong; Li, Bo

    2015-03-01

    Antinuclear antibodies (ANAs) are important biomarkers in the diagnosis of autoimmune diseases in humans; however, the diagnostic performance of ANA in nonclinical safety studies are not well understood. Here, we studied the use of ANAs as potential nonclinical biomarkers for drug-induced autoimmunity (DIA) using a Hep-2 based indirect immunofluorescence assay (IFA). Initially, MRL-fas(lpr)/J mice and HgCl₂-treated rats were used as SLE-positive models. Serum samples obtained from 94 normal mice or 204 normal rats aged one to four months served as the negative control. The IFA effectively distinguished ANAs-positive samples in both species with a cut-off titer of 1:100. Brown Norway rats were treated with 450 mg/kg D-penicillamine for 30 consecutive days. ANAs were generated and corresponded with DIA development. Human Hep-2 cells, mice Neuro 2A cells, and Chinese Hamster Lung cells served as antigen from different species, which were found cross-reactive with ANA-positive serum samples from mice, rats, and humans without any differences in diagnosis. This methodology showed no species-specificity for ANA detection. Furthermore, we found approximately 20 percentage of the mice aged seven to eight months demonstrated age-related ANAs, which was consistent with humans. Overall, our findings demonstrated the use of ANA detection using IFA in the nonclinical diagnosis of murine drug-induced autoimmunity, and age-related ANAs should be considered when aged animals are used.

  8. Use of anti-CD3/CD28 mAb coupled magnetic beads permitting subsequent phenotypic analysis of activated human T cells by indirect immunofluorescence.

    PubMed

    Pène, Jérôme; Rahmoun, Massilva; Temmerman, Stéphane; Yssel, Hans

    2003-12-01

    Functional analysis of T lymphocytes requires in vitro stimulation of these cells under experimental conditions that mimic as closely as possible physiological in vivo stimulation and that involve antigen/T cell receptor (TCR)-mediated activation. Because of the low frequency of antigen-specific T cells in human clinical samples, stimulation with a combination of anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) is a preferred method. Interaction of these mAbs with their ligand results in modulation of the mAb-ligand complex from the cell surface. However, as a result of incomplete modulation, CD3/CD28 mAb complexes often remain at the cell surface, thereby precluding subsequent indirect immunofluorescence and flow cytometry analysis using mouse immunoglobulin (Ig)-specific antibodies. This is of importance in situations in which no specific fluorochrome-conjugated mAbs are available, such as in screening procedures of Ig-containing hybridoma culture supernatants. We propose here the use of CD3/CD28 mAbs, linked to magnetic beads allowing standardization of the activation conditions, optimal activation of T cells and complete modulation of antigen-antibody complexes from the cell surface.

  9. Diagnostic accuracy of indirect immunofluorescence assay for intestinal invasive amebiasis and impact of HIV infection in a non-endemic country.

    PubMed

    Nagata, Naoyoshi; Shimbo, Takuro; Akiyama, Junichi; Niikura, Ryota; Watanabe, Koji; Oka, Shinichi; Uemura, Naomi

    2012-12-01

    To diagnose amebic colitis (AC), serologic tests have the advantage of being inexpensive, noninvasive, and easy to perform, but few studies have investigated their utility, especially in a non-endemic country. A total of 299 symptomatic patients (165 HIV-infected patients) who underwent endoscopy and indirect immunofluorescence (IF) assay were analyzed between 2003 and 2009. The diagnosis of AC was defined as detection of amebic trophozoites from biopsy specimens or intestinal fluid sample via endoscopy. Forty-five patients (29 HIV-infected patients) were diagnosed with AC. The area under the receiver-operating characteristic curve (ROC-AUC) for the IF assay was excellent (0.90), and a cut-off value of 100 provided 89% sensitivity and 87% specificity. ROC-AUC was slightly lower in patients with HIV infection (0.88) than in those without HIV infection (0.94). Among HIV-infected patients, ROC-AUC showed no significant differences between different CD4+ cell counts. The IF assay is useful for diagnosing AC in symptomatic patients with and those without HIV infection. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Autoantibody Levels and Clinical Disease Severity in Patients with Pemphigus: Comparison of Aggregated Anti-desmoglein ELISA Values and Indirect Immunofluorescence Titres.

    PubMed

    Weiss, Doris; Ristl, Robin; Griss, Johannes; Bangert, Christine; Foedinger, Dagmar; Stingl, Georg; Brunner, Patrick M

    2015-05-01

    Detecting serum-autoantibodies by anti-Desmoglein-1 (anti-Dsg1) and anti-Dsg3 ELISAs as well as indirect immunofluorescence (IIF) are established complementary methods to diagnose pemphigus. Whether autoantibody levels also reflect clinical disease activity is still a matter of debate, as head-to-head comparisons of ELISA values and IIF titres with clinical activity over a longer treatment period are scarce. In our retrospective study, we compared aggregated repetitive intra-patient ELISA values and IIF titres with grades of clinical disease (1 = remission, 2 = moderate activity, 3 = exacerbation) in 47 patients suffering from pemphigus vulgaris (PV, n = 36) and pemphigus foliaceus (PF, n=11). We found that anti-Dsg1 ELISA values in PF and mucocutaneous PV as well as anti-Dsg3 ELISA values in PV best reflect disease activity. IIF titres, by contrast, did not show a significant association with disease severity. From these data we conclude that ELISA index values can be a valuable tool to monitor disease in patients with pemphigus, whereas IIF titres reflect clinical activity only insufficiently.

  11. Evaluation of automated multi-parametric indirect immunofluorescence assays to detect anti-neutrophil cytoplasmic antibodies (ANCA) in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA).

    PubMed

    Csernok, Elena; Damoiseaux, Jan; Rasmussen, Niels; Hellmich, Bernhard; van Paassen, Pieter; Vermeersch, Pieter; Blockmans, Daniel; Cohen Tervaert, Jan-Willem; Bossuyt, Xavier

    2016-07-01

    The aim of this multicenter EUVAS study was to evaluate the diagnostic performance of multi-parametric indirect immunofluorescence (IIF) assays to detect anti-neutrophil cytoplasmic antibodies (ANCA) in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). The study included 912 samples from diseased controls and 249 diagnostic samples from GPA (n=183) and MPA (n=66) patients. The performance of two automated multi-parametric assays [Aklides (Medipan/Generic Assays) and EuroPattern (Euroimmun)] combining IIF on cellular and purified antigen substrates was compared with two manual IIF analyses and with commercially available ELISAs for MPO- and PR3-ANCA (Euroimmun). The area under the curve (AUC) of the receiver operating characteristics (ROC) curve to discriminate AAV from controls was 0.925, 0.848, 0.855 and 0.904 for, respectively, the two manual analyses, Aklides and EuroPattern, and 0.959, 0.921 and 0.886 for, respectively, antigen-specific ELISA, antigen-coated beads, and microdot, respectively. Variation in pattern assignment between IIF methods was observed. The performance of IIF depends on the substrate used and the definition of IIF patterns. The performance of automated IIF is improved by multi-parameter testing (combined IIF and antigen-specific testing). Given the variability between IIF methods, the diagnostic importance of this technique is questioned. Copyright © 2016. Published by Elsevier B.V.

  12. Comparison of indirect immunofluorescent-antibody assay, enzyme-linked immunosorbent assay, and Western immunoblot for the diagnosis of Lyme disease in dogs.

    PubMed Central

    Lindenmayer, J; Weber, M; Bryant, J; Marquez, E; Onderdonk, A

    1990-01-01

    Enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent-antibody assay (IFA), and Western immunoblot were used to test serum samples from 128 dogs for the presence of antibody to Borrelia burgdorferi. Sera included 72 samples from dogs suspected of having Lyme disease, 32 samples from dogs residing in areas in which Lyme disease was not considered endemic, and 24 samples from dogs with clinical and serologic evidence of immune-mediated disease (n = 10), Rocky Mountain spotted fever (n = 5), or leptospirosis (n = 9). Results of Western immunoblotting were used as the standard against which performances of ELISA and IFA were measured. ELISA was significantly more sensitive than IFA (84.8 versus 66.7%), although both tests were equally specific (93.5%). Eight samples that were positive by Western immunoblot were simultaneously negative by ELISA and IFA. Of these eight, four were from dogs suspected of having immune-mediated disease, two were from dogs suspected of having leptospirosis, and two were from dogs suspected of having Lyme disease. These results may indicate that sera from dogs with immune-mediated disease, and to a lesser extent sera from those with leptospirosis, cross-react with B. burgdorferi antigens. Alternatively, Western immunoblot results may not truly reflect Lyme disease status, particularly in the case of dogs with immune-mediated diseases. At present, however, the use of Western immunoblotting as a diagnostic standard for dogs offers the best alternative to a clinical definition of disease. PMID:2405018

  13. Development of an indirect immunofluorescence assay for diagnosis of bovine viral diarrhoea virus on ear notch tissue samples in cattle infected persistently.

    PubMed

    Bedeković, Tomislav; Lemo, Nina; Lojkić, Ivana; Cvetnić, Zeljko; Cač, Zeljko; Madić, Josip

    2011-12-01

    Bovine viral diarrhoea virus (BVDV) causes a disease that has a wide range of clinical symptoms in domestic and wild ruminants. It is a major problem in cattle and causes significant economic losses in the cattle industry. The virus infects bovines of all ages and causes both immunosuppression and reproductive, respiratory and digestive disorders. Cattle infected persistently, as a continuing source of the virus and the main factor in transmission of the disease between and among herds, are the main source of BVDV and a primary factor in the epidemiology of the disease. To determine whether a BVDV infection is persistent, two samples should be taken at 3-4 week intervals and tested for the virus antigen. Animal sera, whole blood, organ and ear notch tissue samples can be used for BVDV diagnosis. In ear notch tissue, viral antigen can be detected by an antigen enzyme-linked immunosorbent assay (antigen ELISA), immunohistochemistry (IHC) and reverse-transcription polymerase chain reaction (RT-PCR). This paper describes the development and implementation of an indirect immunofluorescence (IF) method using ear notch tissue samples for diagnosis of cattle infected persistently. Results obtained by this method show that IF is a good alternative to RT-PCR and antigen ELISA and can be a quick and accurate method in diagnosis of BVDV in cattle infected persistently with this virus.

  14. Detection of anti-Leishmania infantum antibodies in sylvatic lagomorphs from an epidemic area of Madrid using the indirect immunofluorescence antibody test.

    PubMed

    Moreno, Inmaculada; Álvarez, Julio; García, Nerea; de la Fuente, Santiago; Martínez, Irene; Marino, Eloy; Toraño, Alfredo; Goyache, Joaquin; Vilas, Felipe; Domínguez, Lucas; Domínguez, Mercedes

    2014-01-31

    An outbreak of human leishmaniasis was confirmed in the southwest of the province of Madrid, Spain, between July 2009 and December 2012. Incidence of Leishmania infection in dogs was unchanged in this period, prompting a search for alternative sylvatic infection reservoirs. We evaluated exposure to Leishmania in serum samples from animals in the area with an indirect immunofluorescence test (IFAT). Using promastigotes from six culture passages and a 1/25 threshold titer, we found anti-Leishmania infantum seroreactivity in 9.3% of cats (4 of 43), 45.7% of rabbits (16/35) and 74.1% of hares (63/85). Use of promastigotes from >10 in vitro passages resulted in a notably IFAT lower titer, suggesting antigenic changes during extended culture. Postmortem inspection of seropositive animals showed no clinical signs of infection. The results clearly suggest that asymptomatic hares were the main reservoir in the outbreak, and corroborate IFAT as a sensitive serological surveillance method to detect such cryptic Leishmania infections.

  15. Application of an indirect immunofluorescent staining method for detection of Salmonella enteritidis in paraffin slices and antigen location in infected duck tissues.

    PubMed

    Yan, Bin; Cheng, An-Chun; Wang, Ming-Shu; Deng, Shu-Xuan; Zhang, Zhen-Hua; Yin, Nian-Chun; Cao, Ping; Cao, Sheng-Yan

    2008-02-07

    To detect Salmonella enteritidis (S. enteritidis) in paraffin slices and antigen location in infected duck tissues. The rabbits were immunized with purified bacillus to obtain S. enteritidis-specific antibody, which were then extracted by the caprylic-ammonium sulphate method, purified through High-Q columns. An indirect immuno-fluorescent staining method (IFA) was established to detect the S. enteritidis antigen in paraffin slices. Detected S. enteritidis in each organ tissue of ducklings experimentally infected with S. enteritidis. The gland of Garder, heart, kidney, spleen, liver, brain, ileum, jejunum, bursa of Fabricius from S. enteritidis experimentally infected ducklings were positive or strongly positive, and the S. enteritidis antigen mainly distributed in the infected cell cytoplasm. IFA is an intuitionist, sensitive and specific method in detecting S. enteritidis antigen in paraffin wax slices, and it is a good method in diagnosis and antigen location of S. enteritidis. We also conclude that the gland of Garder, heart, kidney, spleen, liver, ileum, jejunum are target organs in S. enteritidis infections of duck, and S. enteritidis is an intracellular parasitic bacterium.

  16. Serological diagnosis of autoimmune bullous skin diseases: Prospective comparison of the BIOCHIP mosaic-based indirect immunofluorescence technique with the conventional multi-step single test strategy

    PubMed Central

    2012-01-01

    Background Various antigen-specific immunoassays are available for the serological diagnosis of autoimmune bullous diseases. However, a spectrum of different tissue-based and monovalent antigen-specific assays is required to establish the diagnosis. BIOCHIP mosaics consisting of different antigen substrates allow polyvalent immunofluorescence (IF) tests and provide antibody profiles in a single incubation. Methods Slides for indirect IF were prepared, containing BIOCHIPS with the following test substrates in each reaction field: monkey esophagus, primate salt-split skin, antigen dots of tetrameric BP180-NC16A as well as desmoglein 1-, desmoglein 3-, and BP230gC-expressing human HEK293 cells. This BIOCHIP mosaic was probed using a large panel of sera from patients with pemphigus vulgaris (PV, n = 65), pemphigus foliaceus (PF, n = 50), bullous pemphigoid (BP, n = 42), and non-inflammatory skin diseases (n = 97) as well as from healthy blood donors (n = 100). Furthermore, to evaluate the usability in routine diagnostics, 454 consecutive sera from patients with suspected immunobullous disorders were prospectively analyzed in parallel using a) the IF BIOCHIP mosaic and b) a panel of single antibody assays as commonly used by specialized centers. Results Using the BIOCHIP mosaic, sensitivities of the desmoglein 1-, desmoglein 3-, and NC16A-specific substrates were 90%, 98.5% and 100%, respectively. BP230 was recognized by 54% of the BP sera. Specificities ranged from 98.2% to 100% for all substrates. In the prospective study, a high agreement was found between the results obtained by the BIOCHIP mosaic and the single test panel for the diagnosis of BP, PV, PF, and sera without serum autoantibodies (Cohen’s κ between 0.88 and 0.97). Conclusions The BIOCHIP mosaic contains sensitive and specific substrates for the indirect IF diagnosis of BP, PF, and PV. Its diagnostic accuracy is comparable with the conventional multi-step approach. The highly

  17. Quantitative three-dimensional evaluation of immunofluorescence staining for large whole mount spheroids with light sheet microscopy

    PubMed Central

    Smyrek, I.; Stelzer, E. H. K.

    2017-01-01

    Three-dimensional cell biology and histology of tissue sections strongly benefit from advanced light microscopy and optimized staining procedures to gather the full three-dimensional information. In particular, the combination of optical clearing with light sheet-based fluorescence microscopy simplifies fast high-quality imaging of thick biological specimens. However, verified in toto immunostaining protocols for large multicellular spheroids or for tissue sections have not been published. We present a method for the verification of immunostaining in three-dimensional spheroids. The analysis relies on three criteria to evaluate the immunostaining quality: quality of the antibody stain specificity, signal intensity achieved by the staining procedure and the correlation of the signal intensity with that of a homogeneously dispersed fluorescent dye. We optimized and investigated variations of five immunostaining protocols for three-dimensional cell biology. Our method is an important contribution to three-dimensional cell biology and the histology of tissues since it allows to evaluate the efficiency of immunostaining protocols for large three-dimensional specimens, and to study the distribution of protein expression and cell types within spheroids and spheroid-specific morphological structures without the need of physical sectioning. PMID:28270962

  18. iSERS microscopy guided by wide field immunofluorescence: analysis of HER2 expression on normal and breast cancer FFPE tissue sections.

    PubMed

    Wang, Xin-Ping; Zhang, Yuying; König, Matthias; Papadopoulou, Evanthia; Walkenfort, Bernd; Kasimir-Bauer, Sabine; Bankfalvi, Agnes; Schlücker, Sebastian

    2016-08-15

    Surface-enhanced Raman scattering (SERS) microscopy is an emerging imaging technique for tissue-based cancer diagnostics. Specifically, immuno-SERS (iSERS) microscopy employs antibodies labelled by molecularly functionalized noble metal colloids for antigen localization on tissue specimen. Spectrally resolved iSERS acquisition schemes are typically rather time-consuming when large tissue areas must be scanned. Here, we demonstrate the application of iSERS imaging guided by wide field immunofluorescence (IF) for localization of the human epidermal growth factor receptor 2 (HER2) on breast tissue sections. The addition of unlabelled anti-HER2 primary antibodies to the tissue is followed by the incubation with secondary antibodies labelled with both Alexa-647 (for IF) and hydrophilically stabilized gold nanostars coated with aromatic thiols (for iSERS). False-color iSERS images clearly reveal the different HER2 expression levels on normal and breast cancer tissue, respectively. A series of negative controls confirms that the binding specificity of the secondary antibody is maintained after conjugation to the SERS nanoparticles.

  19. Localization by indirect immunofluorescence of tetrin, actin, and centrin to the oral apparatus and buccal cavity of the macrostomal form of Tetrahymena vorax.

    PubMed

    McLaughlin, Neil B; Buhse, Howard E

    2004-01-01

    We have taken advantage of the size of the macrostomal oral apparatus of Tetrahymena vorax to investigate the immunofluorescent localization of three cytoskeletal proteins--tetrin, actin, and centrin. Tetrin and actin antibodies co-localize to cross-connectives that anchor the membranelles. These antibodies also recognize the coarse filamentous reticulum, a filament associated with the undulating membrane. Actin-specific localization extends beyond the coarse filamentous reticulum-undulating membrane complex into a region called the specialized cytoplasm. A centrin antibody localizes to the fine filamentous reticulum which, along with microtubules of the oral ribs, circumscribes the cytostomal opening. Models of phagocytic contraction based on these data are presented.

  20. Multiple immunofluorescence labeling of formalin-fixed paraffin-embedded tissue.

    PubMed

    Robertson, David; Isacke, Clare M

    2011-01-01

    Multiple immunofluorescent labeling of formalin-fixed paraffin-embedded (FFPE) tissue is not a routinely used method. At least in part, this is due to the perception that the innate autofluorescence of the FFPE material forbids the use of immunofluorescent labeling. As a result, immunohistochemical (immunoperoxidase) staining of FFPE material or cryosectioning methods is used instead. In this chapter, we describe a robust optimized method for high-resolution immunofluorescence labeling of FFPE tissue that involves the combination of antigen retrieval, indirect immunofluorescence, and confocal laser scanning microscopy. Once such samples have been prepared and imaged by confocal microscopy, they can be stored at -20°C for extensive periods (>250 days) and reexamined with minimal loss of quality. As a consequence, this method has the potential to open up the large archival sample collections to multiple immunofluorescent investigations.

  1. Decoupling indirect topographic cross-talk in band excitation piezoresponse force microscopy imaging and spectroscopy

    SciTech Connect

    Mazet, Lucie; Jesse, Stephen; Niu, Gang; Schroeder, Thomas; Schamm-Chardon, Sylvie; Dubourdieu, Catherine; Baddorf, Arthur P.; Kalinin, Sergei V.; Yang, Sang Mo; Okatan, M. Baris

    2016-06-20

    Here, all scanning probe microscopies are subjected to topographic cross-talk, meaning the topography-related contrast in functional images. Here, we investigate the signatures of indirect topographic cross-talk in piezoresponse force microscopy (PFM) imaging and spectroscopy and its decoupling using band excitation (BE) method in ferroelectric BaTiO3 deposited on the Si substrates with free standing nanopillars of diameter 50 nm. Comparison between the single-frequency PFM and BE-PFM results shows that the measured signal can be significantly distorted by topography-induced shifts in the contact resonance frequency and cantilever transfer function. However, with proper correction, such shifts do not affect PFM imaging and hysteresis loop measurements. This suggests the necessity of an advanced approach, such as BE-PFM, for detection of intrinsic sample piezoresponse on the topographically non-uniform surfaces.

  2. Decoupling indirect topographic cross-talk in band excitation piezoresponse force microscopy imaging and spectroscopy

    SciTech Connect

    Mazet, Lucie; Jesse, Stephen; Niu, Gang; Schroeder, Thomas; Schamm-Chardon, Sylvie; Dubourdieu, Catherine; Baddorf, Arthur P.; Kalinin, Sergei V.; Yang, Sang Mo; Okatan, M. Baris

    2016-06-20

    Here, all scanning probe microscopies are subjected to topographic cross-talk, meaning the topography-related contrast in functional images. Here, we investigate the signatures of indirect topographic cross-talk in piezoresponse force microscopy (PFM) imaging and spectroscopy and its decoupling using band excitation (BE) method in ferroelectric BaTiO3 deposited on the Si substrates with free standing nanopillars of diameter 50 nm. Comparison between the single-frequency PFM and BE-PFM results shows that the measured signal can be significantly distorted by topography-induced shifts in the contact resonance frequency and cantilever transfer function. However, with proper correction, such shifts do not affect PFM imaging and hysteresis loop measurements. This suggests the necessity of an advanced approach, such as BE-PFM, for detection of intrinsic sample piezoresponse on the topographically non-uniform surfaces.

  3. Decoupling indirect topographic cross-talk in band excitation piezoresponse force microscopy imaging and spectroscopy

    DOE PAGES

    Mazet, Lucie; Jesse, Stephen; Niu, Gang; ...

    2016-06-20

    Here, all scanning probe microscopies are subjected to topographic cross-talk, meaning the topography-related contrast in functional images. Here, we investigate the signatures of indirect topographic cross-talk in piezoresponse force microscopy (PFM) imaging and spectroscopy and its decoupling using band excitation (BE) method in ferroelectric BaTiO3 deposited on the Si substrates with free standing nanopillars of diameter 50 nm. Comparison between the single-frequency PFM and BE-PFM results shows that the measured signal can be significantly distorted by topography-induced shifts in the contact resonance frequency and cantilever transfer function. However, with proper correction, such shifts do not affect PFM imaging and hysteresismore » loop measurements. This suggests the necessity of an advanced approach, such as BE-PFM, for detection of intrinsic sample piezoresponse on the topographically non-uniform surfaces.« less

  4. Immunofluorescent detection and quantitation of hepatitis A virus in sewage treatment effluent and on agri-food surfaces using scanning confocal microscopy.

    PubMed

    Kukavica-Ibrulj, Irena; Darveau, André; Fliss, Ismaïl

    2003-03-01

    An immunofluorescent (IF) assay was developed for specific detection and quantitation of hepatitis A virus (HAV) in sewage treatment effluent or attached to stainless steel, copper, high density polyethylene and polyvinyl chloride surfaces. Polyclonal antibodies were produced in rabbits and characterized for specificity to HAV. Purified anti-HAV antibodies were used in combination with ALEXA-anti-rabbit conjugate and confocal microscopy for detection and quantitation of HAV in effluent samples. Using this immunological approach, as little as 2 x 10(5) PFU/ml were detected and the signal generated was proportional to the concentration of HAV up to 2 x 10(8) PFU/ml. Counts obtained by IF assay were highly correlated with those of the SYBR Green II nucleic acid labelling method (r(2)=0.995) and conventional plaque assay (r(2)=0.988). The IF assay described is rapid (3 h), sensitive, specific to HAV and suitable for qualitative as well as quantitative studies.

  5. Indirect modulation of nonmagnetic probes for force modulation atomic force microscopy.

    PubMed

    Li, Jie-Ren; Garno, Jayne C

    2009-02-15

    Frequency-dependent changes for phase and amplitude images are demonstrated with test platforms of organosilane ring patterns, using force modulation atomic force microscopy (FM-AFM) with an alternate instrument configuration. The imaging setup using indirect magnetic modulation (IMM) is based on indirect oscillation of soft, nonmagnetic cantilevers, with spring constants <1 N m(-1). The tip is driven to vibrate by the motion of a tip holder assembly which contains ferromagnetic materials. The entire tip assembly is induced to vibrate with the flux of an external ac electromagnetic field, supplied by a wire coil solenoid placed underneath the sample plate. With the use of IMM, dynamic parameters of the driving frequencies and amplitude of the tip motion can be optimized to sensitively map the elastic response of samples. An advantage of this instrument setup is that a magnetic coating is not required to drive the periodic oscillation of the tip. The instrument configuration for IMM may not be practical for intermittent imaging modes, which often work best with stiff cantilevers. However, indirect actuation provides an effective approach for imaging with low force setpoints and is well-suited for dynamic AFM modes using continuous contact imaging.

  6. Comparison of enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and virus isolation for detection of respiratory viruses in nasopharyngeal secretions.

    PubMed Central

    Takimoto, S; Grandien, M; Ishida, M A; Pereira, M S; Paiva, T M; Ishimaru, T; Makita, E M; Martinez, C H

    1991-01-01

    Nasopharyngeal secretions obtained from 94 children with acute respiratory illness were examined for the presence of respiratory syncytial virus (RSV), adenovirus, and influenza virus type A by virus culturing (virus isolation technique [VIT]), immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA). Similar results were obtained in at least two tests for RSV, influenza virus type A, and adenovirus in 92 (97.9%), 88 (93.6%), and 88 (93.6%) cases, respectively. Both rapid virus detection methods showed good specificity for the diagnosis of these virus infections (greater than or equal to 90.7%) and were more sensitive than was VIT for RSV detection. In a more accurate statistical analysis, the indexes of agreement between VIT and ELISA were substantial for RSV (kappa = 0.69; zeta = 5.5; P less than 0.0001), influenza virus type A (kappa = 0.67; zeta = 5.3; P less than 0.0001), and adenovirus (kappa = 0.71; zeta = 6.0; P less than 0.0001), while it was almost perfect for RSV when ELISA was compared with IFA (kappa = 0.88; zeta = 5.7; P less than 0.0001). Although the observed agreement was good in the comparison of these two tests for these three viruses (89%0, the indexes of agreement were moderate in the comparison of IFA and VIT for RSV (K = 0.55; Z = 2.0; P < 0.05), influenza virus type A (K = 0.42; Z = 9.7; P < 0.0001), and adenovirus (K = 0.41; Z = 6.5; P < 0.0001) and of ELISA and IFA for influenza virus type A (K = 0.55; Z = 7.0; P < 0.0001) and adenovirus (K = 0.59; Z = 6.8; P < 0.0001). All of the statistical evaluations demonstrated better agreement between ELISA and VIT for influenza virus type A and adenovirus. PMID:2037663

  7. Development of an indirect immunofluorescence technique for the evaluation of generated antibody titers against Erysipelothrix rhusiopathiae in captive bottlenose dolphins (Tursiops truncatus).

    PubMed

    Bernal-Guadarrama, María José; García-Parraga, Daniel; Fernández-Gallardo, Nuhacet; Zamora-Padrón, Rafael; Pacheco, Víctor; Reyes-Batlle, María; Valladares, Basilio; Lorenzo-Morales, Jacob; Martínez-Carretero, Enrique

    2014-11-01

    Erysipelothrix rhusiopathiae is the causative agent of erysipelas, a disease of many mammalian and avian species, mainly swine and turkeys. In cetaceans, erysipelas is considered to be the most common infection in juvenile individuals, which have not been vaccinated. Moreover, the disease manifest in both forms, the dermatologic and the acute septicemic forms, has been reported in various species of dolphins and whales. It is difficult to diagnose erysipelas by currently available approaches. Moreover, it is mainly based on culture methods and also PCR methods, which are currently being developed. At the present stage, prophylactic approaches are based on antibiotic therapy and vaccination mostly with porcine erysipelas vaccines. In the present study, an Indirect Immuno Fluorescence method for the detection of dolphin antibodies levels against E. rhusiopathiae was developed and applied in two different groups of captive bottlenose dolphins (Tursiops truncatus) from Loro Parque (Tenerife, Canary Islands, Spain) and L'Oceanogràfic de Valencia (Valencia, Spain) in order to check the tittering levels of antibodies after application of porcine erysipelas vaccines in the studied dolphins.

  8. Indirect Immunodetection of Fungal Fragments by Field Emission Scanning Electron Microscopy

    PubMed Central

    Afanou, Komlavi Anani; Straumfors, Anne; Skogstad, Asbjørn; Nayak, Ajay P.; Skaar, Ida; Hjeljord, Linda; Tronsmo, Arne; Green, Brett James

    2015-01-01

    Submicronic fungal fragments have been observed in in vitro aerosolization experiments. The occurrence of these particles has therefore been suggested to contribute to respiratory health problems observed in mold-contaminated indoor environments. However, the role of submicronic fragments in exacerbating adverse health effects has remained unclear due to limitations associated with detection methods. In the present study, we report the development of an indirect immunodetection assay that utilizes chicken polyclonal antibodies developed against spores from Aspergillus versicolor and high-resolution field emission scanning electron microscopy (FESEM). Immunolabeling was performed with A. versicolor fragments immobilized and fixed onto poly-l-lysine-coated polycarbonate filters. Ninety percent of submicronic fragments and 1- to 2-μm fragments, compared to 100% of >2-μm fragments generated from pure freeze-dried mycelial fragments of A. versicolor, were positively labeled. In proof-of-concept experiments, air samples collected from moldy indoor environments were evaluated using the immunolabeling technique. Our results indicated that 13% of the total collected particles were derived from fungi. This fraction comprises 79% of the fragments that were detected by immunolabeling and 21% of the spore particles that were morphologically identified. The methods reported in this study enable the enumeration of fungal particles, including submicronic fragments, in a complex heterogeneous environmental sample. PMID:26092450

  9. Immunofluorescent quantitation of chloroplast proteins.

    PubMed

    Leech, R M; Marrison, J L

    1996-12-01

    Using scanning light microscopy software to detect and measure immunofluorescence in leaf sections Rubisco concentration in situ in chloroplasts has been accurately determined throughout development. The fluorescence measurements were calibrated by comparison with values for Rubisco accumulation obtained from rocket immuno-electrophoresis profiles of soluble protein from isolated cells and from chloroplasts using a purified sample of Rubisco as the standard. It has been shown that in situ immunofluorescence can be used for cytoquantitation of proteins within individual chloroplasts to a sensitivity of 1fg and also for the comparison of the protein levels in adjacent chloroplasts and cells. Several important applications of this new technique are discussed.

  10. High anti–desmoglein 3 antibody ELISA index and negative indirect immunofluorescence result in a patient with pemphigus vulgaris in remission: evaluation of the antibody profile by newly developed methods.

    PubMed

    Nakahara, Tomoko; Takagi, Atsushi; Yamagami, Jun; Kamiya, Koji; Aoyama, Yumi; Iwatsuki, Keiji; Ikeda, Shigaku

    2014-12-01

    IMPORTANCE Pemphigus vulgaris (PV) is a disease that features blistering of the skin and mucous membranes caused by autoantibodies directed against desmoglein 3 (Dsg3) and/or desmoglein 1(Dsg1). Indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA) are 2 methods that are widely used to measure Dsg3 orDsg1 antibody titers in PV. Although the titers of these autoantibodies are generally correlated with disease activity, some patients with a high ELISA index do not have severe symptoms.We encountered a patient with PV in remission,who had a high anti-Dsg3 antibody ELISA index while the IIF result was negative.OBSERVATIONS The anti-Dsg3 antibodies of our patient mainly recognized Ca2+-dependent conformational epitopes and targeted mature Dsg3 protein.We report this case focusing on the discrepancy between ELISA and IIF findings, as well as on the specific characteristics of the patient's autoantibodies evaluated by newly developed methods.CONCLUSIONS AND RELEVANCE This case emphasizes that a discrepancy between disease activity, the ELISA index for Dsg3, and/or IIF findings can occur in PV. Further research on similar patients will be required to elucidate the pathogenic mechanisms in patients with PV who have nonpathogenic antibodies and show a discrepancy between ELISA and IIF.

  11. Development of a recombinant cell-based indirect immunofluorescence assay (RC-IFA) for the determination of autoantibodies against "rings and rods"-associated inosine-5'-monophosphate dehydrogenase 2 in viral hepatitis C.

    PubMed

    Probst, Christian; Radzimski, Christiane; Blöcker, Inga Madeleine; Teegen, Bianca; Bogdanos, Dimitrios P; Stöcker, Winfried; Komorowski, Lars

    2013-03-15

    Autoantibodies against so-called "rings and rods" structures, as determined by indirect immunofluorescence assay (IFA) using the human cell line HEp-2, have been described in chronic hepatitis C virus (HCV) infected patients treated with interferon/ribavirin. Recently, cytidine triphosphate synthase (CTPS) and inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), the enzyme inhibited by ribavirin, were proposed as the target antigens. We wanted to confirm the identification and setup a robust system for autoantibody testing in routine laboratories. CTPS and IMPDH2 were individually expressed in HEK293 cells and the recombinant cells were used in IFA (RC-IFA) to analyze sera from 33 anti-"rings and rods" antibody positive individuals with unknown diagnosis, 50 patients with chronic HCV infection, 100 with autoimmune hepatitis, 50 with primary biliary cirrhosis and 50 healthy blood donors. We found that all sera with anti-"rings and rods" reacted with recombinant IMPDH2 but none with CTPS. In western blot or ELISA, anti-IMPDH2 positive sera reacted only weakly, if at all, with Escherichia coli derived recombinant IMPDH2 indicating that the autoantibody reaction probably depends on the 3-dimensional conformation of the antigen. A rabbit hyperimmune serum raised against bacterially expressed IMPDH2 produced the ring/rods pattern in IFA using HEp-2. We conclude, that IMPDH2 is indeed the main target of anti-"rings and rods" while CTPS is an unlikely target. Moreover, the novel RC-IFA test system allows a standardized semi-quantitative determination of anti-IMPDH2 basing on a defined recombinant antigen. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Bayesian Validation of the Indirect Immunofluorescence Assay and Its Superiority to the Enzyme-Linked Immunosorbent Assay and the Complement Fixation Test for Detecting Antibodies against Coxiella burnetii in Goat Serum

    PubMed Central

    Stenos, John; Vincent, Gemma; Campbell, Angus; Graves, Stephen; Warner, Simone; Devlin, Joanne M.; Nguyen, Chelsea; Stevenson, Mark A.; Wilks, Colin R.; Firestone, Simon M.

    2016-01-01

    Although many studies have reported the indirect immunofluorescence assay (IFA) to be more sensitive in detection of antibodies to Coxiella burnetii than the complement fixation test (CFT), the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the assay have not been previously established for use in ruminants. This study aimed to validate the IFA by describing the optimization, selection of cutoff titers, repeatability, and reliability as well as the DSe and DSp of the assay. Bayesian latent class analysis was used to estimate diagnostic specifications in comparison with the CFT and the enzyme-linked immunosorbent assay (ELISA). The optimal cutoff dilution for screening for IgG and IgM antibodies in goat serum using the IFA was estimated to be 1:160. The IFA had good repeatability (>96.9% for IgG, >78.0% for IgM), and there was almost perfect agreement (Cohen's kappa > 0.80 for IgG) between the readings reported by two technicians for samples tested for IgG antibodies. The IFA had a higher DSe (94.8%; 95% confidence interval [CI], 80.3, 99.6) for the detection of IgG antibodies against C. burnetii than the ELISA (70.1%; 95% CI, 52.7, 91.0) and the CFT (29.8%; 95% CI, 17.0, 44.8). All three tests were highly specific for goat IgG antibodies. The IFA also had a higher DSe (88.8%; 95% CI, 58.2, 99.5) for detection of IgM antibodies than the ELISA (71.7%; 95% CI, 46.3, 92.8). These results underscore the better suitability of the IFA than of the CFT and ELISA for detection of IgG and IgM antibodies in goat serum and possibly in serum from other ruminants. PMID:27122484

  13. Immunofluorescence microscopy of microtubules in intact cell lineages of the moss, Physcomitrella patens. I. Normal and CIPC-treated tip cells.

    PubMed

    Doonan, J H; Cove, D J; Lloyd, C W

    1985-04-01

    Monoclonal antibodies to yeast tubulin have been used to visualize the distribution of microtubules in the intact filamentous protonemata of the moss Physcomitrella patens. Protonemata were prepared for immunofluorescence by fixation in formaldehyde and cells were made permeable with Driselase. Extensive cell files were preserved by 'blotting' the moss onto glutaraldehyde-derivatized coverslips. Problems due to fluorescence from chloroplasts were obviated by extraction with dimethyl sulphoxide and the non-ionic detergent, Nonidet NP40. These improvements allowed us to determine that microtubules were present throughout the cell cycle in the apical dome of caulonemal tip cells, that was a pronounced association of microtubules with the nucleus, that 'astral' microtubules were associated with the mitotic spindle and during anaphase may be involved in reorientation of the spindle before an oblique cytokinesis in caulonemata and that the cytokinetic phragmoplast appeared identical to the structure described for higher plants. Microtubules appeared to converge at the very tip of apical caulonemal cells and this was studied further by treating cells with CIPC--a drug that is known to produce multiple microtubule-organizing centres--and which here produces multiple foci for microtubules at the tip. These observations emphasize the involvement of microtubules in tip growth, alignment of the cell plate and nuclear migration--processes that are fundamental to the morphogenesis of filamentous organisms.

  14. Confocal microscopy with double immunofluorescence staining reveals the functional transient receptor potential vanilloid subtype 1 expressed in myoepithelial cells of human submandibular glands.

    PubMed

    Ding, Qianwen; Zhang, Yan; Cong, Xin; Cai, Zhigang; Han, Jingyan; Su, Yunchao; Wu, Li-Ling; Yu, Guan-Gyan

    2012-05-01

    Myoepithelial cells (MECs) mainly surround acini and intercalated ducts in the human salivary glands. The contraction of MECs provides the expulsive force to promote salivation. We previously found functional transient receptor potential vanilloid subtype 1 (TRPV1) was expressed in rabbit and human submandibular glands and increased saliva secretion. However, it was unknown whether TRPV1 was expressed in MECs of submandibular glands. In this study, we observed the immunoflourescence of TRPV1 was not only located in serous acini and ducts but also surround the basal layer of the acinus and intercalated ducts of human submandibular glands. Double immunofluorescence staining revealed colocalization of TRPV1 with calponin, vimentin, and α-smooth muscle actin, which indicated the myoepithelial expression of TRPV1. Treating submandibular gland tissues with capsaicin, an agonist of TRPV1, substantially increased the phosphorylation of the 20-kDa regulatory light-chain subunit of myosin (MLC(20) ), a crucial molecule for contraction of smooth muscle cells, in MECs. Pretreatment with capsazepine, a specific TRPV1 inhibitor, blocked capsaicin-induced MLC(20) phosphorylation. These results suggest that TRPV1 is expressed in MECs of the human submandibular gland and mediates myoepithelial contraction via a mechanism involving MLC(20) phosphorylation.

  15. Automated analysis of siRNA screens of cells infected by hepatitis C and dengue viruses based on immunofluorescence microscopy images

    NASA Astrophysics Data System (ADS)

    Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

    2008-03-01

    We present an image analysis approach as part of a high-throughput microscopy siRNA-based screening system using cell arrays for the identification of cellular genes involved in hepatitis C and dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in the neighborhood of segmented cell nuclei, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment and single images. In particular, we propose a novel approach for the localization of regions of transfected cells within cell array images, which combines model-based circle fitting and grid fitting. By this scheme we integrate information from single cell array images and knowledge from the complete cell arrays. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behaviour of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

  16. Utility of immunofluorescence in dermatology

    PubMed Central

    Shetty, Varsha M.; Subramaniam, Kumudhini; Rao, Raghavendra

    2017-01-01

    Immunofluorescence (IF) tests have redefined our understanding of many immune-mediated skin diseases, especially autoimmune blistering diseases (AIBDs). Nomenclature of certain AIBDs (for example, linear IgA diseases and IgA pemphigus) has been done based solely on the finding of tissue-bound immunoreactants as detected by IF tests. Direct and indirect are the two major types of IF tests; they are not only useful in the diagnosis but also guide the clinician in the treatment at least in certain AIBDs, as the titer of circulating antibodies as detected by IF reflects the disease activity. In this review, we describe techniques, various types of IF, and its modification. PMID:28217464

  17. New non-invasive method for evaluation of the stratum corneum structure in diseases with abnormal keratinization by immunofluorescence microscopy of desmoglein 1 distribution in tape-stripped samples.

    PubMed

    Oyama, Zuiei; Naoe, Yoshikazu; Kimura, Hiroko; Masunaga, Takuji; Seishima, Mariko; Aoyama, Yumi; Kitajima, Yasuo

    2010-10-01

    The corneodesmosomes in the stratum corneum are critical for the maintenance of stratum corneum integrity. To evaluate the normal and diseased keratinization states in the epidermis, we studied the distribution of desmoglein 1 (DSG1), a major component of corneodesmosomes, in samples of the stratum corneum obtained by tape stripping, a non-invasive method. Samples were collected from lesional skin of four patients with psoriasis and three with lichen planus, and from non-lesional skin of three volunteers. Upper stratum corneum cells were obtained by tape stripping and skin biopsies were obtained from adjacent sites. Tape-stripped samples were examined by immunofluorescence microscopy using anti-DSG1 monoclonal antibody, in combination with histopathology of skin biopsies. In normal human stratum corneum, which shows basket-woven orthokeratosis, DSG1-containing fluorescent dots were distributed on the lateral cell-cell contact areas of plasma membrane, but not on the dorsal/ventral plasma membrane, and formed a well-ordered hexagonal network structure. In psoriatic stratum corneum, fluorescent dots were distributed throughout the cell membrane at ventral aspects of corneocytes as well as at the lateral cell-cell contacts. In lichen planus, fluorescent dots were distributed homogeneously and/or heterogeneously on the ventral surface in some cells. Adjacent cells lacked DSG1 at the lateral cell-cell contacts, but were instead separated by distinctive black-gap lines. These results suggest that the intercellular adhesion by DSG1 may depend on the lateral plasma membrane in normal human stratum corneum, on the dorsal/ventral plasma membrane in lichen planus, and on both lateral and dorsal/ventral plasma membranes in psoriatic stratum corneum. Tape stripping and DSG1 immunofluorescence visualizes adhesion features of corneocytes and has considerable potential for evaluation of abnormal keratinization and the process of healing in response to treatment. © 2010 Japanese

  18. Immunofluorescence detection methods using microspheres

    NASA Astrophysics Data System (ADS)

    Szurdoki, Ferenc; Michael, Karri L.; Agrawal, Divya; Taylor, Laura C.; Schultz, Sandra L.; Walt, David R.

    1999-01-01

    Microsphere-based immunoassays were devised for compounds of agricultural and biomedical interest (e.g., digoxin, theophylline, and zearalenone). Commercially available microspheres with surface functional groups for chemical derivatization were used as solid carriers. After immobilizing the target substances, the surface of the haptenized microspheres was blocked by a protein to reduce aspecific binding. Competitive immunoassays were performed using the functionalized microspheres and antibodies labeled with horseradish peroxidase. Immunofluorescence signal amplification was achieved by enzyme-catalyzed reporter deposition (CARD). An epifluorescence microscope, a CCD camera interfaced with a computer, and microscopy image analysis software were employed for quantitative detection of fluorescent light emitted from individual microspheres. Integration of several such immunoassays and application of an optical encoding method enabled multianalyte determination. These immunoassays can also be utilized in an immunosensor array format. This immunoarray format could facilitate miniaturization and automation of multianalyte immunoassays.

  19. Quantitative studies of immunofluorescent staining*

    PubMed Central

    Beutner, Ernst H.; Sepulveda, Marion R.; Barnett, Eugene V.

    1968-01-01

    Reproducible titres of indirect immunofluorescent (IF) staining with antinuclear factor (ANF)-containing sera could be obtained with different antihuman IgG conjugates by quantitative adjustments of their characteristics. Conversely, one ANF yielded a broad range of ANF titre (80-640) upon appropriate adjustments of the conjugate characteristics. The same and related characteristics of the conjugates also afforded a basis for quantitatively defining the conditions under which non-specific staining (NSS) appeared. The salient characteristics of the anti-IgG conjugates include: (1) their strength of antiglobulin (expressed as units/ml of precipitating antibody or μg antibody N/ml); (2) their apparent fluorescein concentration (in μg F/ml); (3) their protein concentration (in mg/ml). Optical and immunologic sensitivity ratios are calculated from these conjugate characteristics. Optical sensitivity (expressed as fluorescein concentration to protein concentration (F/P) ratios), immunological sensitivities (expressed as units/1% protein) and the dilution employed serve to characterize quantitatively anti-IgG conjugates adequately to define their specific and non-specific staining properties. PMID:4179321

  20. Flow cytometric immunofluorescence of rat anterior pituitary cells

    NASA Technical Reports Server (NTRS)

    Hatfield, J. Michael; Hymer, W. C.

    1985-01-01

    A flow cytometric immunofluorescence technique was developed for the quantification of growth hormone, prolactin, and luteinizing hormone producing cells. The procedure is based on indirect-immunofluorescence of intracellular hormone using an EPICS V cell sorter and can objectively count 50,000 cells in about 3 minutes. It can be used to study the dynamics of pituitary cell populations under various physiological and pharmacological conditions.

  1. Flow cytometric immunofluorescence of rat anterior pituitary cells

    NASA Technical Reports Server (NTRS)

    Hatfield, J. Michael; Hymer, W. C.

    1985-01-01

    A flow cytometric immunofluorescence technique was developed for the quantification of growth hormone, prolactin, and luteinizing hormone producing cells. The procedure is based on indirect-immunofluorescence of intracellular hormone using an EPICS V cell sorter and can objectively count 50,000 cells in about 3 minutes. It can be used to study the dynamics of pituitary cell populations under various physiological and pharmacological conditions.

  2. Immunofluorescence Imaging of DNA Damage Response Proteins

    PubMed Central

    Bennett, Brian T.; Bewersdorf, Jörg; Knight, Kendall L.

    2013-01-01

    Immunofluorescence imaging has provided captivating visual evidence for numerous cellular events, from vesicular trafficking, organelle maturation and cell division to nuclear processes including the appearance of various proteins and chromatin components in distinct foci in response to DNA damaging agents. With the advent of new super-resolution microscope technologies such as 4Pi microscopy, standard immunofluorescence protocols deserve some reevaluation in order to take full advantage of these new technological accomplishments. Here we describe several methodological considerations that will help overcome some of the limitations that may result from the use of currently applied procedures, with particular attention paid to the analysis of possible colocalization of fluorescent signals. We conclude with an example of how application of optimized methods led to a breakthrough in super-resolution imaging of nuclear events occurring in response to DNA damage. PMID:19245833

  3. Microscopy

    Treesearch

    Patricia A. Moss; Les Groom

    2001-01-01

    Microscopy is the study and interpretation of images produced by a microscope. "Interpretation" is the keyword, because the microscope enables one to see structures that are too small or too close together to be resolved by the unaided eye. (The human eye cannot separate two points or lines that are closer together than 0.1 mm.) it is important to...

  4. Difference of EGCg adhesion on cell surface between Staphylococcus aureus and Escherichia coli visualized by electron microscopy after novel indirect staining with cerium chloride.

    PubMed

    Nakayama, Motokazu; Shigemune, Naofumi; Tsugukuni, Takashi; Tokuda, Hajime; Miyamoto, Takahisa

    2011-07-01

    We developed a novel method using indirect staining with cerium chloride for visualization of the catechin derivative epigallocatechin gallate (EGCg) on the surface of particles, i.e., polystyrene beads and bacterial cells, by electron microscopy. The staining method is based on the fact that in an alkaline environment, EGCg produces hydrogen peroxide, and then hydrogen peroxide reacts with cerium, resulting in a cerium hydroperoxide precipitate. This precipitate subsequently reacts with EGCg to produce larger deposits. The amount of precipitate is proportional to the amount of EGCg. Highly EGCg-sensitive Staphylococcus aureus and EGCg-resistant Escherichia coli were treated with EGCg under various pH conditions. Transmission electron microscopy observation showed that the amount of deposits on S. aureus increased with an increase in EGCg concentration. After treating bacterial cells with 0.5mg/mL EGCg (pH 6.0), attachment of EGCg was significantly lower to E. coli than to S. aureus. This is the first report that shows differences in affinity of EGCg to the cell surfaces of Gram-positive and -negative bacteria by electron microscopy.

  5. Rapid Diagnosis of Arbovirus and Arenavirus Infections by Immunofluorescence.

    DTIC Science & Technology

    1980-01-01

    have been prepared with the following viruses: Mayaro, dengue type 3 , dengue type 4 , West Nile, Oropouche and Sicilian phlebotomu fever. Similar slides...for use as antigen in the indirect immunofluorescence (IF) test have been prepared with the following viruses: Mayaro, dengue type 3 , dengue type 4 ...type 2, NGB; dengue type 3 , H 87; dengue type 4 , H 241; JE, Nakayama; Langat, TP 21; Rocio, San Paulo; West Nile, Egypt 101; yellow fever, Asibi

  6. Immunofluorescence and Immunohistochemical Detection of Keratins.

    PubMed

    Stumptner, Cornelia; Gogg-Kamerer, Margit; Viertler, Christian; Denk, Helmut; Zatloukal, Kurt

    2016-01-01

    Reliable detection of keratins in tissues is important for investigating their physiological role and for using keratin expression as a biomarker in medical diagnostics. A particular challenge for the detection of keratins by immunofluorescence microscopy or immunohistochemistry relates to the fact that keratin intermediate filaments are obligatory heteropolymers, which may result in dissociation between RNA and protein expression levels in the event that the homeostasis of the expression of the proper keratin partners is disturbed. Furthermore, variable accessibility of epitopes on keratin polypeptides due to conformational changes may lead to false negative results. Preanalytical effects, such as warm/cold ischemia, fixation, tissue processing, and embedding may result in false negative or inappropriate reactions. An experimental design for how to systematically test preanalytical effects and to validate immunohistochemistry protocols is presented. This kind of evaluation should be performed for each antigen and antibody since the various epitopes recognized by antibodies may behave differently. In this context, one has to be aware that different cell structures may be affected or modified differently by various preanalytical procedures and may thus require different preanalytical and staining protocols. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Investigation of immunofluorescence cross-reactions against Trichinella spiralis by western blot (immunoblot) analysis.

    PubMed Central

    Robert, F; Weil, B; Kassis, N; Dupouy-Camet, J

    1996-01-01

    Immunofluorescence cross-reactions in Trichinella spiralis serodiagnosis are sometimes difficult to identify. We compared the results of an indirect immunofluorescence assay and the profiles obtained by Western blot (immunoblot) analysis for three groups of patients: 10 T. spiralis-infected patients, 10 patients with autoimmune diseases, and 7 patients with parasitic diseases other than trichinellosis. The degree of immunofluorescence cross-reaction was variable. Western blotting allowed us to differentiate Trichinella infection from other parasitic diseases. In 3 of 10 serum samples from patients with autoimmune diseases, bands which had the same sizes as Trichinella bands were observed, and they could correspond to shared epitopes such as heat shock proteins. PMID:8877138

  8. Current concepts of immunofluorescence in oral mucocutaneous diseases

    PubMed Central

    Anuradha, CH; Malathi, N; Anandan, S; Magesh, KT

    2011-01-01

    Aim: To study the immunofluorescence pattern and to assess its reliability as a confirmatory diagnostic test in patients with pemphigus, pemphigoid, lichen planus, and lupus erythematosus and also to assess the disease activity by indirect immunofluorscence (IIF) in patients with pemphigus only. Materials and Methods: Twenty-six patients were included in the study group, out of which, 6 patients were clinically and histopathologically diagnosed as pemphigus, completely free of active lesions were subjected to IIF only to assess the disease activity and were grouped separately. Based on the clinical and provisional diagnosis, the remaining 20 patients who had active lesions were subjected to direct immunofluorscence (DIF) and IIF and were divided into four groups. Biopsy specimens were taken from the periphery of the lesions and were examined by both conventional light microscopic and DIF methods. Five milliliters of venous blood was collected from each patient and were subjected to IIF. Results: Histopathological diagnosis was consistent with direct immunofluorescence study in 15 cases (75%). The various immunofluorescence patterns observed in our study were consistent with those described by various authors in standard textbooks and articles. Conclusion: Histopathology remains gold standard for most of the diseases, it is recognized from this study that not all lesions are amenable to definitive histopathological diagnosis thus; DIF can provide a valuable additional criterion in diagnosis. PMID:22144826

  9. Immunofluorescence Staining — EDRN Public Portal

    Cancer.gov

    Direct immunofluorescence method is used to detect the deposit of immunoglobulins, complement components, fibrinogen, etc. in tissues. This technique is usually performed on frozen sections. The primary antibody is conjugated to fluorescein binds directly with the antigen and can be detected by the fluorescent tag using a fluorescent microscope.

  10. Diagnostic profile on the IFA 40: HEp-20-10 - an immunofluorescence test for reliable antinuclear antibody screening.

    PubMed

    Rohwäder, Edda; Locke, Michael; Fraune, Johanna; Fechner, Kai

    2015-04-01

    Indirect immunofluorescence assay is the recommended gold standard to test for antinuclear antibodies (ANA), which are important biomarkers for systemic rheumatic autoimmune diseases. It is internationally accepted that indirect immunofluorescence assay ANA screening is most sensitive on human epithelial (HEp-2) cells. The cells present a multitude of antigens that display distinguishable localization patterns in interphase and mitotic cells in indirect immunofluorescence analysis. Here, we present the IFA 40: HEp-20-10 test kit (Euroimmun AG, Lübeck, Germany), which is cleared for sale on the US market by the FDA. The test has been designed for qualitative and semiquantitative screening of ANA in human sera. It uses the commonly applied 1:40 cutoff dilution and the enhanced HEp-20-10 cell line for more efficient pattern recognition and has been validated in various studies and by method comparison. The IFA 40: HEp-20-10 test fulfills the essential criteria for reliable application in autoimmune diagnostics.

  11. Evolution of Bergmann glia in developing human fetal cerebellum: a Golgi, electron microscopic and immunofluorescent study.

    PubMed

    Choi, B H; Lapham, L W

    1980-05-26

    Astrogliogenesis in the human fetal cerebellum was examined in 46 cerebella obtained from hysterotomy specimens ranging between 9 and 20 weeks of ovulation age. By correlating the results obtained by rapid Golgi and Golgi-Cox methods, the indirect immunofluorescence technique for glial fibrillary acidic protein, and electron microscopy, it was possible to ensure identification of cells and obtain a comprehensive view of the ontogenesis of cerebellar astroglia, in particular Bergmann fibers. Radial fibers were present at 9 weeks of ovulation age, with features of astroglial differentiation. In the cerebellar hemisphere radial fibers arising near the ventricular zone did not reach all the way to the pial surface but terminated in vascular walls of the intermediate zone. A second set of glial cells located in the intermediate zone gave rise to long, tapering processes oriented radially to the pia, some reaching to the pial surface and terminating there in conical swellings. Radial glia with these features were observed in cerebella at all fetal ages examined, indicating their availability for guidance of external granular cells as they migrate inward. With advancing fetal age, the segment of those radial glia traversing the molecular layer demonstrated an increasing resemblance to Bergmann fibers, though the cell bodies giving rise to these processes were still located below the Purkinje cells. Transitional forms between radial glial processes and fibers beginning to resemble Bergmann fibers were observed in numerous specimens impregnated with the Golgi methods. Astrogliogenesis in human fetal cerebellum occurs earlier than formerly believed, and Bergmann fibers are a final stage in the development of a defined group of radial glia in the cerebellum.

  12. X-ray microscopy of live biological micro-organisms

    NASA Astrophysics Data System (ADS)

    Raja Al-Ani, Ma'an Nassar

    Real-time, compact x-ray microscopy has the potential to benefit many scientific fields, including microbiology, pharmacology, organic chemistry, and physics. Single frame x-ray micro-radiography, produced by a compact, solid-state laser plasma source, allows scientists to use x-ray emission for elemental analysis, and to observe biological specimens in their natural state. In this study, x-ray images of mouse kidney tissue, live bacteria, Pseudomonas aeruginosa and Burkholderia cepacia, and the bacteria's interaction with the antibiotic gentamicin, are examined using x-ray microscopy. For the purposes of comparing between confocal microscopy and x-ray microscopy, we introduced to our work the technique of gold labeling. Indirect immunofluorescence staining and immuno-gold labeling were applied on human lymphocytes and human tumor cells. Differential interference contrast microscopy (DIC) showed the lymphocyte body and nucleus, as did x-ray microscopy. However, the high resolution of x-ray microscopy allows us to differentiate between the gold particles bound to the antibodies and the free gold. A compact, tabletop Nd: glass laser is used in this study to produce x-rays from an Yttrium target. An atomic force microscope is used to scan the x-ray images from the developed photo-resist. The use of compact, tabletop laser plasma sources, in conjunction with x-ray microscopy, is a new technique that has great potential as a flexible, user-friendly scientific research tool.

  13. Autofluorescence and non-specific immunofluorescent labeling in frozen bovine intestinal tissue sections: Solutions for multi-color immunofluorescence experiments

    USDA-ARS?s Scientific Manuscript database

    Autofluorescence and non-specific immunofluorescent labeling are common challenges associated with immunofluorescence experiments. Autofluorescence typically demonstrates a broad emission spectrum, increasing the potential for overlap with experiments that utilize multiple fluorophores. During immun...

  14. A Versatile Method for Immunofluorescent Staining of Cells Cultured on Permeable Membrane Inserts.

    PubMed

    Gillespie, Jenni L; Anyah, Anwuli; Taylor, John M; Marlin, Jerry W; Taylor, Tracey A H

    2016-09-12

    BACKGROUND Obtaining high-quality images of cellular structures via immunofluorescence staining is critical for cellular localization studies. Often, these studies cannot be performed in parallel with certain oncology, virology, pharmacokinetic, and drug absorption studies due to model system technicalities requiring the cells to be cultured on porous membranes rather than glass or plastic. MATERIAL AND METHODS Here, we report a method of immunofluorescent staining of cells cultured on permeable membranes. RESULTS As proof of principle, HeLa cells grown on Transwell® membrane supports were stained with fluorescently labeled antibodies using this modified immunofluorescence staining method and visualized by fluorescent microscopy. CONCLUSIONS This protocol is a convenient alternative to staining cells on glass coverslips, thereby expanding the scope and applications of this important research tool.

  15. Autofluorescence and Nonspecific Immunofluorescent Labeling in Frozen Bovine Intestinal Tissue Sections: Solutions for Multicolor Immunofluorescence Experiments.

    PubMed

    Jenvey, Caitlin J; Stabel, Judith R

    2017-09-01

    Autofluorescent compounds present in intestinal tissue often hinder the ability to utilize multiple, spectrally different, fluorophores. In addition, fixatives and blocking solutions may contribute to background autofluorescence or nonspecific immunofluorescent labeling. During immunofluorescence protocol development, autofluorescent pigments were observed in frozen bovine mid-ileal intestinal tissue sections. Coagulant fixatives, normal serum blocking, histochemical stains Sudan Black B (SBB) and 3,3'-diaminobenzidine (DAB), and spectral separation using imaging software were compared for their ability to reduce autofluorescence, as well as their effect on immunofluorescent labeling. Fluorescent pigments of frozen bovine mid-ileal intestinal tissue sections, most likely caused by eosinophils and lipofuscin, were masked successfully with a combination of DAB and SBB. Little to no statistical differences were observed for all other methods investigated; however, tissue fixed with 1:1 acetone methanol and 10% horse serum diluted in 0.05 M Tris buffer demonstrated lower mean fluorescence intensities. Spectral separation of specific immunofluorescent labeling from background autofluorescence is a simple method for removing unwanted fluorescence; however, successful separation is dependent on tissue and labeling quality.

  16. Immunofluorescence staining with frozen mouse or chick embryonic tissue sections.

    PubMed

    Wang, Hui; Matise, Michael P

    2013-01-01

    Immunofluorescence (IF), a form of immunohistochemistry (IHC) with specific applications, is commonly used for both basic research and clinical studies, including diagnostics, and involves visualizing the cellular distribution of target molecules (e.g., proteins, DNA, and small molecules) using a microscope capable of exciting and detecting fluorochrome compounds that emit light at specific, largely nonoverlapping wavelengths. The procedure for carrying out IF varies according to the tissue type and methods for processing and preparing tissue (e.g., fixative used to preserve tissue morphology and antigenicity). The protocol presented here provides a general guideline for multichannel IF staining using frozen embryonic mouse or chicken tissue sectioned on a cryostat. In general, the procedure involves the following: (1) fixing freshly dissected tissues in a 4 % paraformaldehyde solution buffered in the physiological pH range, (2) cryopreservation of tissue in a 30 % sucrose solution, (3) embedding and sectioning tissue in Optimal Cutting Temperature (OCT) matrix compound, (4) direct or indirect detection of the target antigen/s using fluorochrome-conjugated antibodies.

  17. New method of antibody detection by indirect immunoperoxidase plaque staining for serodiagnosis of African swine fever.

    PubMed Central

    Pan, I C; Huang, T S; Hess, W R

    1982-01-01

    An indirect immunoperoxidase plaque-staining method was developed for detecting antibody to African swine fever virus infection. In both sensitivity and specificity, the test was comparable to indirect immunofluorescence. Because it has all of the desirable features of the indirect immunofluorescence test and may also be readily used for testing large numbers of sera, the indirect immunoperoxidase plaque-staining method can be used as a single and final serodiagnostic test in a large-scale survey of African swine fever. The indirect immunoperoxidase plaque-staining method should be applicable to other viruses that can be adapted to and grown in cell cultures. Images PMID:6185528

  18. Rapid Identification of Dengue Virus Serotypes Using Monoclonal Antibodies in an Indirect Immunofluorescence Test.

    DTIC Science & Technology

    1982-06-18

    encephalitis(TBH-28), West Nile(E-101), Yellow fever(French neurotropic and 17D strains), and Zika . Two Sandfly Fever viruses (213452 and Candiru) were... viruses of the Phlebovirus genus (Table 1). Several monoclonal antibody preparations reacted solely with dengue virus serotypes. Two preparations (13E7 and...others (20,21,22) was a superior host for dengue viruses , yielding an average of about 3 logl0 more infectious virus . Its superiority as a medium for

  19. Indirect inversions

    NASA Astrophysics Data System (ADS)

    Sergienko, Olga

    2013-04-01

    Since Doug MacAyeal's pioneering studies of the ice-stream basal traction optimizations by control methods, inversions for unknown parameters (e.g., basal traction, accumulation patterns, etc) have become a hallmark of the present-day ice-sheet modeling. The common feature of such inversion exercises is a direct relationship between optimized parameters and observations used in the optimization procedure. For instance, in the standard optimization for basal traction by the control method, ice-stream surface velocities constitute the control data. The optimized basal traction parameters explicitly appear in the momentum equations for the ice-stream velocities (compared to the control data). The inversion for basal traction is carried out by minimization of the cost (or objective, misfit) function that includes the momentum equations facilitated by the Lagrange multipliers. Here, we build upon this idea, and demonstrate how to optimize for parameters indirectly related to observed data using a suite of nested constraints (like Russian dolls) with additional sets of Lagrange multipliers in the cost function. This method opens the opportunity to use data from a variety of sources and types (e.g., velocities, radar layers, surface elevation changes, etc.) in the same optimization process.

  20. Quantitative studies of immunofluorescent staining. VII. Quantitative reference standard slide for standardization of fluorescence microscopes.

    PubMed

    Rostami, R; Beutner, E H; Kumar, V

    1992-01-01

    We evaluated a quantitative reference standard (QRS) slide with 10-microns beads with different concentrations of Coulter Electronics green dye No. 1. Microfluorospectrophotometric readings of the QRS slides provided quantitative comparisons between the sensitivity of the different fluorescence microscopes. The visual comparisons between beads with a range of intensities of fluorescence of Coulter Electronics green dye No. 1 indicated that the end points vary with different optical systems as do the end points in standard indirect immunofluorescent titrations. Fluorescent emission wavelength of QRS slide beads gave the same peak as fluorescein-labeled beads; both differed from the peak of orange beads of an 'Optical Standard' slide. Since shelf life studies show no changes in fluorescence intensity of beads over a period of 27 months or longer, QRS slide beads can afford a device for standardization of fluorescence microscopes used for immunofluorescent tests.

  1. Direct Immunofluorescence in Oral Lichen Planus

    PubMed Central

    Okuma, Nis; Thanakun, Supanee; Laothumthut, Titikarn

    2015-01-01

    Introduction Oral lichen planus (OLP) is a common immune-mediated oral mucosal disease. Diagnosis of OLP depends mainly on both clinical and histopathological features. Direct immunofluorescence (DIF) is a useful investigation method to distinguish between similar lesions and to confirm diagnosis in cases of uncharacterized features. Aim The purpose of this study was to evaluate the prevalence and pattern of DIF in a group of Thai patients with OLP. Materials and Methods Records of clinically and histologically diagnosed OLP patients attending the Oral Medicine Clinic, Faculty of Dentistry, Mahidol University, Bangkok, Thailand were consecutively reviewed for DIF results. The DIF patterns in these patients were analysed. Results There were 82 atrophic and/or erosive OLP patients with a mean age of 51.6 years. Male to female ratio was 1:5. Of these, 82.9% showed positive DIF. Buccal mucosa was superior to the gingiva and palate in terms of sensitivity for DIF. All specimens except one (98.5%) demonstrated deposition of fibrinogen at the basement membrane zone (BMZ) in a shaggy pattern. The most common DIF pattern was shaggy fibrinogen at BMZ with IgM deposition on the colloid bodies (CB) (35.3%) followed by shaggy fibrinogen along BMZ (27.9%). Conclusion The prevalence of positive DIF in Thai OLP patients was 82.9%. The most common finding was shaggy fibrinogen at BMZ. The typical pattern was shaggy fibrinogen along BMZ with or without positive IgM at CB. DIF pattern could be evaluated for the diagnosis of OLP lacking clinical and/or histopathological characteristic features. PMID:26436043

  2. Tyramide Signal Amplification for Immunofluorescent Enhancement.

    PubMed

    Faget, Lauren; Hnasko, Thomas S

    2015-01-01

    Enzyme-linked signal amplification is a key technique used to enhance the immunohistochemical detection of protein, mRNA, and other molecular species. Tyramide signal amplification (TSA) is based on a catalytic reporter deposit in close vicinity to the epitope of interest. The advantages of this technique are its simplicity, enhanced sensitivity, high specificity, and compatibility with modern multi-label fluorescent microscopy. Here, we describe the use of a TSA kit to increase the signal of enhanced green fluorescent protein (eGFP) expressed under the control of Slc17a6 regulatory elements in the brain of a transgenic mouse. The labeling procedure consists of 6 basic steps: (1) tissue preparation, (2) blocking of nonspecific epitopes, (3) binding with primary antibody, (4) binding with horseradish peroxidase-conjugated secondary antibody, (5) reacting with fluorescent tyramide substrate, and (6) imaging of the signal. The procedures described herein detail these steps and provide additional guidance and background to assist novice users.

  3. Epstein-Barr virus viral capsid antigen titer by immunofluorescence with microplates: new semiautomated method based on the microtiter system.

    PubMed Central

    Lamy, M E; Favart, A M; Burtonboy, G; Arana, A

    1977-01-01

    A semiautomated method of an indirect immunofluorescence technique for the titration of antibodies directed against viral capsid antigens of Epstein-Barr virus has been developed with Microtiter system units. By this method, a technician is able to titrate some hundred samples daily. The technique is safe, easy, and reproducible. The various procedures are described, and the sensitivity of the test is discussed. Images PMID:195981

  4. A novel protocol of whole mount electro-immunofluorescence staining.

    PubMed

    Liu, Hongshan; Kao, Winston W Y

    2009-01-01

    To develop a new method of whole mount immunostaining that improves the penetration of staining reagents into the cornea and decreases non-specific binding and background. Adult mouse corneas were fixed overnight in 4% paraformaldehyde or a mixture of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at 4 degrees C. After washing with 0.1% Triton X-100, corneas were embedded in 1% solidified agarose in a plastic column and fluorescent staining reagents, e.g., FITC-IgG (Fluorescein isothiocyanate-immunoglobulinG) conjugates in 0.5% solidified agarose was overlaid onto the specimens. The column was directionally immersed in a submarine gel electrophoresis apparatus filled with Tris-glycine buffer (TGB, pH=7.4) and electrophoresed at 4-10 mA for 10-24 h. For comparison, conventional protocols of immune fluorescent staining were also employed. The outcomes were evaluated by confocal microscopy. Antibody conjugates recognizing extracellular matrix (ECM) components, integral membrane protein, and intracellular structural proteins were used in whole mount corneas. The images of confocal laser scanning microscopy (CLSM) displayed a uniform distribution pattern of keratocan in corneal stroma, which is similar to that of section-staining. Anti-beta-tubulin antibodies bound to microtubes that are distributed within the whole cell body of superficial corneal epithelium cells and stromal keratocytes, but it was found perinuclear of corneal epithelial wing layers and endothelium; integral membrane protein, FAK (focal adhesion kinase), specifically labeled stromal cells of keratectomy corneas that healed for three weeks. In comparison, conventional protocols of immune fluorescent staining using the same antibody conjugates were also employed but did not yield satisfactory results. It was found that IgG conjugates examined did not readily penetrate into stroma and/or intact corneal epithelium. Phalloidin is a small molecule that can readily penetrate

  5. In situ identification of bacterial species in marine microfouling films by using an immunofluorescence technique.

    PubMed Central

    Zambon, J J; Huber, P S; Meyer, A E; Slots, J; Fornalik, M S; Baier, R E

    1984-01-01

    An immunofluorescence technique was developed for the in situ identification of specific bacteria in marine microfouling films. Microorganisms adherent to glass plates after 30 days of immersion in a synthetic seawater system were cultured and classified by biochemical tests, flagellar arrangement, and the API 20E system. All isolates were gram-negative aerobic or facultative motile rods, predominantly Pseudomonas spp. Rabbit antisera to the five dominant organisms including Achromobacter spp., Comamonas terrigena, P. putrefaciens, a yellow-pigmented Pseudomonas sp., and Vibrio alginolyticus were prepared. These antisera were shown to be species specific in indirect immunofluorescence assays against a battery of 26 marine isolates from 14 bacterial species, with the exception of antisera to the Pseudomonas spp, which cross-reacted with each other but not with test bacteria of other genera. These immunofluorescent reagents enabled the in situ identification of all five bacterial species in microfouling films. Low-surface-energy test plates had smaller numbers of adherent bacteria in microfouling films than medium-surface-energy test plates, suggesting that the degree of microfouling may be influenced by the surface energy. In addition, the reagents could identify up to 39% of the attached bacteria in microfouling films spontaneously formed on steel plates in flow cells deployed in different areas of the Atlantic Ocean. The microbial composition of the ocean-formed films varied with the geographical area of their formation. The present results indicate that immunofluorescence techniques may provide a rapid and reliable means to identify, in situ, specific bacteria in marine microfouling films. PMID:6393875

  6. A comparison between immunofluorescence staining on smears from Membrana nictitans (M3 test), immunohistopathology and routine pathology in cats with suspected feline infectious peritonitis (FIP).

    PubMed

    Hök, K

    1991-01-01

    An indirect immunofluorescence method using smears from membrana nictitans (M3 test) to diagnose feline corona virus (FCV) infection was compared with immunohistopathology (using indirect immunofluorescence assay (IFFA) performed on organs (IFO], and routine pathology (RP) in cats with suspected feline infectious peritonitis (FIP). A close correlation between the 2 immunofluorescence methods (IFO and M3) was observed. Although the M3 test requires samples from only 1 organ per animal, both the sensitivity and specificity were high (80%), when compared to IFO (using samples from an average of 5 organs per animal). In 21% of the cats with suspected FIP typical pathological lesions were found. As the M3 test is relatively easy to perform, it could reduce work-load of pathology laboratories and provide valuable data for clinical and epidemiological use.

  7. Technical report: immunofluorescence and TUNEL staining of celloidin embedded human temporal bone tissues.

    PubMed

    Markaryan, Adam; Nelson, Erik G; Tretiakova, Maria; Hinojosa, Raul

    2008-07-01

    The large archival human temporal bone collections of the world have been fixed in formalin and embedded in celloidin. These treatments have created challenges to the use of contemporary probes, which are routinely used in the evaluation of fresh and frozen tissues, for the analysis of archival temporal bone tissues. Formalin alters the configuration of proteins and can obscure antigens by modifying the epitopes recognized by antibodies. Celloidin embedding provides superior support of the delicate membranous structures of the inner ear to maintain tissue integrity during sectioning, however, inadequate removal of celloidin may limit tissue permeability resulting in poor penetration of large molecules. Methods are described in this manuscript that have allowed reproducible immunofluorescence and TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) staining results in these archival tissues. To our knowledge, successful immunofluorescence staining of type I collagen, immunofluorescence staining of cytochrome c oxidase subunit III (COX III), and TUNEL staining in archival human temporal bone tissues with confocal microscopy has not been previously reported. These results demonstrate the utility of developing techniques to evaluate the existing collections of archival temporal bones which remain our greatest source of tissue for investigating the causes of ear diseases.

  8. The role of electron microscopy for the diagnosis of glomerulopathies.

    PubMed

    Sementilli, Angelo; Moura, Luiz Antonio; Franco, Marcello Fabiano

    2004-05-06

    Electron microscopy has been used for the morphological diagnosis of glomerular diseases for more than three decades and its value has been widely emphasized. However, recent reports have analyzed the routine use of electron microscopy critically. Its use in other areas of diagnosis such as tumor diseases has declined considerably; in addition, in view of the unavoidable financial pressure for the reduction of costs due to investigations and diagnostic routines, the selection of cases for electron microscopy has been quite rigorous. To identify the glomerular diseases that depend on electron microscopy for a final diagnosis, by means of reviewing renal biopsies performed over a 12-year period. Prospective Hospital Ana Costa, Hospital Guilherme Alvaro and Serviço de Anatomia Patológica de Santos, Santos, São Paulo, Brazil. 200 consecutive renal biopsies obtained from private hospitals and the teaching hospital from 1979 to 1991 were studied. All cases were analyzed via light microscopy, immunofluorescence and electron microscopy. The diagnosis was first made via light microscopy plus immunofluorescence and then via electron microscopy. Electron microscopy was diagnostic or essential for diagnosis in 10.0% of the cases, corresponding to 3.4% of primary glomerulopathies and 100% of hereditary glomerulopathies. Electron microscopy was contributory (useful) to the diagnosis in 5.5% of the cases, confirming the preliminary diagnosis formulated on the basis of clinical and laboratory data and light microscopy plus immunofluorescence findings. We obtained a 7.5% rate of discordant immunofluorescence, which was considered as such when negative immunofluorescence findings were not confirmed by electron microscopy. The final diagnosis with the use of light microscopy plus immunofluorescence alone was 77.0%. It was possible to diagnose with certainty a great percentage of glomerulopathies (82.5-90% of the cases) based on the light microscopy and immunofluorescence findings

  9. Distinguishing Epidermolysis Bullosa Acquisita From Bullous Pemphigoid Without Direct Immunofluorescence.

    PubMed

    Gardner, Kerry M; Crawford, Richard I

    2017-07-01

    It has been postulated that periodic acid-Schiff staining of basement membrane can predict direct immunofluorescence patterns seen in epidermolysis bullosa acquisita and bullous pemphigoid. It has also been suggested that the type of inflammatory infiltrate or presence of fraying of basal keratinocytes may differentiate these two conditions. In this study, we aimed to confirm these observations. We reviewed 13 cases of direct immunofluorescence-confirmed epidermolysis bullosa acquisita and 19 cases of direct immunofluorescence-confirmed bullous pemphigoid, all with a subepidermal blister in the routinely processed specimen. The gold standard for diagnosis of epidermolysis bullosa acquisita vs bullous pemphigoid was taken to be identification of immune deposits on the dermal side ('floor' for epidermolysis bullosa acquisita) or the epidermal side ('roof' for bullous pemphigoid) of the salt-split direct immunofluorescence specimen. Our tests to distinguish epidermolysis bullosa acquisita from bullous pemphigoid on the routinely processed biopsy included periodic acid-Schiff basement membrane on the blister roof, neutrophilic infiltrate, lack of eosinophilic infiltrate, and absence of keratinocyte fraying. Sensitivity and specificity for each test were as follows: periodic acid-Schiff staining of roof (sensitivity 25%, specificity 95%), neutrophilic infiltrate (sensitivity 54%, specificity 74%), lack of eosinophilic infiltrate (sensitivity 92%, specificity 68%), and absence of keratinocyte fraying (sensitivity 62%, specificity 58%). Features in the routinely processed biopsy were unable to reliably distinguish between epidermolysis bullosa acquisita and bullous pemphigoid. Direct immunofluorescence on salt-split skin remains the standard for differentiation.

  10. Immunofluorescence detection of pea protein in meat products.

    PubMed

    Petrášová, Michaela; Pospiech, Matej; Tremlová, Bohuslava; Javůrková, Zdeňka

    2016-08-01

    In this study we developed an immunofluorescence method to detect pea protein in meat products. Pea protein has a high nutritional value but in sensitive individuals it may be responsible for causing allergic reactions. We produced model meat products with various additions of pea protein and flour; the detection limit (LOD) of the method for pea flour was 0.5% addition, and for pea protein it was 0.001% addition. The repeatabilities and reproducibilities for samples both positive and negative for pea protein were all 100%. In a blind test with model products and commercial samples, there was no statistically significant difference (p > 0.05) between the declared concentrations of pea protein and flour and the immunofluorescence method results. Sensitivity was 1.06 and specificity was 1.00. These results show that the immunofluorescence method is suitable for the detection of pea protein in meat products.

  11. A simple solution for antibody signal enhancement in immunofluorescence and triple immunogold assays.

    PubMed

    Rosas-Arellano, Abraham; Villalobos-González, Juan B; Palma-Tirado, Lourdes; Beltrán, Felipe A; Cárabez-Trejo, Alfonso; Missirlis, Fanis; Castro, Maite A

    2016-10-01

    Immunolocalization techniques are standard in biomedical research. Tissue fixation with aldehydes and cell membrane permeabilization with detergents can distort the specific binding of antibodies to their high affinity epitopes. In immunofluorescence protocols, it is desirable to quench the sample's autofluorescence without reduction of the antibody-dependent signal. Here we show that adding glycine to the blocking buffer and diluting the antibodies in a phosphate saline solution containing glycine, Triton X-100, Tween20 and hydrogen peroxide increase the specific antibody signal in tissue immunofluorescence and immunogold electron microscopy. This defined antibody signal enhancer (ASE) solution gives similar results to the commercially available Pierce Immunostain Enhancer (PIE). Furthermore, prolonged tissue incubation in resin and fixative and application of ASE or PIE are described in an improved protocol for triple immunogold electron microscopy that is used to show co-localization of GABA-A ρ2 and dopamine D2 receptors in GFAP-positive astrocytes in the mouse striatum. The addition of glycine, Triton X-100, Tween20 and hydrogen peroxide during antibody incubation steps is recommended in immunohistochemistry methods.

  12. 21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... consist of rabiesvirus antisera conjugated with a fluorescent dye used to identify rabiesvirus...

  13. 21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... consist of rabiesvirus antisera conjugated with a fluorescent dye used to identify rabiesvirus...

  14. 21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... consist of rabiesvirus antisera conjugated with a fluorescent dye used to identify rabiesvirus...

  15. Rapid Diagnosis of Arbovirus and Arenavirus Infections by Immunofluorescence.

    DTIC Science & Technology

    1983-01-31

    Lassa fever virus ...Crimean Hemorrhagic fever -Cong viruses Ebola virus Immunofluorescence Marburg virus ELISA Rift Valley fever virus Equatorial Africa 20. ABSTRACT Ctf ae...Cong, Rift Valley fever , Ebola, Lassa fever , and Marburg viruse was continued. In Sudan, prevalence rates wore Marburg 0.2%, CCHF 0.7%, Lassa 3.9%,

  16. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents...

  17. 21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460 Rabiesvirus...

  18. 21 CFR 866.3460 - Rabiesvirus immuno-fluorescent reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Rabiesvirus immuno-fluorescent reagents. 866.3460 Section 866.3460 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460 Rabiesvirus...

  19. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Mycobacterium tuberculosis immunofluorescent reagents. 866.3370 Section 866.3370 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents...

  20. Immunofluorescence Patterns in Selected Dermatoses, Including Blistering Skin Diseases Utilizing Multiple Fluorochromes

    PubMed Central

    Abreu-Velez, Ana Maria; Calle-Isaza, Juliana; Howard, Michael S.

    2015-01-01

    Background: Autoimmune vesiculobullous disorders represent a heterogeneous group of dermatoses whose diagnosis is made based on clinical history, histologic features, and immunopathologic features. The most commonly used techniques for the diagnosis of these diseases are direct and indirect immunofluorescence (DIF and IIF), including salt-split processing. NaCl split skin is used to determine the level of blister formation, and the localization of autoantibodies relative to the split. Classically, immunofluorescence has been performed with one fluorochrome in the diagnosis of autoimmune bullous skin diseases. Aims: To compare DIF and IIF of the skin, using a single fluorochrome versus multiple fluorochromes. Materials and Methods: We studied 20 autoimmune skin disease cases using fluorescein isothiocyanate (FITC) alone, in comparison to multiple fluorochromes (with or without DNA counterstaining). Results: The use of multiple fluorochromes helped to simultaneously visualize reactivity in multiple skin areas, in contrast to using FITC alone. Conclusions: Using multiple fluorochromes allows simultaneous labeling of two or more antigens within the same cell/or tissue section, assists in colocalization of unknown antigens with known molecules, and helps in ruling out “background” staining. PMID:26605203

  1. IgG antibodies in patients with pemphigus vulgaris before and after diagnosing with immunofluorescence.

    PubMed

    Azimi, Hamideh; Majidi, Jafar; Estakhri, Rasul; Goldust, Mohamad

    2013-06-15

    Pemphigus is defined as a group of chronic self-immune vesicular diseases histologically recognized by inter-epidermic vesicles resulting from acantholysis. The aim of this study was to evaluate the precipitant and circulative IgG antibodies in patients with pemphigus vulgaris before and after treating with immunofluorescence. Sixty-two patients (34 females and 28 males) with clinically and pathologically confirmed P.V. were studied prospectively over a one year period of time during which direct and indirect immunofluorescent tests were performed before and after treatment. They had mild or moderate forms of disease. All patients received prednisolon 1-2 mg/kg/day and Azathioprine 2-3 mg/kg/day or methylpredisolon (1 g day(-1) for 4 days) and cyclophosphamide (500 mg/first day) pulse therapy due to general condition. Thirty- four females and 28 males enrolled, the mean age were 39.5 years (SD = 12.7). Before treatment, 10 and 52 cases were positive for skin depositing + or ++) and circulatory IgG (1/20 -1/60), respectively. Two to 3 month later, 37 were IgG positive with titers 1/20 to 1/160. The correlation between circulatory IgG before and after treatment was weakly positive (p = 0.05, r = 0.415). In the present study, treatment methods used for patients suffering from pemphigus vulgaris were not successful in significantly decreasing the circulative autoantibodies levels.

  2. Automated tests of ANA immunofluorescence as throughput autoantibody detection technology: strengths and limitations.

    PubMed

    Meroni, Pier Luigi; Bizzaro, Nicola; Cavazzana, Ilaria; Borghi, Maria Orietta; Tincani, Angela

    2014-03-03

    Anti-nuclear antibody (ANA) assay is a screening test used for almost all autoimmune rheumatic diseases, and in a number of these cases, it is a diagnostic/classification parameter. In addition, ANA is also a useful test for additional autoimmune disorders. The indirect immunofluorescence technique on monolayers of cultured epithelial cells is the current recommended method because it has higher sensitivity than solid phase assays. However, the technique is time-consuming and requires skilled operators. Automated ANA reading systems have recently been developed, which offer the advantage of faster and much easier performance as well as better harmonization in the interpretation of the results. Preliminary validation studies of these systems have given promising results in terms of analytical specificity and reproducibility. However, these techniques require further validation in clinical studies and need improvement in their recognition of mixed or less common staining patterns.

  3. Immunofluorescent studies on human spermatozoa. III. Immunoglobulin classes of human spermatozoal antibodies

    PubMed Central

    Hansen, K. Brogaard

    1972-01-01

    Antibodies in human sera against four different antigens of human spermatozoa discovered by means of an indirect two-layer immunofluorescence technique (IFT) were characterized by determination of the class of immunoglobulins to which they belonged. A three-layer IFT using monospecific antisera against human IgG, IgA or IgM as the second layer was employed together with fractionation of sera on Sephadex G-200 or DEAE-cellulose followed by testing of the concentrated pools in a two-layer IFT. The study revealed that antibodies against the antigen in the front part of the acrosome were primarily IgM and those against the antigen in the tail primarily IgG. Antibodies against antigens in the equatorial segment and the postnuclear cap showed a varying predominance of these two immunoglobulins. Spermatozoal antibody as IgA was found only in small amounts. PMID:4558409

  4. Toward a working definition of C3 glomerulopathy by immunofluorescence.

    PubMed

    Hou, Jean; Markowitz, Glen S; Bomback, Andrew S; Appel, Gerald B; Herlitz, Leal C; Barry Stokes, M; D'Agati, Vivette D

    2014-02-01

    Precise immunofluorescence criteria for C3 glomerulopathy remain to be defined. Here we tested hierarchical immunofluorescence criteria with varying stringency for C3 glomerulopathy in a cohort with dense deposit disease as the gold standard and then applied these criteria to analyze the incidence of C3 glomerulopathy in membranoproliferative glomerulonephritis (MPGN) types 1 and 3. Among 319 archived cases of primary MPGN types 1-3, immunofluorescence reports were retrospectively coded as glomerular deposits of the following: C3 only; C3 dominant with trace or 1+ immunoglobulin (Ig)M only; and C3 dominant and at least two orders of intensity stronger than any combination of IgG, IgM, IgA, and C1q. The most restrictive criteria of 'C3 only' captured only half of the cases with dense deposit disease (compared with 8% of type 1 and 10% of type 3). Adding the most liberal definition identified 88% of those with dense deposit disease (compared with 31% of type 1 and 39% of type 3). The unaccounted 12% had stronger intensity of Ig staining, but it never exceeded the intensity of C3. Among MPGN type 3, 90% of C3 glomerulopathy cases were the Strife and Anders variant. Repeat biopsies in C3 glomerulopathy revealed a change in immunofluorescence pattern in 10 of 23 biopsies. The prevalence of low serum C3 and/or low C4 did not significantly differ among the three immunofluorescence criteria. Thus, 'C3 only' is an impractical definition of C3 glomerulopathy, and we propose a definition of C3 dominant and at least two orders of magnitude more intense than any other immune reactant, which requires validation by alternative pathway evaluation. These criteria provide a framework for identifying patients most likely to benefit from investigations of alternative complement pathway dysregulation.

  5. Fourier-ring descriptor to characterize rare circulating cells from images generated using immunofluorescence microscopy.

    PubMed

    Emerson, Tegan; Kirby, Michael; Bethel, Kelly; Kolatkar, Anand; Luttgen, Madelyn; O'Hara, Stephen; Newton, Paul; Kuhn, Peter

    2015-03-01

    We address the problem of subclassification of rare circulating cells using data driven feature selection from images of candidate circulating tumor cells from patients diagnosed with breast, prostate, or lung cancer. We determine a set of low level features which can differentiate among candidate cell types. We have implemented an image representation based on concentric Fourier rings (FRDs) which allow us to exploit size variations and morphological differences among cells while being rotationally invariant. We discuss potential clinical use in the context of treatment monitoring for cancer patients with metastatic disease.

  6. Removal of Drosophila muscle tissue from larval fillets for immunofluorescence analysis of sensory neurons and epidermal cells

    PubMed Central

    Tenenbaum, Conrad M.; Gavis, Elizabeth R.

    2017-01-01

    SHORT ABSTRACT Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall. LONG ABSTRACT Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development. PMID:27842373

  7. A protocol for immunofluorescence staining of floating neurospheres.

    PubMed

    Sasaki, Ryo; Aoki, Shunsuke; Yamato, Masayuki; Uchiyama, Hiroto; Wada, Keiji; Ogiuchi, Hideki; Okano, Teruo; Ando, Tomohiro

    2010-07-26

    This protocol describes the immunofluorescence staining of floating neurospheres in culture plates. Although this protocol is similar to conventional immunofluorescence staining, the staining procedure of floating neurospheres in multiwell culture plates and the washing procedure are different. Neurospheres in culture plates are transferred to a 12-well plate using a 200-1000microL pipette. The spheres are precipitated by gravity for 3min. Then, the 12-well plate is tilted slightly, and the culture medium is aspirated by the pipette. After aspiration, the spheres are visually verified to be at the bottom of the wells. PBS (400microL) is added to the well for washing the spheres. This procedure is repeated three times. This protocol is easier than a conventional procedure using cryostat sections and can give clear sphere structures. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

  8. Indirection and computer security.

    SciTech Connect

    Berg, Michael J.

    2011-09-01

    The discipline of computer science is built on indirection. David Wheeler famously said, 'All problems in computer science can be solved by another layer of indirection. But that usually will create another problem'. We propose that every computer security vulnerability is yet another problem created by the indirections in system designs and that focusing on the indirections involved is a better way to design, evaluate, and compare security solutions. We are not proposing that indirection be avoided when solving problems, but that understanding the relationships between indirections and vulnerabilities is key to securing computer systems. Using this perspective, we analyze common vulnerabilities that plague our computer systems, consider the effectiveness of currently available security solutions, and propose several new security solutions.

  9. RGB method in immunofluorescence investigations on stem cells

    NASA Astrophysics Data System (ADS)

    Riccio, Massimo; Resca, Elisa; Bertoni, Laura; Cavani, Francesco; Sena, Paola; Ferretti, Marzia; Baldini, Andrea; Palumbo, Carla; De Pol, Anto

    2011-03-01

    Colour is not related to a particular discipline, but it is transversely present in many circles and in almost all the aspects of life. It has a special value in art, but also as far as other disciplines are concerned, like the sciences, the colour is at the basis of some of their intrinsic significances and it often needed to allow the interpretation of some of their phenomena as well. As regards the development of cell biology knowledge, colour acquired more and more importance in revealing the observations of the researchers. A field in which the methods based on the colours are particularly employed is the immunofluorescence, used to identify specific proteins in cells and tissues. These techniques combine the fluorochrome properties with specific molecules, i.e. antibodies, directed against particular substances to investigate, for example a specific protein. In single immunofluorescence analysis, the signal from an excited fluorochrome corresponds to a particular protein. In multiple immunofluorescence analysis, two or more signals are simultaneously detected to show the localization of different proteins on the same sample. The three primary colours red, green and blue were currently assigned to the signals from immunofluorescence-processed samples and visualized by the RGB method. In the present work, different examples of RGB applications in immunocytochemical investigations are showed: the first concerns the multiple analysis of three markers, localized in different loci of the cell plasma membrane; the second is related to the co-localization of two signals in the same site of specific subcellular structures. In this case the secondary colours, obtained by overlapping the primary ones, demonstrate the specific co-presence of two proteins in the same site. With the present paper, the authors wish to underline the relevant role of colours also in those areas in which colours are the means not the end.

  10. Dual immunofluorescence staining of proteoglycans in human temporal bones.

    PubMed

    Markaryan, Adam; Nelson, Erik G; Kohut, Robert I; Hinojosa, Raul

    2011-07-01

    Immunofluorescence staining methods have been developed to study the distribution of macromolecules in archival formalin-fixed celloidin-embedded human temporal bone tissues. The aim of this study was to investigate the feasibility of utilizing this approach to evaluate the codistribution of more than one molecule of interest in a single tissue section. Retrospective study of proteoglycan codistribution in archival human temporal bone tissues. The chondroitin sulfate and keratan sulfate proteoglycans were selected for evaluating this methodology. Human tissues with known proteoglycan staining patterns were studied as controls. Thirty-one formalin-fixed celloidin-embedded archival human temporal bones were evaluated, and the observations in 11 specimens are described. A dual immunofluorescence staining method was developed using primary antibodies of differing isotypes and secondary antibodies labeled with fluorophores having nonoverlapping emission characteristics. The specificity of the dual immunofluorescence technique for chondroitin sulfate and keratan sulfate proteoglycans was demonstrated in control tissues and confirmed through inhibition studies. The normal human tectorial membrane exhibited intense chondroitin sulfate staining. Cochlear and vestibular hair cells exhibited predominantly keratan sulfate staining. Keratan sulfate staining predominated in spiral ganglion cell bodies and fibers. Alterations in the normal distribution pattern of proteoglycans were observed in cases of presbycusis and otosclerosis. The dual immunofluorescence staining methodology can be used to study archival formalin-fixed celloidin-embedded human temporal bone tissues. This technique may be applied to the evaluation of other molecules in archival human temporal bone tissues and lead to improvement in our understanding of the function of these molecules and their role in disease processes. Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.

  11. Rapid Diagnosis of Arbovirus and Arenavirus Infections by Immunofluorescence.

    DTIC Science & Technology

    1982-02-01

    Hemorrhagic Fever-Congo viruses Ebola virus Immunofluorescence Marburg virus ELISA Rift Valley Fever virus Equatorial Africa 20. AWsrNAC(ouingm ,.rm . IN me& &O...Lassa fever, and Marburg viruses was begun. Positi.e reactiorito Ebola virus have been observed with sera from military recruits from southern Sudan...geographic region. .- One hundred and fifty-eight (158) bunyavirus supergroup viruses have been screened for reaction on KHF virus -infected spot-slides

  12. Immunofluorescent staining of influenza virus antigen in fixed and paraffin-embedded tissue of experimentally infected hamsters.

    PubMed

    Lück, P C; Helbig, J H; Witzleb, W

    1989-01-01

    Sections of formalin-fixed, paraffin-embedded tissue of experimentally influenza virus-infected hamsters were treated with 0.25% trypsin and tested for virus antigen by indirect immunofluorescent staining. The results were comparable to those obtained with aceton-fixed cryo-microtome sections. As far as we know, this is the first description of influenza virus demonstration in formalin-fixed, paraffin-embedded tissue after reactivation by trypsin-treatment. This technique may be useful for influenza virus detection in human autopsy cases. It allows an etiological diagnosis even when fresh tissue for cryocut sections or virus cultivation is not available.

  13. Magneto immunofluorescence assay for diagnosis of celiac disease.

    PubMed

    Kergaravat, Silvina V; Beltramino, Luis; Garnero, Nidia; Trotta, Liliana; Wagener, Marta; Fabiano, Silvia N; Pividori, Maria Isabel; Hernandez, Silvia R

    2013-10-10

    A magneto immunofluorescence assay for the detection of anti-transglutaminase antibodies (ATG2) in celiac disease was developed. The ATG2 were recognized by transglutaminase enzyme immobilized on the magnetic beads and then the immunological reaction was revealed by antibodies labeled with peroxidase. The fluorescent response of the enzymatic reaction with o-phenylenediamine and H2O2 as substrates was correlated with anti-transglutaminase titer, showing EC50 and LOD values of 1:11,600 and 1:74,500 of antibody titers, respectively. A total number of 29 sera samples from clinically confirmed cases of celiac disease and 19 negative control samples were tested by the novel magneto immunofluorescence assay. The data were submitted to the receiver-operating characteristic plot (ROC) analysis which indicated that 8.1 U was the most effective cut-off value to discriminate correctly between celiac and non-celiac patients. The immunofluorescence assay exhibited a sensitivity of 96.6%, a specificity of 89.5% and an efficiency 93.8% compared with the commercial optical ELISA kit.

  14. Research Techniques Made Simple: Immunofluorescence Antigen Mapping in Epidermolysis Bullosa.

    PubMed

    Has, Cristina; He, Yinghong

    2016-07-01

    Inherited epidermolysis bullosa is a group of genetic blistering diseases with a broad spectrum of clinical severity and molecular defects. Epidermolysis bullosa results from mutations in genes encoding proteins involved in cell-cell and cell-matrix adhesion in the epidermis. Immunofluorescence antigen mapping makes use of monoclonal antibodies against proteins of the dermal-epidermal junction zone to determine the layer of skin where cleavage occurs and the relative protein abundance. It allows the diagnosis of the type and subtype of inherited epidermolysis bullosa and sheds light on molecular mechanisms underlying the disease. Immunofluorescence mapping steps include obtaining a skin biopsy sample, processing the biopsy material, antigen-antibody interaction on tissue, washing, incubation with fluorescently conjugated secondary antibodies, mounting, observation under a fluorescence microscope, and interpretation. A minimal antibody panel allows discrimination of the main epidermolysis bullosa subtypes. Extended panels can be used depending on the diagnostic or scientific question to be addressed. Immunofluorescence mapping contributed to significant progress in understanding epidermolysis bullosa, including identification of new underlying genetic mutations, mutation mechanisms, and the presence of revertant mosaicism. It is also an important tool in the assessment of the efficacy of experimental therapeutic approaches. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Spatial Mixture Modelling for Unobserved Point Processes: Examples in Immunofluorescence Histology

    PubMed Central

    Ji, Chunlin; Merl, Daniel; Kepler, Thomas B.; West, Mike

    2010-01-01

    We discuss Bayesian modelling and computational methods in analysis of indirectly observed spatial point processes. The context involves noisy measurements on an underlying point process that provide indirect and noisy data on locations of point outcomes. We are interested in problems in which the spatial intensity function may be highly heterogenous, and so is modelled via flexible nonparametric Bayesian mixture models. Analysis aims to estimate the underlying intensity function and the abundance of realized but unobserved points. Our motivating applications involve immunological studies of multiple fluorescent intensity images in sections of lymphatic tissue where the point processes represent geographical configurations of cells. We are interested in estimating intensity functions and cell abundance for each of a series of such data sets to facilitate comparisons of outcomes at different times and with respect to differing experimental conditions. The analysis is heavily computational, utilizing recently introduced MCMC approaches for spatial point process mixtures and extending them to the broader new context here of unobserved outcomes. Further, our example applications are problems in which the individual objects of interest are not simply points, but rather small groups of pixels; this implies a need to work at an aggregate pixel region level and we develop the resulting novel methodology for this. Two examples with with immunofluorescence histology data demonstrate the models and computational methodology. PMID:21037943

  16. Spatial Mixture Modelling for Unobserved Point Processes: Examples in Immunofluorescence Histology.

    PubMed

    Ji, Chunlin; Merl, Daniel; Kepler, Thomas B; West, Mike

    2009-12-04

    We discuss Bayesian modelling and computational methods in analysis of indirectly observed spatial point processes. The context involves noisy measurements on an underlying point process that provide indirect and noisy data on locations of point outcomes. We are interested in problems in which the spatial intensity function may be highly heterogenous, and so is modelled via flexible nonparametric Bayesian mixture models. Analysis aims to estimate the underlying intensity function and the abundance of realized but unobserved points. Our motivating applications involve immunological studies of multiple fluorescent intensity images in sections of lymphatic tissue where the point processes represent geographical configurations of cells. We are interested in estimating intensity functions and cell abundance for each of a series of such data sets to facilitate comparisons of outcomes at different times and with respect to differing experimental conditions. The analysis is heavily computational, utilizing recently introduced MCMC approaches for spatial point process mixtures and extending them to the broader new context here of unobserved outcomes. Further, our example applications are problems in which the individual objects of interest are not simply points, but rather small groups of pixels; this implies a need to work at an aggregate pixel region level and we develop the resulting novel methodology for this. Two examples with with immunofluorescence histology data demonstrate the models and computational methodology.

  17. Characterization of instrumentation and calibrators for quantitative microfluorometry for immunofluorescence tests.

    PubMed Central

    Kaplan, D S; Picciolo, G L

    1989-01-01

    The current method for measuring reagents for immunofluorescence microscopy involves a subjective evaluation of the endpoint (titer) with a negative or positive (1+ to 4+) scale. Variability is due to the biological constituents of the reagent, the observer, and the instrumentation. To have reliable methods for evaluation of performance of these products, we are developing a quantitative method that uses photometric measurements of microscopically observed epifluorescence of slide preparations. A computer-controlled microscope-photometer converts the light intensity into a voltage measurement. Our goal is to replace the subjective endpoint determination with an objective, quantitative method. The instrumentation and its operating characteristics are presented in this paper. Selected commercially available fluorescent materials were evaluated as calibrators for the instrumentation. These materials showed consistency in measurement and thus demonstrated their suitability for various levels of calibration. It is possible that they will prove useful as a reference standard for interlaboratory comparisons. PMID:2654179

  18. Image analysis to quantify histological and immunofluorescent staining of ex vivo skin and skin cell cultures.

    PubMed

    McMullen, R L; Bauza, E; Gondran, C; Oberto, G; Domloge, N; Farra, C Dal; Moore, D J

    2010-04-01

    Image processing steps and analysis techniques were developed for the quantification of photomicrographs obtained from light and fluorescence microscopy. The substrates examined were either skin cell cultures, such as normal human keratinocytes (NHK) or fibroblasts, or ex vivo skin sections. Examples of the analyses are provided for the comparison of skincare active ingredient treated samples vs. placebo to demonstrate the utility of the methods to quantify and provide numerical data for a procedure that is typically qualitative in nature and based on observations by a histologist. Quantifiable experiments that are discussed include: Fontana Masson staining for melanin expression; Nile red staining to detect cellular lipid droplets; nuclei staining with diamidino-phenylindole (DAPI); and immunofluorescent staining of protein expression with a primary antibody directed against the protein (antigen) and a secondary antibody tagged with a fluorescent dye (Alexa Fluor 488) against the primary antibody.

  19. Quick fluorescent in situ hybridization protocol for Xist RNA combined with immunofluorescence of histone modification in X-chromosome inactivation.

    PubMed

    Yue, Minghui; Charles Richard, John Lalith; Yamada, Norishige; Ogawa, Akiyo; Ogawa, Yuya

    2014-11-26

    Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell level to detect the spatial dynamics of RNA localization with simultaneous insight into the localization of proteins, epigenetic modifications and other details which can be highlighted by immunofluorescence. X-chromosome inactivation is a paradigm for long non-coding RNA (lncRNA)-mediated gene silencing. X-inactive specific transcript (Xist) lncRNA accumulation (called an Xist cloud) on one of the two X-chromosomes in mammalian females is a critical step to initiate X-chromosome inactivation. Xist RNA directly or indirectly interacts with various chromatin-modifying enzymes and introduces distinct epigenetic landscapes to the inactive X-chromosome (Xi). One known epigenetic hallmark of the Xi is the Histone H3 trimethyl-lysine 27 (H3K27me3) modification. Here, we describe a simple and quick immuno-FISH protocol for detecting Xist RNA using RNA FISH with multiple oligonucleotide probes coupled with immunofluorescence of H3K27me3 to examine the localization of Xist RNA and associated epigenetic modifications. Using oligonucleotide probes results in a shorter incubation time and more sensitive detection of Xist RNA compared to in vitro transcribed RNA probes (riboprobes). This protocol provides a powerful tool for understanding the dynamics of lncRNAs and its associated epigenetic modification, chromatin structure, nuclear organization and transcriptional regulation.

  20. Automated evaluation of autoantibodies on human epithelial-2 cells as an approach to standardize cell-based immunofluorescence tests

    PubMed Central

    2010-01-01

    Introduction Analysis of autoantibodies (AAB) by indirect immunofluorescence (IIF) is a basic tool for the serological diagnosis of systemic rheumatic disorders. Automation of autoantibody IIF reading including pattern recognition may improve intra- and inter-laboratory variability and meet the demand for cost-effective assessment of large numbers of samples. Comparing automated and visual interpretation, the usefulness for routine laboratory diagnostics was investigated. Methods Autoantibody detection by IIF on human epithelial-2 (HEp-2) cells was conducted in a total of 1222 consecutive sera of patients with suspected systemic rheumatic diseases from a university routine laboratory (n = 924) and a private referral laboratory (n = 298). IIF results from routine diagnostics were compared with a novel automated interpretation system. Results Both diagnostic procedures showed a very good agreement in detecting AAB (kappa = 0.828) and differentiating respective immunofluorescence patterns. Only 98 (8.0%) of 1222 sera demonstrated discrepant results in the differentiation of positive from negative samples. The contingency coefficients of chi-square statistics were 0.646 for the university laboratory cohort with an agreement of 93.0% and 0.695 for the private laboratory cohort with an agreement of 90.6%, P < 0.0001, respectively. Comparing immunofluorescence patterns, 111 (15.3%) sera yielded differing results. Conclusions Automated assessment of AAB by IIF on HEp-2 cells using an automated interpretation system is a reliable and robust method for positive/negative differentiation. Employing novel mathematical algorithms, automated interpretation provides reproducible detection of specific immunofluorescence patterns on HEp-2 cells. Automated interpretation can reduce drawbacks of IIF for AAB detection in routine diagnostics providing more reliable data for clinicians. PMID:20214808

  1. Oral Mucocutaneous Lesions – A Comparative Clinicopathological and Immunofluorescence Study

    PubMed Central

    Rameshkumar, Annasamy; Varghese, Alex Kumaranthara; Dineshkumar, Thayalan; Ahmed, Shaheen; Venkatramani, Janaki; Sugirtharaj, G

    2015-01-01

    Background: Oral mucosa is often affected by many diseases including mucocutaneous disorders. The diagnoses of these disorders are primarily based on history, clinical features, and histopathology. For the past few years’ immunofluorescence techniques emerged as an important tool to study the pathogenesis and in the diagnosis of oral mucocutaneous and vesiculobullous disorders. The present study was designed to carry out retrospective and prospective analysis of oral mucocutaneous lesions to elucidate the clinicopathologic features and its immunofluorescence findings. Materials and Methods: A total of 70 subjects with oral mucocutaneous lesions were retrieved from the oral pathology files of Tamil Nadu Govt. Dental College and their clinical features were evaluated, and the histopathology was also evaluated with the help of hematoxylin and eosin stained sections. For the prospective study, biopsy from 12 subjects with oral mucocutaneous lesions was subjected to routine histopathological examination and DIF to evaluate the consistency of the methods. Results: In the retrospective analysis of 70 subjects with oral mucocutaneous lesions in relation to clinical features and histopathology, most of the findings were similar to the previous studies except for few criteria like male predilection in lichen planus and mucous membrane pemphigoid (MMP) and more prevalence of pemphigus vulgaris than MMP (2:1). In the prospective analysis of 12 subjects, the histopathological diagnosis was consistent with DIF study in 66% of cases. Conclusion: The diagnostic efficiency of oral mucocutaneous lesions can be improved by modern tools like DIF studies in addition to traditional methods like clinical and histopathology. PMID:25878481

  2. The detection of platelet isoantibodies by membrane immunofluorescence.

    PubMed

    van der Schans, G S; Veenhoven, W A; Snijder, J A; Nieweg, H O

    1977-07-01

    A membrane ummunofluorescence test for the detection of platelet isoantibodies is described. Gel filtration of the incubation mixture was incorporated in the procedure and proved effective for the removal of serum proteins from the platelet suspension. With this technique isoantibodies were found in the serum of 13 out of a group of 16 patients who had received multiple transfusions. The results were checked by measuring the uptake of 125I-labeled anti-IgG fraction by gel-filtered platelets. Subsequently the membrane immunofluorescence method was also compared with established techniques described for the detection of isoantibodies such as the microtest for lymphocytotoxicity and a complement-fixation method and the procedures based on the release of labeled serotonin, the phagocytosis of chromium-tagged platelets, the increase of platelet factor 3 activity, and on platelet aggregation. We had the opportunity to investigate the serum of one patient for the presence of isoantibodies against platelets from HLA identical siblings both before and after the administration of their platelets. On the basis of this experience it is concluded that the membrane immunofluorescence test for platelet isoantibodies is a relatively simple method with a high degree of specificity and adequate sensitivity.

  3. [Comparison of the indirect immunofluorescent (IFAT), ELISA test and the comercial Chagatek test for anti-Trypanosoma cruzi antibodies detection].

    PubMed

    Enciso, Clara; Montilla, Marleny; Santacruz, María M; Nicholls, Rubén Santiago; Rodríguez, Adriana; Mercado, Marcela; Puerta, Concepción

    2004-03-01

    Chagas disease is a public health problem in Colombia, particularly in the eastern region. Because of human migration from rural areas to urban centers, the possibility of transfusional transmission becomes increasingly important. However the risk can be minimized by a careful screening of blood donors by means of serological tests. Colombian blood banks use comercial, foreign serological tests for screening for T. cruzi infection. The purpose of the current study was to compare the IFAT and ELISA tests (both use antigen obtained from Colombian strains) with the comercially available Chagatek tests. Sera of blood donors were classified in two groups on the basis of the IFAT: group I, 15 positive patients and group II, 14 negative patients. Sera from each group were tested by the ELISA and Chagatek tests. The ELISA test detected 100% of the patients as positive in group I and 7% (1/14) of patients as positive in group II. The Chagatek test detected 93% (14/15) of the patients as positive in group I and 50% (7/14) in group II. The kappa index for concordance between the ELISA and IFAT tests was 0.93 (95% C.I.: 0.80-1.00); between IFAT and Chagatek 0.43 (95% C.I.: 0.26-0.62), and between ELISA and Chagatek 0.49 (95% C.I.: 0.31-0.67). These results highlighted the importance of using autochtonous Colombian strains as antigens in screening tests for blood donors.

  4. [Serologic cross-reactions to Leishmania infantum using indirect immunofluorescence in HIV+ and HIV- patients with active tuberculosis].

    PubMed

    López-Vélez, R; Turientes, M C; Gómez-Mampaso, E

    1998-03-01

    Clinical presentation of disseminated tuberculosis and visceral leishmaniosis can be very similar, mainly in those infected with HIV, being serology a useful tool in making a differential diagnosis. Cross-reactions of IFAT serodiagnosis of visceral leishmaniosis with other diseases are well known, but few data is available with tuberculosis. Detection of serum antibodies against Leishmania, using a commercial IFAT kit, was attempted in sera of 51 patients with active pulmonar and/or extrapulmonar tuberculosis (25 HIV+ and 26 HIV-). Overall cross-reactions was found in 19.6% patients without significative differences in between 2 groups, but differences in positive serum titres was observed: one at 1/256, three at 1/160, and one at 1/80 dilution, in the HIV+ group, whereas all 5 patients in HIV- group cross-reacted at 1/80 dilution. Recognition of specific leishmanial antigenic bands by serum antibodies of patients with tuberculosis were not clearly defined by Western-blot. IFAT technique for leishmaniosis cross-react in 20% of patients with tuberculosis.

  5. Immunofluorescence on paraffin-embedded sections in evaluation of immune complex deposits in renal biopsy specimens.

    PubMed

    Wagrowska-Danilewicz, Małgorzata; Danilewicz, Marian

    2009-01-01

    The data focused on the value of immunofluorescence on paraffin-embedded sections are controversial, and it is still difficult to obtain reproducible results. The aim of our study was to evaluate the usefulness of immunofluorescence on paraffin-embedded renal section in detecting immune complex deposits in IgA nephropathy (n = 24), membranous glomerulopathy (n = 22) and lupus nephritis (n = 24). Our study revealed that direct immunofluorescence on paraffin-embedded sections pre-treated with proteinase K for 30 or 60 min is a less sensitive method than immunofluorescence on frozen sections; therefore a number of glomerulopathies may be overlooked. Immunofluorescence on paraffin sections showed dominant or co-dominant fluorescence of Riga only in 41.7% of cases of Riga nephropathy. In the studied glomerulopathies the number of positive immunofluorescences of IgA, IgG, IgM and C3 was significantly lower in immunofluorescence on paraffin sections in comparison with findings obtained from immunofluorescence on frozen sections. Irrespective of glomerular disease the rate of agreement between immunofluorescence on paraffin sections and immunofluorescence on frozen sections with respect to the presence of IgA was 56.5%, IgM - 44.4%, IgG - 73.9%, and C3 - 51.5%. In conclusion, our study revealed that immunofluorescence on paraffin sections cannot replace immunofluorescence on frozen sections in the assessment of human renal biopsies, and must be interpreted with great caution.

  6. Autoradiography and Immunofluorescence Combined for Autecological Study of Single Cell Activity with Nitrobacter as a Model System1

    PubMed Central

    Fliermans, C. B.; Schmidt, E. L.

    1975-01-01

    Specific detection of a particular bacterium by immunofluorescence was combined with estimation of its metabolic activity by autoradiography. The nitrifying bacteria Nitrobacter agilis and N. winogradskyi were used as a model system. Nitrobacter were incubated with NaH14CO3 and 14CO2 prior to study. The same preparations made for autoradiograms were stained with fluorescent antibodies specific for the Nitrobacter species. Examination by epifluorescence and transmitted dark-field microscopy revealed Nitrobacter cells with and without associated silver grains. Direct detection and simultaneous evaluation of metabolic activity of Nitrobacter was demonstrated in pure cultures, in a simple mixed culture, and in a natural soil. Images PMID:1103733

  7. Direct immunofluorescence on hair follicles--present and future perspectives.

    PubMed

    Alexandru, Adina; Zurac, Sabina; Salavastru, Carmen M; Andrei, Razvan; Tebeica, Tiberiu; Staniceanu, Florica; Tiplica, George S

    2013-06-01

    Direct immunofluorescence (DIF) is an important tool for evaluating bullous autoimmune and connective tissue disorders. We report 21 cases of pemphigus vulgaris, bullous pemphigoid and lupus erythematosus that were investigated by performing DIF on scalp hair follicles. The study was done using a simplified technique of preparing the hairs for DIF testing. The anagen hairs tested positive in pemphigus vulgaris patients while the telogen hairs were negative. In bullous pemphigoid and lupus erythematosus cases hair DIF presented negative results.Hair DIF has the potential of taking the place of skin or mucosal DIF in pemphigus patients if performed on anagen hair follicles. The technique used to perform hair DIF is important in obtaining reliable results and eliminating the possibility of generating false-negative testing. Larger studies are needed in order to validate this method.

  8. Immunofluorescence versus ELISA for the detection of antinuclear antigens.

    PubMed

    Rondeel, Jan M M

    2002-05-01

    Determining the presence and specificity of antinuclear antigens (ANA) is a challenge to a laboratory involved in the diagnosis of connective tissue disease (CTD). The immunofluorescent technique (IF), once considered the gold standard, is more and more displaced by ELISA. ELISA can be fully automated and the interpretation does not require the extensive experience needed in IF. However, literature in which both techniques are compared does not give unequivocal conclusions that ELISA indeed performs better. The clue as to which technique is best in the cascade testing of ANA, is given by its clinical value, not only by its technical and logistic performance. Selective test ordering is strongly recommended to increase the predictive value of these tests. The pros and cons of both techniques are discussed.

  9. The Use of Direct Immunofluorescence in Frontal Fibrosing Alopecia.

    PubMed

    Donati, Aline; Gupta, Aditya K; Jacob, Carolina; Cavelier-Balloy, Benedicte; Reygagne, Pascal

    2017-08-01

    Frontal fibrosing alopecia (FFA) differs from lichen planopilaris (LPP) in many clinical aspects, but histology fails to distinguish between these entities. Direct immunofluorescence (DIF) is a diagnostic technique used for autoimmune diseases, including those affecting skin and hair. To characterize DIF patterns in patients with FFA. Data was collected retrospectively from FFA cases presenting to the Centre de Santé Sabouraud Hair Clinic in Paris from November 2013 to November 2014. Of 149 patients with FFA, 44 cases underwent DIF. Thirteen cases showed positive results with DIF. Patterns characteristic of LPP and lupus erythematosus were observed, with nearly half showing nonspecific staining. DIF patterns in patients with FFA were variable. This diagnostic technique should be used with caution in cases of cicatricial alopecia, particularly FFA.

  10. Direct immunofluorescence for immunobullous and other skin diseases.

    PubMed

    Ghanadan, Alireza; Saghazadeh, Amene; Daneshpazhooh, Maryam; Rezaei, Nima

    2015-05-01

    A swift glance at ample evidence currently available about the assay clearly illustrates that the development of direct immunofluorescence (DIF), in which direct fluorescent antibodies are utilized to identify the target antigen, has been of immense importance. The immunoreactant deposits have been delineated by the DIF assay in three main locations, including throughout the epidermis, at the dermoepidermal junction (also known as the basement membrane zone) and in and/or around blood vessel walls. DIF testing can be conducted on several specimen sources, which are categorized according to feasibility of collection into invasive (e.g., skin) and non-invasive (e.g., hair). This review was intended to indicate that inspection of immunoreactant deposits via DIF is highly instrumental in diagnosing and monitoring the immunobullous and other diseases of the skin.

  11. Indirect decentralized learning control

    NASA Technical Reports Server (NTRS)

    Longman, Richard W.; Lee, Soo C.; Phan, M.

    1992-01-01

    The new field of learning control develops controllers that learn to improve their performance at executing a given task, based on experience performing this specific task. In a previous work, the authors presented a theory of indirect learning control based on use of indirect adaptive control concepts employing simultaneous identification and control. This paper develops improved indirect learning control algorithms, and studies the use of such controllers in decentralized systems. The original motivation of the learning control field was learning in robots doing repetitive tasks such as on an assembly line. This paper starts with decentralized discrete time systems, and progresses to the robot application, modeling the robot as a time varying linear system in the neighborhood of the nominal trajectory, and using the usual robot controllers that are decentralized, treating each link as if it is independent of any coupling with other links. The basic result of the paper is to show that stability of the indirect learning controllers for all subsystems when the coupling between subsystems is turned off, assures convergence to zero tracking error of the decentralized indirect learning control of the coupled system, provided that the sample time in the digital learning controller is sufficiently short.

  12. Peroxisomes in Saccharomyces cerevisiae: immunofluorescence analysis and import of catalase A into isolated peroxisomes.

    PubMed Central

    Thieringer, R; Shio, H; Han, Y S; Cohen, G; Lazarow, P B

    1991-01-01

    To isolate peroxisomes from Saccharomyces cerevisiae of a quality sufficient for in vitro import studies, we optimized the conditions for cell growth and for cell fractionation. Stability of the isolated peroxisomes was monitored by catalase latency and sedimentability of marker enzymes. It was improved by (i) using cells that were shifted to oleic acid medium after growth to stationary phase in glucose precultures, (ii) shifting the pH from 7.2 to 6.0 during cell fractionation, and (iii) carrying out equilibrium density centrifugation with Nycodenz containing 0.25 M sucrose throughout the gradient. A concentrated peroxisomal fraction was used for in vitro import of catalase A. After 2 h of incubation, 62% of the catalase was associated with, and 16% was imported into, the organelle in a protease-resistant fashion. We introduced immunofluorescence microscopy for S. cerevisiae peroxisomes, using antibodies against thiolase, which allowed us to identify even the extremely small organelles in glucose-grown cells. Peroxisomes from media containing oleic acid were larger in size, were greater in number, and had a more intense fluorescence signal. The peroxisomes were located, sometimes in clusters, in the cell periphery, often immediately adjacent to the plasma membrane. Systematic immunofluorescence observations of glucose-grown S. cerevisiae demonstrated that all such cells contained at least one and usually several very small peroxisomes despite the glucose repression. This finding fits a central prediction of our model of peroxisome biogenesis: peroxisomes form by division of preexisting peroxisomes; therefore, every cell must have at least one peroxisome if additional organelles are to be induced in that cell. Images PMID:1986244

  13. [A method for combining Fluoro-Jade B staining and immunofluorescent staining].

    PubMed

    Zuo, Xia-Lin; Jin, Ji-Zi; Liu, Dan-Dan; Sun, Wei-Wen; Xu, En

    2016-05-01

    To explore a method for combining Fluoro-Jade B (FJB) staining with immunofluorescent staining in rats with focal cortical infarction. Permanent distal middle cerebral artery occlusion (dMCAO) was induced in rats by electrocoagulation. The rat models were randomized into two groups, and frozen sections of the brain tissues from each group were stained with FJB followed by immunofluorescent staining or in the reverse order. FJB staining followed by immunofluorescence staining clearly visualized both FJB-positive and immunofluorescence-positive cells in the frozen sections, but the staining protocol in the reverse sequence failed to clearly show the immunofluorescence-positive cells. FJB staining prior to immunofluorescence staining does not affect the staining effect of protein immunofluorescent staining and better visualizes the positive cells.

  14. The Reliability of a Novel Automated System for ANA Immunofluorescence Analysis in Daily Clinical Practice.

    PubMed

    Alsuwaidi, Mohammed; Dollinger, Margit; Fleck, Martin; Ehrenstein, Boris

    2016-01-01

    Automated interpretation (AI) systems for antinuclear antibody (ANA) analysis have been introduced based on assessment of indirect immunofluorescence (IIF) patterns. The diagnostic performance of a novel automated IIF reading system was compared with visual interpretation (VI) of IIF in daily clinical practice to evaluate the reduction of workload. ANA-IIF tests of consecutive serum samples from patients with suspected connective tissue disease were carried out using HEp-2 cells according to routine clinical care. AI was performed using a visual analyser (Zenit G-Sight, Menarini, Germany). Agreement rates between ANA results by AI and VI were calculated. Of the 336 samples investigated, VI yielded 205 (61%) negative, 42 (13%) ambiguous, and 89 (26%) positive results, whereas 82 (24%) were determined to be negative, 176 (52%) ambiguous, and 78 (24%) positive by AI. AI displayed a diagnostic accuracy of 175/336 samples (52%) with a kappa coefficient of 0.34 compared to VI being the gold standard. Solely relying on AI, with VI only performed for all ambiguous samples by AI, would have missed 1 of 89 (1%) positive results by VI and misclassified 2 of 205 (1%) negative results by VI as positive. The use of AI in daily clinical practice resulted only in a moderate reduction of the VI workload (82 of 336 samples: 24%).

  15. Immunofluorescence studies of a possible prethymic T-cell differentiation in congenitally athymic (nude) mice.

    PubMed

    Loor, F; Roelants, G E

    1975-06-30

    A rabbit antimouse brain theta reagent was made specific for cells of the T lineage by absorption in vivo in nude mice. When used in double fluorescence together with an antimouse immunoglobulin reagent, four types of cells were found in spleen and lymph nodes of both normal and nude mice: Ig+thetaBr-, Ig-thetaBr+, Ig-thetaBr-, and Ig+thetaBr+. The data show that about 20% of nude mouse spleen lymphocytes are definitely of T lineage (Ig-thetaBr+). On these cells, the detection of the "thetaBr" determinant, which is identical or very close to the "theta" determinant, depends on the large amplification produced by indirect immunofluorescence, which suggests a low density of theta antigen. Similar experiments suggest the presence of cells that express some TL antigen in the spleen of nudes made congenic to a TL+ strain (BALB/c). It is proposed that the T-cell precursor that will further differentiate in the thymus already expresses a low density of theta and, in TL+ strains, TL antigen.

  16. Detection of Babesia divergens in southern Norway by using an immunofluorescence antibody test in cow sera

    PubMed Central

    2010-01-01

    Background The incidence of bovine babesiosis, caused by Babesia divergens (Apicomplexa: Piroplasmida) has decreased markedly since the 1930 s, but may re-emerge as a consequence of climate change and changes in legislation and pasturing practices. This is a potentially serious disease, with both economical and animal welfare consequences. Therefore, there is a need to survey the distribution of B. divergens. Methods We tested sera from 306 healthy pastured cows from 24 farms along the southern Norwegian coast by using an indirect immunofluorescence IgG antibody test (IFAT). Fractions of seropositive cows were compared by calculating 95% CI. Results The results of this test showed that 27% of the sera were positive for B. divergens antibodies. The fraction of antibody-positive sera that we detected showed a two-humped distribution, with a high fraction of positives being found in municipalities in the western and eastern parts of the study area, while the municipalities between these areas had few or no positive serum samples. Conclusions Neither the farmers' observations nor the Norwegian Dairy Herd Recording System give an adequate picture of the distribution of bovine babesiosis. Serological testing of cows by using IFAT is a convenient way of screening for the presence of B. divergens in an area. PMID:20925923

  17. Electron Microscopy.

    ERIC Educational Resources Information Center

    Beer, Michael

    1980-01-01

    Reviews technical aspects of structure determination in biological electron microscopy (EM). Discusses low dose EM, low temperature microscopy, electron energy loss spectra, determination of mass or molecular weight, and EM of labeled systems. Cites 34 references. (CS)

  18. Indirect microbial detection

    NASA Technical Reports Server (NTRS)

    Wilkins, J. R.

    1980-01-01

    Indirect method for detection of microbial growth utilizes flow of charged particles across barrier that physically separated growing cells from electrodes and measures resulting difference in potential between two platinum electrodes. Technique allows simplified noncontact monitoring of all growth in highly infectious cultures or in critical biochemical studies.

  19. Indirect decentralized repetitive control

    NASA Technical Reports Server (NTRS)

    Lee, Soo Cheol; Longman, Richard W.

    1993-01-01

    Learning control refers to controllers that learn to improve their performance at executing a given task, based on experience performing this specific task. In a previous work, the authors presented a theory of indirect decentralized learning control based on use of indirect adaptive control concepts employing simultaneous identification and control. This paper extends these results to apply to the indirect repetitive control problem in which a periodic (i.e., repetitive) command is given to a control system. Decentralized indirect repetitive control algorithms are presented that have guaranteed convergence to zero tracking error under very general conditions. The original motivation of the repetitive control and learning control fields was learning in robots doing repetitive tasks such as on an assembly line. This paper starts with decentralized discrete time systems, and progresses to the robot application, modeling the robot as a time varying linear system in the neighborhood of the desired trajectory. Decentralized repetitive control is natural for this application because the feedback control for link rotations is normally implemented in a decentralized manner, treating each link as if it is independent of the other links.

  20. Comparison of Genital Chlamydia trachomatis Infection Incidence Between Women With Infertility and Healthy Women in Iran Using PCR and Immunofluorescence Methods.

    PubMed

    Marashi, Seyed Mahmoud Amin; Moulana, Zahra; Imani Fooladi, Abbas Ali; Mashhadi Karim, Mohammad

    2014-04-01

    For a long time, infertility has been one of the most sequels in medical sciences with microbial agents as one group of its causes. The possible etiological role of Chlamydia trachomatis in infertility was suggested years ago, but it has not yet been proved completely. To decrease the severe involvements of C. trachomatis infections, screening by efficient diagnostic methods are necessary. In this study we attempted to determine the incidence of C. trachomatis in infertile women and compared this with healthy women. This case-control study was performed on 150 infertile women with unknown causes and without physiological deficiency for infertility. The control group consisted of 200 fertile safe and impregnated women. Presence of C. trachomatis in the two groups was examined by direct and indirect immunofluorescence tests and PCR. C. trachomatis was detected by direct immunofluorescence method in 23 (15.3%) infertile women compared and 7 (3.5%) healthy controls. Using indirect immunofluorescence tests, a positive test titer of 1:16 as well as the above results were detected in 34 (22.6%) of the infertile cases and 9 (4.5%) of the controls. C. trachomatis was detected by PCR method in 48 (32%) infertile women and 13 (8.7%) among the controls. The results of our study suggest that there is a significant association between C. trachomatis infection and female infertility.

  1. Paraffin immunofluorescence in the renal pathology laboratory: more than a salvage technique.

    PubMed

    Messias, Nidia C; Walker, Patrick D; Larsen, Christopher P

    2015-06-01

    Immunofluorescence studies on paraffin-embedded tissue after Pronase digestion (paraffin immunofluorescence) is used as a salvage technique in renal pathology, when frozen tissue for routine immunofluorescence is inadequate. We have recently found that it is also useful in rare cases in which the immune deposits are 'masked' on routine immunofluorescence, giving false-negative staining by routine immunofluorescence and positive staining by paraffin immunofluorescence. This study aims to evaluate the role of paraffin immunofluorescence in clinical practice with emphasis on its utility to avoid misdiagnosis of cases with masked immune complex deposits. Paraffin immunofluorescence was used in 304 (6.1%) of 4969 native biopsies reviewed from our files. In 207 (68.1%) cases, paraffin immunofluorescence was used as a salvage technique. It was necessary for diagnosis in 24 (11.6%) and had a significant contribution in 63 (30.4%) of these cases. Paraffin immunofluorescence was used to evaluate masked deposits in 97 (31.9%) cases. In 61 (62.9%) of these cases it was used to evaluate masked immune complex glomerular deposits, and in 36 cases (37.1%) it was used to evaluate masked paraproteins. Of the cases where immune complex deposits were sought, paraffin immunofluorescence was necessary for diagnosis in 16 (26.2%) cases and had a significant contribution in 4 (6.6%) cases. Fourteen of the 20 cases with masked deposits had C3 dominant stain by routine immunofluorescence, which could have been misdiagnosed as C3 glomerulopathy. Overall, paraffin immunofluorescence was necessary or had a significant contribution to diagnosis in >1/3 of the cases and is a valuable technique in renal pathology.

  2. IFDOTMETER: A New Software Application for Automated Immunofluorescence Analysis.

    PubMed

    Rodríguez-Arribas, Mario; Pizarro-Estrella, Elisa; Gómez-Sánchez, Rubén; Yakhine-Diop, S M S; Gragera-Hidalgo, Antonio; Cristo, Alejandro; Bravo-San Pedro, Jose M; González-Polo, Rosa A; Fuentes, José M

    2016-04-01

    Most laboratories interested in autophagy use different imaging software for managing and analyzing heterogeneous parameters in immunofluorescence experiments (e.g., LC3-puncta quantification and determination of the number and size of lysosomes). One solution would be software that works on a user's laptop or workstation that can access all image settings and provide quick and easy-to-use analysis of data. Thus, we have designed and implemented an application called IFDOTMETER, which can run on all major operating systems because it has been programmed using JAVA (Sun Microsystems). Briefly, IFDOTMETER software has been created to quantify a variety of biological hallmarks, including mitochondrial morphology and nuclear condensation. The program interface is intuitive and user-friendly, making it useful for users not familiar with computer handling. By setting previously defined parameters, the software can automatically analyze a large number of images without the supervision of the researcher. Once analysis is complete, the results are stored in a spreadsheet. Using software for high-throughput cell image analysis offers researchers the possibility of performing comprehensive and precise analysis of a high number of images in an automated manner, making this routine task easier.

  3. An immunofluorescent method for characterization of Barrett's esophagus cells.

    PubMed

    Inge, Landon J; Fowler, Aaron J; Bremner, Ross M

    2014-07-20

    Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One of the primary risk factors of EAC is Barrett's esophagus (BE), a metaplastic change of the normal squamous esophagus in response to chronic heartburn. Despite the well-established connection between EAC and BE, interrogation of the molecular events, particularly altered signaling pathways involving progression of BE to EAC, are poorly understood. Much of this is due to the lack of suitable in vitro models available to study these diseases. Recently, immortalized BE cell lines have become commercially available allowing for in vitro studies of BE. Here, we present a method for immunofluorescent staining of immortalized BE cell lines, allowing in vitro characterization of cell signaling and structure after exposure to therapeutic compounds. Application of these techniques will help develop insight into the mechanisms involved in BE to EAC progression and provide potential avenues for treatment and prevention of EAC.

  4. Indirect resin composites

    PubMed Central

    Nandini, Suresh

    2010-01-01

    Aesthetic dentistry continues to evolve through innovations in bonding agents, restorative materials, and conservative preparation techniques. The use of direct composite restoration in posterior teeth is limited to relatively small cavities due to polymerization stresses. Indirect composites offer an esthetic alternative to ceramics for posterior teeth. This review article focuses on the material aspect of the newer generation of composites. This review was based on a PubMed database search which we limited to peer-reviewed articles in English that were published between 1990 and 2010 in dental journals. The key words used were ‘indirect resin composites,’ composite inlays,’ and ‘fiber-reinforced composites.’ PMID:21217945

  5. Immunofluorescence assays (IFA) and enzyme-linked immunosorbent assays (ELISA) in autoimmune disease diagnostics--technique, benefits, limitations and applications.

    PubMed

    Bayer, P M; Fabian, B; Hübl, W

    2001-01-01

    Autoimmune diseases are relatively frequent disease complexes, affecting approximately five to seven percent of the population. After cardio-vascular and malignant diseases they come third in mortality. As the clinical diagnosis of rheumatic autoimmune diseases is difficult, laboratory tests are helpful in differential diagnosis and for verification of the clinical diagnosis. The most commonly used assay is the determination of ANAs (anti nuclear antibodies) by indirect immunofluorescence (IFA). However, this method lacks reliable standardisation and is very dependable on the qualification of the observer. Enzyme Immunoassays (EIA) and Immunoblotting techniques, on the contrary, attain good standardisation and comparability. However, the latter methods are limited to the presentation of defined autoantibodies only. There is a need to select a suitable strategy for the use of laboratory parameters in order to support the clinical diagnosis more efficiently. A possible strategy is to replace IFA as a first line screening-step by second-generation ANA-EIA kits.

  6. Confocal laser scanning microscopy and 3-D reconstructions of neuronal structures in human brain cortex.

    PubMed

    Belichenko, P V; Dahlström, A

    1995-09-01

    Human brain material was studied with Lucifer yellow (LY) microinjections, indirect Texas red immunofluorescence, and confocal laser scanning microscopy (CLSM). The scanned images were transferred to a Silicon Graphics (SG) IRIS computer equipped with software for reconstructing the 3-D architecture of cells. By employing dual channel CLSM (Bio-Rad MRC 600), LY-injected cells and Texas red immunofluorescence could be studied simultaneously. Autopsy material with 2- to 48-h postmortem delays (6 control and 2 Rett's syndrome cases) as well as biopsy material (14 cases with therapy-resistant partial epilepsy--TRPE--undergoing neurosurgery) were used. In each specimen, 100-200 pyramidal and nonpyramidal neurons were visualized by LY microinjection. Single neurons were imaged and 2-D reconstructions of each neuron were made using z-projections of serial optical images; 3-D reconstructions and rotations were computed using the SG workstation, with VoxelView software from Vital Images (UK), and stored in a "neuronal library" on laser or magnetic optical disks. In Ret's syndrome cases and in patients with TRPE various abnormalities in the dendritic geometry of pyramidal and nonpyramidal cells have been found. The combination of LY injections with immunofluorescence allows the investigation of transmitter-related substances around the LY-injected cells. Using antibodies to synaptic vesicle proteins, presynaptic elements docking onto individual spines have been demonstrated. This approach may contribute to the understanding of different neurological and psychiatric disorders and may be useful in the Mapping of the Human Brain project. It may also be integrated with functional imaging by PET scan and with the human genome project.

  7. Analytical Microscopy

    SciTech Connect

    Not Available

    2006-06-01

    In the Analytical Microscopy group, within the National Center for Photovoltaic's Measurements and Characterization Division, we combine two complementary areas of analytical microscopy--electron microscopy and proximal-probe techniques--and use a variety of state-of-the-art imaging and analytical tools. We also design and build custom instrumentation and develop novel techniques that provide unique capabilities for studying materials and devices. In our work, we collaborate with you to solve materials- and device-related R&D problems. This sheet summarizes the uses and features of four major tools: transmission electron microscopy, scanning electron microscopy, the dual-beam focused-ion-beam workstation, and scanning probe microscopy.

  8. Immunofluorescence identifies distinct subsets of endothelial cells in the human liver

    PubMed Central

    Strauss, Otto; Phillips, Anthony; Ruggiero, Katya; Bartlett, Adam; Dunbar, P. Rod

    2017-01-01

    As well as systemic vascular endothelial cells, the liver has specialised sinusoidal endothelial cells (LSEC). LSEC dysfunction has been documented in many diseased states yet their phenotype in normal human liver has not been comprehensively assessed. Our aim was to improve characterisation of subsets of endothelial cells and associated pericytes in the human liver. Immunofluorescence microscopy was performed on normal human liver tissue samples to assess endothelial and structural proteins in a minimum of three donors. LSEC are distributed in an acinar pattern and universally express CD36, but two distinctive subsets of LSEC can be identified in different acinar zones. Type 1 LSEC are CD36hiCD32−CD14−LYVE-1− and are located in acinar zone 1 of the lobule, while Type 2 LSEC are LYVE-1+CD32hiCD14+CD54+CD36mid-lo and are located in acinar zones 2 and 3 of the lobule. Portal tracts and central veins can be identified using markers for systemic vascular endothelia and pericytes, none of which are expressed by LSEC. In areas of low hydrostatic pressure LSEC are lined by stellate cells that express the pericyte marker CD146. Our findings identify distinctive populations of LSEC and distinguish these cells from adjacent stellate cells, systemic vasculature and pericytes in different zones of the liver acinus. PMID:28287163

  9. Pathological Gleason prediction through gland ring morphometry in immunofluorescent prostate cancer images

    NASA Astrophysics Data System (ADS)

    Scott, Richard; Khan, Faisal M.; Zeineh, Jack; Donovan, Michael; Fernandez, Gerardo

    2016-03-01

    The Gleason score is the most common architectural and morphological assessment of prostate cancer severity and prognosis. There have been numerous quantitative techniques developed to approximate and duplicate the Gleason scoring system. Most of these approaches have been developed in standard H and E brightfield microscopy. Immunofluorescence (IF) image analysis of tissue pathology has recently been proven to be extremely valuable and robust in developing prognostic assessments of disease, particularly in prostate cancer. There have been significant advances in the literature in quantitative biomarker expression as well as characterization of glandular architectures in discrete gland rings. In this work we leverage a new method of segmenting gland rings in IF images for predicting the pathological Gleason; both the clinical and the image specific grade, which may not necessarily be the same. We combine these measures with nuclear specific characteristics as assessed by the MST algorithm. Our individual features correlate well univariately with the Gleason grades, and in a multivariate setting have an accuracy of 85% in predicting the Gleason grade. Additionally, these features correlate strongly with clinical progression outcomes (CI of 0.89), significantly outperforming the clinical Gleason grades (CI of 0.78). This work presents the first assessment of morphological gland unit features from IF images for predicting the Gleason grade.

  10. Easy performance of 6-color confocal immunofluorescence with 4-laser line microscopes.

    PubMed

    Eissing, Nathalie; Heger, Lukas; Baranska, Anna; Cesnjevar, Robert; Büttner-Herold, Maike; Söder, Stephan; Hartmann, Arndt; Heidkamp, Gordon F; Dudziak, Diana

    2014-09-01

    Confocal laser scanning microscopy is an advanced technique for imaging tissue samples in vitro and in vivo at high optical resolution. The development of new fluorochrome variants do not only make it possible to perform multicolor flow cytometry of single cells, but in combination with high resolution laser scanning systems also to investigate the distribution of cells in lymphoid tissues by confocal immunofluorescence analyses, thus allowing the distinction of various cell populations directly in the tissue. Here, we provide a protocol for the visualization of at least six differently fluorochrome-labeled antibodies at the same time using a conventional confocal laser scanning microscope with four laser lines (405 nm, 488 nm, 555 nm, and 639 nm laser wavelength) in both murine and human tissue samples. We further demonstrate that compensation correction algorithms are not necessary to reduce spillover of fluorochromes into other channels when the used fluorochromes are combined according to their specific emission bands and the varying Stokes shift for co-excited fluorochromes with the same laser line.

  11. AMP deaminase histochemical activity and immunofluorescent isozyme localization in rat skeletal muscle

    NASA Technical Reports Server (NTRS)

    Thompson, J. L.; Sabina, R. L.; Ogasawara, N.; Riley, D. A.

    1992-01-01

    The cellular distribution of AMP deaminase (AMPda) isozymes was documented for rat soleus and plantaris muscles, utilizing immunofluorescence microscopy and immunoprecipitation methods. AMPda is a ubiquitous enzyme existing as three distinct isozymes, A, B and C, which were initially purified from skeletal muscle, liver (and kidney), and heart, respectively. AMPda-A is primarily concentrated subsarcolemmally and intermyofibrillarly within muscle cells, while isozymes B and C are concentrated within non-myofiber elements of muscle tissue. AMPda-B is principally associated with connective tissues surrounding neural elements and the muscle spindle capsule, and AMPda-C is predominantly associated with circulatory elements, such as arterial and venous walls, capillary endothelium, and red blood cells. These specific localizations, combined with documented differences in kinetic properties, suggest multiple functional roles for the AMPda isozymes or temporal segregation of similar AMPda functions. Linkage of the AMPda substrate with adenosine production pathways at the AMP level and the localization of isozyme-C in vascular tissue suggest a regulatory role in the microcirculation.

  12. Semi-Automated, Occupationally Safe Immunofluorescence Microtip Sensor for Rapid Detection of Mycobacterium Cells in Sputum

    PubMed Central

    Soelberg, Scott D.; Weigel, Kris M.; Hiraiwa, Morgan; Cairns, Andrew; Lee, Hyun-Boo; Furlong, Clement E.; Oh, Kieseok; Lee, Kyong-Hoon; Gao, Dayong; Chung, Jae-Hyun; Cangelosi, Gerard A.

    2014-01-01

    An occupationally safe (biosafe) sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60°C for 5 to 10 min. Samples spiked with M. tuberculosis complex cells showed approximately 106-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between Mycobacterium species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for M. tuberculosis, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure. PMID:24465845

  13. AMP deaminase histochemical activity and immunofluorescent isozyme localization in rat skeletal muscle

    NASA Technical Reports Server (NTRS)

    Thompson, J. L.; Sabina, R. L.; Ogasawara, N.; Riley, D. A.

    1992-01-01

    The cellular distribution of AMP deaminase (AMPda) isozymes was documented for rat soleus and plantaris muscles, utilizing immunofluorescence microscopy and immunoprecipitation methods. AMPda is a ubiquitous enzyme existing as three distinct isozymes, A, B and C, which were initially purified from skeletal muscle, liver (and kidney), and heart, respectively. AMPda-A is primarily concentrated subsarcolemmally and intermyofibrillarly within muscle cells, while isozymes B and C are concentrated within non-myofiber elements of muscle tissue. AMPda-B is principally associated with connective tissues surrounding neural elements and the muscle spindle capsule, and AMPda-C is predominantly associated with circulatory elements, such as arterial and venous walls, capillary endothelium, and red blood cells. These specific localizations, combined with documented differences in kinetic properties, suggest multiple functional roles for the AMPda isozymes or temporal segregation of similar AMPda functions. Linkage of the AMPda substrate with adenosine production pathways at the AMP level and the localization of isozyme-C in vascular tissue suggest a regulatory role in the microcirculation.

  14. Immunofluorescence studies on autoantibodies to steroid-producing cells, and to germline cells in endocrine disease and infertility

    PubMed Central

    Sotsiou, Flora; Bottazzo, G. F.; Doniach, Deborah

    1980-01-01

    This study was aimed at comparing the clinical significance of antibodies to steroid-producing cells with reactions to gonadal germline cells in patients with autoimmune polyendocrine diseases and isolated infertility or amenorrhoea respectively. Indirect immunofluorescence was used on human adrenal, ovary and testis. The gonad substrates were compared with rat, rabbit and monkey glands. 152 adrenal-positive sera were selected from 1030 that had been tested on adrenal cortex. Antibodies to steroid-producing cells in the gonads were found in fifty of these 152 selected cases and were studied in detail. When using human gonads as substrates, steroid-producing-cell antibodies were never detected in the absence of adrenal cortical immunofluorescence, though false-positive reactions were sometimes obtained on rat or rabbit gonads. Adrenal antibodies as well as those to steroid-producing cells were most frequent in Addisonian cases having one or more additional endocrine disease. The frequency of both types of antibody was lower in patients with Addison's disease and no other disorder but showing evidence of `polyendocrine serology'. Both antibodies were found least frequently when adrenalitis was unassociated with clinical or subclinical autoimmunity in other organs. We were able to confirm the immunofluorescence patterns described by other authors on adrenal gland and gonads, as well as the independent rise or fall in titre of these two types of antibodies in individual cases with time. Prolonged follow-up of forty-two Addisonian patients showed that adrenal antibodies disappeared in seven instances (17%). Ovum and sperm antibodies were found in about 25% of infertility cases and a smaller proportion of polyendocrine patients. Germline cell antibodies were rarely associated with other organ-specific reactions. In two cases, amenorrhoea was due to partial pituitary deficiency and the sera of the patients contained antibodies to pituitary prolactin-cells. Testicular

  15. 3D multiplexed immunoplasmonics microscopy

    NASA Astrophysics Data System (ADS)

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-01

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed

  16. Neuromyelitis optica immunoglobulin G in Chinese patients detected by immunofluorescence assay on a monkey brain substrate.

    PubMed

    Long, Youming; Hu, Xueqiang; Peng, Fuhua; Lu, Zhengqi; Wang, Yuge; Yang, Yu; Qiu, Wei

    2012-01-01

    Serum neuromyelitis optica immunoglobulin G (NMO-IgG) is used as a biomarker to differentiate between neuromyelitis optica (NMO) and multiple sclerosis (MS). However, the original assay is expensive and complex and shows low sensitivity. Here, we investigated the potential of NMO-IgG detection using an indirect immunofluorescence (IIF) assay on monkey brains. NMO-IgG seroprevalence was determined in 168 samples by an IIF assay on a monkey brain substrate. The data were compared with those from a standard mouse brain IIF assay using McNemar and kappa tests. Thirty-one of 50 (62%) NMO patients, 7 of 18 (38.9%) longitudinally extensive transverse myelitis patients, 6 of 57 (10.5%) MS patients, and 5 of 10 (50%) optic neuritis patients were seropositive for NMO-IgG. None of the acute partial transverse myelitis patients (n = 3) or healthy controls (n = 20) was positive. Thus, the sensitivity of the test was 62% for the patients with clinically definite NMO. The specificity was 89.5%, considering the 57 MS patients as the control group. The modified IIF assay on monkey brains and the standard IIF assay based on mouse brains were not significantly different (McNemar test; p = 1.000). The two assays were concordant in 39 seropositive samples and 100 seronegative samples (kappa test; kappa = 0.592, p < 0.0001). Although the modified IIF monkey brain assay was no better than the standard mouse brain IIF assay, we affirmed that NMO-IgG is a sensitive and specific biomarker to differentiate between NMO and MS. Copyright © 2011 S. Karger AG, Basel.

  17. Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

    PubMed Central

    Pinne, Marija; Haake, David

    2011-01-01

    Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular localization can provide insights into the functional role of bacterial proteins. Surface localization of bacterial proteins is a key step towards identification of virulence factors involved in mechanisms of pathogenicity. Methods for fractionating leptospiral membranes 2-5 may be selective for a certain class of outer-membrane proteins (OMPs), such as lipoproteins vs. transmembrane OMPs, and therefore lead to misclassification. This likely is due to structural differences and how they are associated to the outer membrane. Lipoproteins are associated with membranes via a hydrophobic interaction between the N-terminal lipid moiety (three fatty acids) and the lipid bilayer phospholipids 6, 7. In contrast, transmembrane OMPs are typically integrated into the lipid bilayer by amphipathic β-sheets arranged in a barrel-like structure 8, 9. In addition, presence of a protein in the outer-membrane does not necessarily guarantee that the protein or its domains are exposed on the surface. Spirochetal outer membranes are known to be fragile and therefore necessitate methods involving gentle manipulation of cells and inclusion of sub-surface protein controls to assess the integrity of the outer membrane. Here, we present an immunofluorescence assay (IFA) method to directly assess surface exposure of proteins on intact leptospires. This method is based on recognition of leptospiral surface proteins by antigen-specific antibodies. Herein, antibodies specific for OmpL5410 are detetcted aftero binding to native, surface exposed epitopes. Comparison of antibody reactivity to intact versus permeabilized cells enables evaluation of cellular distribution and whether or not a protein is selectively present on leptospiral surface. The integrity of outer membrane should be assessed using antibody to one or more subsurface proteins

  18. Bioechnology of indirect liquefaction

    SciTech Connect

    Datta, R.; Jain, M.K.; Worden, R.M.; Grethlein, A.J.; Soni, B.; Zeikus, J.G.; Grethlein, H.

    1990-05-07

    The project on biotechnology of indirect liquefaction was focused on conversion of coal derived synthesis gas to liquid fuels using a two-stage, acidogenic and solventogenic, anaerobic bioconversion process. The acidogenic fermentation used a novel and versatile organism, Butyribacterium methylotrophicum, which was fully capable of using CO as the sole carbon and energy source for organic acid production. In extended batch CO fermentations the organism was induced to produce butyrate at the expense of acetate at low pH values. Long-term, steady-state operation was achieved during continuous CO fermentations with this organism, and at low pH values (a pH of 6.0 or less) minor amounts of butanol and ethanol were produced. During continuous, steady-state fermentations of CO with cell recycle, concentrations of mixed acids and alcohols were achieved (approximately 12 g/l and 2 g/l, respectively) which are high enough for efficient conversion in stage two of the indirect liquefaction process. The metabolic pathway to produce 4-carbon alcohols from CO was a novel discovery and is believed to be unique to our CO strain of B. methylotrophicum. In the solventogenic phase, the parent strain ATCC 4259 of Clostridium acetobutylicum was mutagenized using nitrosoguanidine and ethyl methane sulfonate. The E-604 mutant strain of Clostridium acetobutylicum showed improved characteristics as compared to parent strain ATCC 4259 in batch fermentation of carbohydrates.

  19. Array tomography: high-resolution three-dimensional immunofluorescence.

    PubMed

    Micheva, Kristina D; O'Rourke, Nancy; Busse, Brad; Smith, Stephen J

    2010-11-01

    Array tomography, which is described in this article, is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Although the fabrication procedures can be relatively difficult, the high resolution, depth invariance, and molecular discrimination offered by array tomography justify the effort involved. Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture.

  20. Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea.

    PubMed

    Montgomery, Scott C; Cox, Brandon C

    2016-01-01

    The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

  1. Specific immunofluorescent staining of pathogenic treponemes with a monoclonal antibody.

    PubMed Central

    Ito, F; Hunter, E F; George, R W; Pope, V; Larsen, S A

    1992-01-01

    Two hybrid cell lines which produced mouse monoclonal antibody to the DAL-1 street strain of Treponema pallidum subsp. pallidum were established. These monoclonal antibodies strongly reacted with T. pallidum subsp. pallidum (Nichols strain, DAL-1, and two other street strains, strains MN-1 and MN-3) and T. pallidum subsp. pertenue by indirect microimmunofluorescent antibody and enzyme-linked immunosorbent assay techniques, but they did not react with normal rabbit testicular tissue. These monoclonal antibodies did not react with nonpathogenic treponemes, such as T. phagedenis Reiter, T. denticola MRB, T. refringens Noguchi, or other spirochetes, such as Borrelia burgdorferi and Leptospira interrogans serovar pomona in microimmunofluorescent antibody smear slides or in Western blots (immunoblots). While unlabeled antibodies are useful for investigating the antigenic structures of T. pallidum, we labeled these monoclonal antibodies with fluorescein isothiocyanate and used them for diagnosing syphilis by direct staining of lesion exudate or T. pallidum subsp. pallidum in formalin-fixed tissues from patients suspected of having syphilis. Both monoclonal antibodies were directed against antigens of T. pallidum subsp. pallidum with a molecular weight of 37,000 as determined by the Western blotting technique. Images PMID:1374079

  2. Indirect fluorescent-antibody and quantitative agar-gel immunodiffusion tests for the serological diagnosis of paracoccidioidomycosis.

    PubMed

    Restrepo, A; Moncada, L H

    1972-07-01

    The value of various serological tests in the diagnosis of paracoccidioidomycosis was studied. Quantitative agar-gel immunodiffusion and indirect immunofluorescent tests were performed, and the results were compared with those of complement fixation and qualitative agar-gel procedures. The quantitative immunodiffusion procedure was found to serve as the simplest and safest quantitative test that could be performed for evaluation purposes, whereas the indirect fluorescent-antibody test gave nonspecific reactions and, as such, proved unsuitable.

  3. Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues.

    PubMed

    Kajimura, Junko; Ito, Reiko; Manley, Nancy R; Hale, Laura P

    2016-02-01

    Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH3/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies.

  4. Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues

    PubMed Central

    Kajimura, Junko; Ito, Reiko; Manley, Nancy R.; Hale, Laura P.

    2015-01-01

    Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH3/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies. PMID:26392518

  5. Correlative Microscopy

    USDA-ARS?s Scientific Manuscript database

    Microscopy and Imaging offers many opportunities to collaborate and cooperate with scientists in many different fields nationally and internationally. Images have proven to be very important components in basic research, product development and understanding structure/function relationships in addit...

  6. Correlative microscopy.

    PubMed

    Loussert Fonta, Céline; Humbel, Bruno M

    2015-09-01

    In recent years correlative microscopy, combining the power and advantages of different imaging system, e.g., light, electrons, X-ray, NMR, etc., has become an important tool for biomedical research. Among all the possible combinations of techniques, light and electron microscopy, have made an especially big step forward and are being implemented in more and more research labs. Electron microscopy profits from the high spatial resolution, the direct recognition of the cellular ultrastructure and identification of the organelles. It, however, has two severe limitations: the restricted field of view and the fact that no live imaging can be done. On the other hand light microscopy has the advantage of live imaging, following a fluorescently tagged molecule in real time and at lower magnifications the large field of view facilitates the identification and location of sparse individual cells in a large context, e.g., tissue. The combination of these two imaging techniques appears to be a valuable approach to dissect biological events at a submicrometer level. Light microscopy can be used to follow a labelled protein of interest, or a visible organelle such as mitochondria, in time, then the sample is fixed and the exactly same region is investigated by electron microscopy. The time resolution is dependent on the speed of penetration and fixation when chemical fixatives are used and on the reaction time of the operator for cryo-fixation. Light microscopy can also be used to identify cells of interest, e.g., a special cell type in tissue or cells that have been modified by either transfections or RNAi, in a large population of non-modified cells. A further application is to find fluorescence labels in cells on a large section to reduce searching time in the electron microscope. Multiple fluorescence labelling of a series of sections can be correlated with the ultrastructure of the individual sections to get 3D information of the distribution of the marked proteins: array

  7. Role of Immunofluorescence in Adult Onset Nephrotic Syndrome-A Study in a Tertiary Care Centre of Western India.

    PubMed

    Rane, Sharada; Mutyal, Prerana; Dcunha, Nicholas; Parkhi, Mayur; Jadhav, Meenal

    2017-05-01

    Study of renal Immunofluorescence (IF) is an ancillary but essential technique in evaluation of renal biopsies in glomerulopathies and also it enlightens on the pathogenesis of nephrotic syndrome. To determine the role of IF in evaluating definite subtyping and diagnosis of adult onset nephrotic syndrome and attempting clinicopathological correlation. A total of 52 patients of adult onset nephrotic syndrome were evaluated clinically and with pertinent investigations; and subjected to USG guided percutaneous renal biopsy which was processed and stained for light microscopy and for immunofluorescence by direct method (DIF) using antibodies against IgG, IgM, IgA and C3. The predominant age group affected was 18-47 years (78.85%). Male:female ratio was 1:0.63. The most frequent glomerular lesion encountered was Focal Segmental Glomerulosclerosis (FSGS, 36.54%) followed by lupus nephritis (26.92%) and IgA nephropathy (9.62%). The most common glomerular lesion in males was FSGS and that in females was lupus nephritis. DIF was mainly coarsely granular whenever present. One case of lupus nephritis and diabetic nephropathy, showed non specific IF. It was negative in all cases of FSGS, Minimal Change Disease (MCD) and renal amyloidosis. The IF helped in differentiating eight cases that were normal on light microscopy as IgA nephropathy (n=5) and authentic MCD (n=3). It helped in endorsing 19 cases of FSGS to be a progression of MCD. It was also of help in sub-typing all cases of Membranoproliferative Glomerulonephritis (MPGN) (n=3) as MPGN-Type I. IF was of great help in diagnosing exact type of glomerulopathy in adult onset nephrotic syndrome and provided insight in its pathogenesis.

  8. Epicardium: interstitial Cajal-like cells (ICLC) highlighted by immunofluorescence

    PubMed Central

    Suciu, L; Popescu, L M; Regalia, T; Ardelean, A; Manole, C G

    2009-01-01

    Abstract During the last few years, there is an increasing interest in the role of the epicardium in cardiac development, myocardial remodelling or repair and regeneration. Several types of cells were described in the subepicardial loose connective tissue, beneath the epicardial mesothe-lium. We showed previously (repeatedly) the existence of interstitial Cajal-like cells (ICLCs) in human and mammalian myocardium, either in atria or in ventricles. Here, we describe ICLCs in adult mice epicardium and primary culture as well as in situ using frozen sections. The identification of ICLCs was based on phase contrast microscopy and immunophenotyping. We found cells with characteristic morphologic aspects: spindle-shaped, triangular or polygonal cell body and typical very long (tens to hundreds micrometres) and very thin cyto-plasmic processes, with a distinctive ‘beads-on-a-string’ appearance. The dilations contain mitochondria, as demonstrated by MitoTracker Green FM labelling of living cells. Epicardial ICLCs were found positive for c-kit/CD117 and/or CD34. However, we also observed ICLCs positive for c-kit and vimentin. In conclusion, ICLCs represent a distinct cell type in the subendocardium, presumably comprising at least two subpopulations: (i) c-kit/CD34-positive and (ii) only c-kit-positive. ICLCs might be essential as progenitor (or promoter) cells for developing cardiomyocyte lineages in normal and/or injured heart. PMID:19382907

  9. Expansion Microscopy

    PubMed Central

    Chen, Fei; Tillberg, Paul W.; Boyden, Edward S.

    2014-01-01

    In optical microscopy, fine structural details are resolved by using refraction to magnify images of a specimen. Here we report the discovery that, by synthesizing a swellable polymer network within a specimen, it can be physically expanded, resulting in physical magnification. By covalently anchoring specific labels located within the specimen directly to the polymer network, labels spaced closer than the optical diffraction limit can be isotropically separated and optically resolved, a process we call expansion microscopy (ExM). Thus, this process can be used to perform scalable super-resolution microscopy with diffraction-limited microscopes. We demonstrate ExM with effective ~70 nm lateral resolution in both cultured cells and brain tissue, performing three-color super-resolution imaging of ~107 μm3 of the mouse hippocampus with a conventional confocal microscope. PMID:25592419

  10. Variations of attractors and wavelet spectra of the immunofluorescence distributions for women in the pregnant period

    NASA Astrophysics Data System (ADS)

    Galich, Nikolay E.

    2008-07-01

    Communication contains the description of the immunology data treatment. New nonlinear methods of immunofluorescence statistical analysis of peripheral blood neutrophils have been developed. We used technology of respiratory burst reaction of DNA fluorescence in the neutrophils cells nuclei due to oxidative activity. The histograms of photon count statistics the radiant neutrophils populations' in flow cytometry experiments are considered. Distributions of the fluorescence flashes frequency as functions of the fluorescence intensity are analyzed. Statistic peculiarities of histograms set for women in the pregnant period allow dividing all histograms on the three classes. The classification is based on three different types of smoothing and long-range scale averaged immunofluorescence distributions, their bifurcation and wavelet spectra. Heterogeneity peculiarities of long-range scale immunofluorescence distributions and peculiarities of wavelet spectra allow dividing all histograms on three groups. First histograms group belongs to healthy donors. Two other groups belong to donors with autoimmune and inflammatory diseases. Some of the illnesses are not diagnosed by standards biochemical methods. Medical standards and statistical data of the immunofluorescence histograms for identifications of health and illnesses are interconnected. Peculiarities of immunofluorescence for women in pregnant period are classified. Health or illness criteria are connected with statistics features of immunofluorescence histograms. Neutrophils populations' fluorescence presents the sensitive clear indicator of health status.

  11. Positron microscopy

    SciTech Connect

    Hulett, L.D. Jr.; Xu, J.

    1995-02-01

    The negative work function property that some materials have for positrons make possible the development of positron reemission microscopy (PRM). Because of the low energies with which the positrons are emitted, some unique applications, such as the imaging of defects, can be made. The history of the concept of PRM, and its present state of development will be reviewed. The potential of positron microprobe techniques will be discussed also.

  12. What to do with high autofluorescence background in pancreatic tissues - an efficient Sudan black B quenching method for specific immunofluorescence labelling.

    PubMed

    Erben, Till; Ossig, Rainer; Naim, Hassan Y; Schnekenburger, Jürgen

    2016-09-01

    High levels of autofluorescence in tissue samples can entirely mask specific labellings with fluorophores and thus impair immunofluorescence histochemistry. In pancreatic tissue samples we observed autofluorescence as a common problem often mediated by the fixation and processing procedure. Using epifluorescence microscopy, we analysed the intensity and spatial distribution of autofluorescence in formalin-fixed, paraffin-embedded human pancreatic tissues and developed an efficient quenching method to reduce the unwanted light emission. The optimized quenching protocol using Sudan black B reduced the unequally distributed tissue autofluorescence to a low and intensity-equalized background level. Quantitative image analysis demonstrated autofluorescence suppression by 65-95%, depending on the selected fluorescence filter setups. The procedure did not affect specific immunofluorescence labelling or tissue integrity. As a clear result of Sudan black B treatment, a tremendous improvement of the signal-to-noise ratio was achieved, allowing a reliable detection and quantification of specific fluorescent labels. Other tissue treatment methods, such as cupric sulphate, toluidine blue and ultraviolet irradiation, or combinations of these with Sudan black B, were not as efficient. The easy-to-perform Sudan black B technique improves dramatically qualitative and quantitative fluorescence analysis of critical pancreatic tissue sections and rescues even overfixed tissues for immunofluorescence application. © 2016 John Wiley & Sons Ltd.

  13. Serotonin receptor of type 6 (5-HT6) in human prefrontal cortex and hippocampus post-mortem: an immunohistochemical and immunofluorescence study.

    PubMed

    Marazziti, Donatella; Baroni, Stefano; Pirone, Andrea; Giannaccini, Gino; Betti, Laura; Testa, Giovanna; Schmid, Lara; Palego, Lionella; Borsini, Franco; Bordi, Fabio; Piano, Ilaria; Gargini, Claudia; Castagna, Maura; Catena-Dell'osso, Mario; Lucacchini, Antonio

    2013-01-01

    Given the paucity of data on the distribution of serotonin (5-HT) receptors of type 6 (5-HT(6)) in the human brain, the aim of this study was to investigate their distribution in postmortem human prefrontal cortex, striatum and hippocampus by either immunohistochemical or immunofluorescence techniques. The brain samples were obtained from 6 subjects who had died for causes not involving primarily or secondarily the CNS. The 5-HT(6) receptor distribution was explored by the [(125)I]SB-258585 binding to brain membranes followed by immunohistochemical and immunofluorescence evaluations. A specific [(125)I]SB-258585 binding was detected in all the regions under investigation, whilst the content in the hippocampus and cortex being about 10-30 times lower than in the striatum. Immunohistochemistry and double-label immunofluorescence microscopy experiments, carried out in the prefrontal cortex and hippocampus only, since data in the striatum were already published, showed the presence of 5-HT(6) receptors in both pyramidal and glial cells of prefrontal cortex, while positive cells were mainly pyramidal neurons in the hippocampus. The heterogeneous distribution of 5-HT(6) receptors provides a preliminary explanation of how they might regulate different functions in different brain areas, such as, perhaps, brain trophism in the cortex and neuronal firing in the hippocampus. This study, taking into account all the limitations due to the postmortem model used, represents the starting point to explore the 5-HT(6) receptor functionality and its sub-cellular distribution. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Immunofluorescence on paraffin embedded renal biopsies: Experience of a tertiary care center with review of literature

    PubMed Central

    Singh, Geetika; Singh, Lavleen; Ghosh, Ranajoy; Nath, Devajit; Dinda, Amit Kumar

    2016-01-01

    AIM To describe the technique of immunofluorescence on paraffin embedded tissue sections and discuss the potential pitfalls with an in depth review of literature. METHODS Immunofluorescence is integral to diagnostic renal pathology. Immunofluorescence on paraffin embedded renal biopsies (IF-P) after enzyme treatment has been described in literature, however has not found widespread use in renal pathology laboratories. In our laboratory proteinase K digestion of paraffin embedded renal biopsy material was standardized and applied prospectively in cases where immunofluorescence on fresh frozen tissue was non contributory or not possible. Diagnostic utility was assessed and in a cohort of cases comparison of intensity of staining with routine immunofluorescence was performed. RESULTS Over the 5-year study period, of the 3141 renal biopsies received IF-P was performed on 246 cases (7.7%) and was interpretable with optimal digestion in 214 cases (6.8%). It was of diagnostic utility in the majority of cases, which predominantly included glomerular disease. Non-diagnostic IF-P was found in membranous nephropathy (2 of 11 cases), membranoproliferative glomerulonephritis (2 of 32 cases), lupus nephritis (1 of 25 cases), post infectious glomerulonephritis (1 of 11 cases) and chronic glomerulonephritis (3 of 8 cases). Comparing cases with both routine IF and IF-P, 35 of 37 showed either equal intensity or a minor difference in intensity of staining (1+) for the diagnostic immunoglobulin/complement. Technically assessment of immunofluorescence on the paraffin embedded tissue was found to be easier with clearly observed morphology, however a false positive staining pattern was observed in under-digested tissue. CONCLUSION As a “salvage” technique, immunofluorescence on paraffin embedded renal biopsies is of great diagnostic utility, however not without pitfalls. PMID:27648410

  15. Prognostic Value of Glomerular Collagen IV Immunofluorescence Studies in Male Patients with X-Linked Alport Syndrome

    PubMed Central

    Gangemi, Concetta; Giannakakis, Kostas; Crisafi, Antonella; Faraggiana, Tullio; Fallerini, Chiara; Renieri, Alessandra; Muda, Andrea Onetti; Emma, Francesco

    2013-01-01

    Summary Background and objectives X-linked Alport syndrome (X-AS) is caused by mutations of the COL4A5 gene, which encodes for the collagen IV α5 chain (α5[COLIV]), resulting in structural and functional abnormalities of the glomerular basement membrane (GBM) and leading to CKD. The aim of the present study was to evaluate the prognostic value of residual collagen IV chain expression in the GBM of patients with X-AS. Design, setting, participants, & measurements The medical records of 22 patients with X-AS from 21 unrelated families collected between 1987 and 2009 were reviewed (median age at last follow-up, 19.9 years; range, 5.4–35.1 years); GBM expression of α1, α3, and α5(COLIV) chains was assessed by immunofluorescence microscopy. Results GBM distribution of the α5(COLIV) chain was diffuse in 1 and segmental or absent in 21 of the 22 patients; the expression of the α3(COLIV) chain was diffuse in 5 of 22 patients and segmental or absent in 17 of 22 patients. Patients with diffuse staining for the α3(COLIV) chain presented with proteinuria significantly later (median age, 16.9 versus 6.1 years; P=0.02) and reached an estimated GFR < 90 ml/min per 1.73 m2 at an older age (median age, 27.0 versus 14.9 years; P=0.01) compared with patients with segmental or absent staining. Two thirds of patients with abnormal α3(COLIV) expression by immunofluorescence studies had null or truncating COL4A5 mutations, as opposed to none of the 4 tested patients with diffuse α3(COLIV) chain glomerular distribution. Conclusions These results indicate that maintained expression of the α3(COLIV) chain is an early positive prognostic marker in patients with X-linked Alport symdrome. PMID:23371956

  16. Detection by an immunofluorescence test of Encephalitozoon intestinalis spores in routinely formalin-fixed stool samples stored at room temperature.

    PubMed

    Moura, H; Sodre, F C; Bornay-Llinares, F J; Leitch, G J; Navin, T; Wahlquist, S; Bryan, R; Meseguer, I; Visvesvara, G S

    1999-07-01

    Of the several microsporidia that infect humans, Enterocytozoon bieneusi is known to cause a gastrointestinal disease whereas Encephalitozoon intestinalis causes both a disseminated and an intestinal disease. Although several different staining techniques, including the chromotrope technique and its modifications, Uvitex 2B, and the quick-hot Gram-chromotrope procedure, detect microsporidian spores in fecal smears and other clinical samples, they do not identify the species of microsporidia. A need for an easily performed test therefore exists. We reevaluated 120 stool samples that had been found positive for microsporidia previously, using the quick-hot Gram-chromotrope technique, and segregated them into two groups on the basis of spore size. We also screened the smears by immunofluorescence microscopy, using a polyclonal rabbit anti-E. intestinalis serum at a dilution of 1:400. Spores in 29 (24.1%) of the 120 samples fluoresced brightly, indicating that they were E. intestinalis spores. No intense background or cross-reactivity with bacteria, yeasts, or other structures in the stool samples was seen. Additionally, the numbers of spores that fluoresced in seven of these samples were substantially smaller than the numbers of spores that were present in the stained smears, indicating that these samples were probably derived from patients with mixed infections of Enterocytozoon bieneusi and E. intestinalis. Because a 1:400 dilution of this serum does not react with culture-grown Encephalitozoon hellem, Encephalitozoon cuniculi, or Vittaforma corneae or with Enterocytozoon bieneusi spores in feces, we concluded that an immunofluorescence test using this serum is a good alternative for the specific identification of E. intestinalis infections.

  17. Detection by an Immunofluorescence Test of Encephalitozoon intestinalis Spores in Routinely Formalin-Fixed Stool Samples Stored at Room Temperature

    PubMed Central

    Moura, H.; Sodre, F. C.; Bornay-Llinares, F. J.; Leitch, G. J.; Navin, T.; Wahlquist, S.; Bryan, R.; Meseguer, I.; Visvesvara, G. S.

    1999-01-01

    Of the several microsporidia that infect humans, Enterocytozoon bieneusi is known to cause a gastrointestinal disease whereas Encephalitozoon intestinalis causes both a disseminated and an intestinal disease. Although several different staining techniques, including the chromotrope technique and its modifications, Uvitex 2B, and the quick-hot Gram-chromotrope procedure, detect microsporidian spores in fecal smears and other clinical samples, they do not identify the species of microsporidia. A need for an easily performed test therefore exists. We reevaluated 120 stool samples that had been found positive for microsporidia previously, using the quick-hot Gram-chromotrope technique, and segregated them into two groups on the basis of spore size. We also screened the smears by immunofluorescence microscopy, using a polyclonal rabbit anti-E. intestinalis serum at a dilution of 1:400. Spores in 29 (24.1%) of the 120 samples fluoresced brightly, indicating that they were E. intestinalis spores. No intense background or cross-reactivity with bacteria, yeasts, or other structures in the stool samples was seen. Additionally, the numbers of spores that fluoresced in seven of these samples were substantially smaller than the numbers of spores that were present in the stained smears, indicating that these samples were probably derived from patients with mixed infections of Enterocytozoon bieneusi and E. intestinalis. Because a 1:400 dilution of this serum does not react with culture-grown Encephalitozoon hellem, Encephalitozoon cuniculi, or Vittaforma corneae or with Enterocytozoon bieneusi spores in feces, we concluded that an immunofluorescence test using this serum is a good alternative for the specific identification of E. intestinalis infections. PMID:10364604

  18. Prognostic value of glomerular collagen IV immunofluorescence studies in male patients with X-linked Alport syndrome.

    PubMed

    Massella, Laura; Gangemi, Concetta; Giannakakis, Kostas; Crisafi, Antonella; Faraggiana, Tullio; Fallerini, Chiara; Renieri, Alessandra; Muda, Andrea Onetti; Emma, Francesco

    2013-05-01

    X-linked Alport syndrome (X-AS) is caused by mutations of the COL4A5 gene, which encodes for the collagen IV α5 chain (α5[COLIV]), resulting in structural and functional abnormalities of the glomerular basement membrane (GBM) and leading to CKD. The aim of the present study was to evaluate the prognostic value of residual collagen IV chain expression in the GBM of patients with X-AS. The medical records of 22 patients with X-AS from 21 unrelated families collected between 1987 and 2009 were reviewed (median age at last follow-up, 19.9 years; range, 5.4-35.1 years); GBM expression of α1, α3, and α5(COLIV) chains was assessed by immunofluorescence microscopy. GBM distribution of the α5(COLIV) chain was diffuse in 1 and segmental or absent in 21 of the 22 patients; the expression of the α3(COLIV) chain was diffuse in 5 of 22 patients and segmental or absent in 17 of 22 patients. Patients with diffuse staining for the α3(COLIV) chain presented with proteinuria significantly later (median age, 16.9 versus 6.1 years; P=0.02) and reached an estimated GFR < 90 ml/min per 1.73 m(2) at an older age (median age, 27.0 versus 14.9 years; P=0.01) compared with patients with segmental or absent staining. Two thirds of patients with abnormal α3(COLIV) expression by immunofluorescence studies had null or truncating COL4A5 mutations, as opposed to none of the 4 tested patients with diffuse α3(COLIV) chain glomerular distribution. These results indicate that maintained expression of the α3(COLIV) chain is an early positive prognostic marker in patients with X-linked Alport symdrome.

  19. Quantification of DNA adducts in individual cells by immunofluorescence: effects of variation in DNA conformation.

    PubMed

    Frank, Adrian J; Tilby, Michael J

    2003-02-15

    Previously reported detection of melphalan-DNA adducts by immunofluorescent staining indicated considerable intercell variation in fluorescence levels. Investigations were undertaken to determine whether this variation reflected actual intercell differences in adduct levels. Melphalan-treated CCRF-CEM leukaemia cells were analysed by the trapped-in-agarose DNA immunostaining (TARDIS) method using fluorescein immunofluorescence and Hoechst dye-DNA fluorescence. Increasing the time of DNA denaturation in alkali affected the staining intensity, in agreement with known adduct properties, but failed to reduce intercell heterogeneity. To test the hypothesis that heterogeneity resulted from variation in levels of DNA strand breaks, drug-treated cells were exposed to ionising radiation. An increase in level and reduction in heterogeneity of immunofluorescence were observed, optimal at 10 Gy. When samples were irradiated after lysis, 1 Gy was optimal. At the optimal doses, irradiation before or after lysis resulted in similar levels of DNA strand breaks. Our conclusions are as follows: (a) There was no major intercell variation in the number of adducts other than from variation in DNA content. (b) Detection of melphalan, and possibly other adducts, by immunofluorescence can be markedly influenced by the level of strand breaks present in the DNA. (c) Samples analysed for melphalan adducts by immunofluorescence should be irradiated to minimise errors due to this factor. Copyright 2003 Elsevier Science (USA)

  20. Immunofluorescence of rabbit corneas after collagen cross-linking treatment with riboflavin and ultraviolet A.

    PubMed

    Esquenazi, Salomon; He, Jiucheng; Li, Na; Bazan, Haydee E P

    2010-04-01

    To assess ultrastructural modifications in keratocytes and inflammatory cell response in rabbit corneas after riboflavin and ultraviolet A exposure using immunofluorescence microscopy. Twenty adult New Zealand albino rabbits weighing 2.0–3.0 kg were used in this study. Two rabbits served as controls.The animals had their epithelia removed and were cross-linked with riboflavin 0.1% solution (10 mg riboflavin-5-phosphate in 10 mL of 20% dextran-T-500) applied every 3 minutes for 30 minutes, and exposed to ultraviolet A (360 nm, 3 mW/cm2) for 30 minutes. Four rabbits were humanely euthanized at each time point of 1, 3, and 11 days and at 3 and 5 weeks after the procedure. Immunohistochemistry studies of thin sections of each cornea were performed using terminal deoxynucleotyl transferase–mediated uridine triphosphate biotin nick-end labeling staining, alpha smooth muscle actin (a-SMA),CD-3, myeloperoxidase antibodies, and 4',6-diamidino-2-phenylindole(DAPI) counterstaining. In another experiment, 6 additional rabbits were treated as above, and after 10 days of cross-linking, 5 mL of lipopolysaccharide endotoxin (1 mg/mL) was injected in the midstroma. Cross-linked corneas showed early stromal edema. By 5 weeks, complete resolution of the edema and a pronounced highly-organized anterior 200-mm fluorescent zone was observed. Terminal deoxynucleotyl transferase mediated uridine triphosphate biotin nick-end labeling staining showed keratocyte death by both necrosis and apoptosis between days 1 and 3 after cross-linking. At day 1,the limbal area close to the cross-linking zone showed some inflammatory cells and a-SMA–positive cells, indicative of the presence of myofibroblasts. By day 3, some myofibroblasts had migrated to the area beneath the cross-linked stroma. Between days 3 and 5 weeks, there was an increase in a-SMA staining in the area surrounding the cross-linked stroma. The area of cross-linking remained acellular up to 5 weeks. Collagen cross

  1. Direct vs. Indirect Moral Enhancement.

    PubMed

    Schaefer, G Owen

    2015-09-01

    Moral enhancement is an ostensibly laudable project. Who wouldn't want people to become more moral? Still, the project's approach is crucial. We can distinguish between two approaches for moral enhancement: direct and indirect. Direct moral enhancements aim at bringing about particular ideas, motives or behaviors. Indirect moral enhancements, by contrast, aim at making people more reliably produce the morally correct ideas, motives or behaviors without committing to the content of those ideas, motives and/or actions. I will argue, on Millian grounds, that the value of disagreement puts serious pressure on proposals for relatively widespread direct moral enhancement. A more acceptable path would be to focus instead on indirect moral enhancements while staying neutral, for the most part, on a wide range of substantive moral claims. I will outline what such indirect moral enhancement might look like, and why we should expect it to lead to general moral improvement.

  2. Surface-biofunctionalized multicore/shell CdTe@SiO(2) composite particles for immunofluorescence assay.

    PubMed

    Jing, Lihong; Li, Yilin; Ding, Ke; Qiao, Ruirui; Rogach, Andrey L; Gao, Mingyuan

    2011-12-16

    Strongly fluorescent multicore/shell structured CdTe@SiO(2) composite particles of ∼ 50 nm were synthesized via the reverse microemulsion method by using CdTe quantum dots co-stabilized by thioglycolic acid and thioglycerol. The optical stability of the CdTe@SiO(2) composite particles in a wide pH range, under prolonged UV irradiation in pure water, or in different types of physiological buffers was systematically investigated. Towards immunofluorescence assay, both poly(ethylene glycol) (PEG) and carboxyl residues were simultaneously grafted on the surface of the silanol-terminated CdTe@SiO(2) composite particles upon further reactions with silane reagents bearing a PEG segment and carboxyl group, respectively, in order to suppress the nonspecific interactions of the silica particles with proteins and meanwhile introduce reactive moieties to the fluorescent particles. Agarose gel electrophoresis, dynamic light scattering and conventional optical spectroscopy were combined to investigate the effectiveness of the surface modifications. Via the surface carboxyl residue, various antibodies were covalently conjugated to the fluorescent particles and the resultant fluorescent probes were used in detecting cancer cells through both direct fluorescent antibody and indirect fluorescent antibody assays, respectively.

  3. Immunofluorescence localization of dissociation supernatant and extracellular matrix components in Lytechinus pictus sectioned embryos. M.S. Thesis

    NASA Technical Reports Server (NTRS)

    Garciaflack, Ana Leticia

    1988-01-01

    Indirect immunofluorescence was used to localize specific extracellular components in embryos of the sea urchin Lytechinus pictus. Hyalin and S2 (a group of components found in the disaggregation supernatant from Strongylocentrotus purpuratus blastulae) were uniformly present at all stages (unfertilized up to 32 hr) except hyalin could not be detected at the 12 hour early blastula stage. Laminin was found in 16 cell, 32 cell, 6 hour, 18 hour, 24 hour, and 32 hour stages, with especially bright fluorescence at 18 hours. Collagen I was present at all stages (freshly fertilized up to 32 hour) except little was detected at 12 hours. Fibronectin was uniformly present in blastocoelar fibers stained with anto-collagen I and anti-fibronectin. These results were compared with those for S. purpuratus to produce an overview of the localization of specific extracellular matrix components during development of two species of sea urchins. The results set the stage for future studies that will examine the function of these components at the various developmental stages.

  4. Antikinetochore and antitopoisomerase I antibodies in systemic scleroderma: comparative study using immunoblotted recombinant antigens, immunofluorescence, and double immunodiffusion.

    PubMed

    Jarzabek-Chorzelska, M; Blaszczyk, M; Kolacinska-Strasz, Z; Chorzelski, T; Jabłońska, S; Maul, G G

    1990-01-01

    In 135 patients with systemic scleroderma, we compared three different methods to determine antinuclear autoantibody (ANA) specificity: indirect immunofluorescence, double immunodiffusion, and, employing recombinant antigens, immunoblotting using both marker autoantigens of this disease. A characteristic Scl-70 antibody pattern was found on HEp-2 cells in 83.8% of the patients, double immunodiffusion was positive for the Scl-70 antibodies in 81.9%, and immunoblot with the recombinant topoisomerase I (Topo I) was positive in 71% of the patients. For the centromere autoantibodies we found a high concordance between the anticentromere antibody (ACA) pattern on HEp-2 cells (27 patients positive) and the detection of recombinant kinetochore in immunoblotting (26 patients positive). The three testing techniques gave comparable results, except that the Topo I recombinant antigen used in immunoblotting reacted strongly with fewer than expected of the known Scl-70-positive sera. However, a method using recombinant antigens expressing all epitopes (rather than one of the epitopes of Topo I) will undoubtedly become the method of choice for detecting antibodies in systemic scleroderma. Using the immunoblotting technique with the recombinant antigens we detected in four patients antibodies against both Topo I and kinetochore. More severe symptoms of systemic scleroderma were found in patients who had both antibodies. The combined presence of both marker autoantibodies is therefore not as rare as previously reported and may predict severe disease.

  5. Immunofluorescence localization of dissociation supernatant and extracellular matrix components in Lytechinus pictus sectioned embryos. M.S. Thesis

    NASA Technical Reports Server (NTRS)

    Garciaflack, Ana Leticia

    1988-01-01

    Indirect immunofluorescence was used to localize specific extracellular components in embryos of the sea urchin Lytechinus pictus. Hyalin and S2 (a group of components found in the disaggregation supernatant from Strongylocentrotus purpuratus blastulae) were uniformly present at all stages (unfertilized up to 32 hr) except hyalin could not be detected at the 12 hour early blastula stage. Laminin was found in 16 cell, 32 cell, 6 hour, 18 hour, 24 hour, and 32 hour stages, with especially bright fluorescence at 18 hours. Collagen I was present at all stages (freshly fertilized up to 32 hour) except little was detected at 12 hours. Fibronectin was uniformly present in blastocoelar fibers stained with anto-collagen I and anti-fibronectin. These results were compared with those for S. purpuratus to produce an overview of the localization of specific extracellular matrix components during development of two species of sea urchins. The results set the stage for future studies that will examine the function of these components at the various developmental stages.

  6. Surface-biofunctionalized multicore/shell CdTe@SiO2 composite particles for immunofluorescence assay

    NASA Astrophysics Data System (ADS)

    Jing, Lihong; Li, Yilin; Ding, Ke; Qiao, Ruirui; Rogach, Andrey L.; Gao, Mingyuan

    2011-12-01

    Strongly fluorescent multicore/shell structured CdTe@SiO2 composite particles of ~ 50 nm were synthesized via the reverse microemulsion method by using CdTe quantum dots co-stabilized by thioglycolic acid and thioglycerol. The optical stability of the CdTe@SiO2 composite particles in a wide pH range, under prolonged UV irradiation in pure water, or in different types of physiological buffers was systematically investigated. Towards immunofluorescence assay, both poly(ethylene glycol) (PEG) and carboxyl residues were simultaneously grafted on the surface of the silanol-terminated CdTe@SiO2 composite particles upon further reactions with silane reagents bearing a PEG segment and carboxyl group, respectively, in order to suppress the nonspecific interactions of the silica particles with proteins and meanwhile introduce reactive moieties to the fluorescent particles. Agarose gel electrophoresis, dynamic light scattering and conventional optical spectroscopy were combined to investigate the effectiveness of the surface modifications. Via the surface carboxyl residue, various antibodies were covalently conjugated to the fluorescent particles and the resultant fluorescent probes were used in detecting cancer cells through both direct fluorescent antibody and indirect fluorescent antibody assays, respectively.

  7. The logic of indirect speech

    PubMed Central

    Pinker, Steven; Nowak, Martin A.; Lee, James J.

    2008-01-01

    When people speak, they often insinuate their intent indirectly rather than stating it as a bald proposition. Examples include sexual come-ons, veiled threats, polite requests, and concealed bribes. We propose a three-part theory of indirect speech, based on the idea that human communication involves a mixture of cooperation and conflict. First, indirect requests allow for plausible deniability, in which a cooperative listener can accept the request, but an uncooperative one cannot react adversarially to it. This intuition is supported by a game-theoretic model that predicts the costs and benefits to a speaker of direct and indirect requests. Second, language has two functions: to convey information and to negotiate the type of relationship holding between speaker and hearer (in particular, dominance, communality, or reciprocity). The emotional costs of a mismatch in the assumed relationship type can create a need for plausible deniability and, thereby, select for indirectness even when there are no tangible costs. Third, people perceive language as a digital medium, which allows a sentence to generate common knowledge, to propagate a message with high fidelity, and to serve as a reference point in coordination games. This feature makes an indirect request qualitatively different from a direct one even when the speaker and listener can infer each other's intentions with high confidence. PMID:18199841

  8. Use of commercially available rabbit monoclonal antibodies for immunofluorescence double staining.

    PubMed

    Bzorek, Michael; Stamp, Inger Merete; Petersen, Bodil Laub; Frederiksen, Lisbeth

    2008-07-01

    Immunohistochemistry, that is, the use of polyclonal and monoclonal antibodies to detect cell and tissue antigens at a microscopical level is a powerful tool for both research and diagnostic purposes. Especially in the field of hematologic disease, there is often a need to detect several antigens synchronously, and we report here a fast and easy technique for demonstrating more than 1 antigen in 1 slide using immunofluorescence. We have used commercially available rabbit monoclonal antibodies (Cyclin D1, CD3, CD5, CD23, etc.) paired with mouse monoclonal antibodies (CD7, CD20, CD79a, Pax-5, etc.) for double immunofluorescence labeling on paraffin-embedded tissue sections. Commercially available rabbit monoclonal antibodies in combination with mouse monoclonal antibodies proved useful in double immunofluorescence labeling on paraffin-embedded tissue, and all combinations used yielded excellent results.

  9. Evaluation of cell lines and immunofluorescence and plaque assay procedures for quantifying reoviruses in sewage.

    PubMed Central

    Ridinger, D N; Spendlove, R S; Barnett, B B; George, D B; Roth, J C

    1982-01-01

    Twelve continuous cell lines were tested to determine their sensitivity to reovirus types 1, 2, and 3 isolated from sewage. Madin-Darby bovine kidney (MDBK), rhesus monkey kidney (LLC-MK2), and human embryonic intestinal (intestinal 407) cells were most sensitive, respectively. In a similar study, MDBK cells were more sensitive than LLC-MK2 and Buffalo green monkey kidney (BGM) cells to sewage-isolated, protamine-precipitated reoviruses which had not been serotyped and had no previous cell contact. Sewage-isolated, protamine-precipitated reoviruses were also used in conjunction with MDBK cells in a comparative evaluation of immunofluorescent cell count and plaque assay procedures. The immunofluorescence assay is more sensitive and more rapid than the plaque assay. Reoviruses in excess of 10(4)/liter of raw sewage were detected by the immunofluorescent cell count assay. PMID:7044308

  10. Immunofluorescent staining of rat gastric parietal cells by human antibody unrelated to pernicious anaemia

    PubMed Central

    Muller, H. K.; McGiven, A. R.; Nairn, R. C.

    1971-01-01

    Immunofluorescence tests on 94 human sera reacting with rat gastric parietal cells revealed that 41 (44%) of the sera contained antibody to a rat parietal cell antigen that was distinct from the pernicious anaemia autoantigen. Ten of the sera contained antibodies to both parietal cell antigens. The remaining 53 (56%) sera contained only parietal cell antibodies of the pernicious anaemia type. We recommend that mouse gastric mucosa, which does not react with the heterologous rat parietal cell antibody, replace rat gastric mucosa for immunofluorescence diagnostic tests. PMID:4929573

  11. Identification of claudins by western blot and immunofluorescence in different cell lines and tissues.

    PubMed

    González-Mariscal, Lorenza; Garay, Erika; Quirós, Miguel

    2011-01-01

    Claudins are integral proteins of the TJ. Each epithelia in the organism expresses a unique set of claudins that determines the degree of sealing of the paracellular pathway and the ionic selectivity of the tissue. TJs are dynamic structures whose organization and composition change in response to alterations in the environment as well as under physiological and pathological conditions. Changes in claudin expression and subcellular distribution can be analyzed in western blot and immunofluorescence experiments, employing a wide array of available specific antibodies against claudins. In this chapter, we describe in detail protocols used for western blot and immunofluorescence detection of claudins in epithelial cell lines and in various tissue samples.

  12. Method for collecting and immobilizing individual cumulus cells enabling quantitative immunofluorescence analysis of proteins.

    PubMed

    Appeltant, R; Maes, D; Van Soom, A

    2015-07-01

    Most immunofluorescence methods rely on techniques dealing with a very large number of cells. However, when the number of cells in a sample is low (e.g., when cumulus cells must be analyzed from individual cumulus-oocyte complexes), specific techniques are required to conserve, fix, and analyze cells individually. We established and validated a simple and effective method for collecting and immobilizing low numbers of cumulus cells that enables easy and quick quantitative immunofluorescence analysis of proteins from individual cells. To illustrate this technique, we stained proprotein of a disintegrin and metalloproteinase with thrombospondin-like repeats-1 (proADAMTS-1) and analyzed its levels in individual porcine cumulus cells.

  13. Indirect Reciprocity; A Field Experiment.

    PubMed

    van Apeldoorn, Jacobien; Schram, Arthur

    2016-01-01

    Indirect reciprocity involves cooperative acts towards strangers, either in response to their kindness to third parties (downstream) or after receiving kindness from others oneself (upstream). It is considered to be important for the evolution of cooperative behavior amongst humans. Though it has been widely studied theoretically, the empirical evidence of indirect reciprocity has thus far been limited and based solely on behavior in laboratory experiments. We provide evidence from an online environment where members can repeatedly ask and offer services to each other, free of charge. For the purpose of this study we created several new member profiles, which differ only in terms of their serving history. We then sent out a large number of service requests to different members from all over the world. We observe that a service request is more likely to be rewarded for those with a profile history of offering the service (to third parties) in the past. This provides clear evidence of (downstream) indirect reciprocity. We find no support for upstream indirect reciprocity (in this case, rewarding the service request after having previously received the service from third parties), however. Our evidence of downstream indirect reciprocity cannot be attributed to reputational effects concerning one's trustworthiness as a service user.

  14. Indirect Reciprocity; A Field Experiment

    PubMed Central

    van Apeldoorn, Jacobien; Schram, Arthur

    2016-01-01

    Indirect reciprocity involves cooperative acts towards strangers, either in response to their kindness to third parties (downstream) or after receiving kindness from others oneself (upstream). It is considered to be important for the evolution of cooperative behavior amongst humans. Though it has been widely studied theoretically, the empirical evidence of indirect reciprocity has thus far been limited and based solely on behavior in laboratory experiments. We provide evidence from an online environment where members can repeatedly ask and offer services to each other, free of charge. For the purpose of this study we created several new member profiles, which differ only in terms of their serving history. We then sent out a large number of service requests to different members from all over the world. We observe that a service request is more likely to be rewarded for those with a profile history of offering the service (to third parties) in the past. This provides clear evidence of (downstream) indirect reciprocity. We find no support for upstream indirect reciprocity (in this case, rewarding the service request after having previously received the service from third parties), however. Our evidence of downstream indirect reciprocity cannot be attributed to reputational effects concerning one’s trustworthiness as a service user. PMID:27043712

  15. Indirect Reciprocity under Incomplete Observation

    PubMed Central

    Nakamura, Mitsuhiro; Masuda, Naoki

    2011-01-01

    Indirect reciprocity, in which individuals help others with a good reputation but not those with a bad reputation, is a mechanism for cooperation in social dilemma situations when individuals do not repeatedly interact with the same partners. In a relatively large society where indirect reciprocity is relevant, individuals may not know each other's reputation even indirectly. Previous studies investigated the situations where individuals playing the game have to determine the action possibly without knowing others' reputations. Nevertheless, the possibility that observers of the game, who generate the reputation of the interacting players, assign reputations without complete information about them has been neglected. Because an individual acts as an interacting player and as an observer on different occasions if indirect reciprocity is endogenously sustained in a society, the incompleteness of information may affect either role. We examine the game of indirect reciprocity when the reputations of players are not necessarily known to observers and to interacting players. We find that the trustful discriminator, which cooperates with good and unknown players and defects against bad players, realizes cooperative societies under seven social norms. Among the seven social norms, three of the four suspicious norms under which cooperation (defection) to unknown players leads to a good (bad) reputation enable cooperation down to a relatively small observation probability. In contrast, the three trustful norms under which both cooperation and defection to unknown players lead to a good reputation are relatively efficient. PMID:21829335

  16. The Future of Indirect Evidence

    PubMed Central

    Efron, Bradley

    2009-01-01

    Familiar statistical tests and estimates are obtained by the direct observation of cases of interest: a clinical trial of a new drug, for instance, will compare the drug’s effects on a relevant set of patients and controls. Sometimes, though, indirect evidence may be temptingly available, perhaps the results of previous trials on closely related drugs. Very roughly speaking, the difference between direct and indirect statistical evidence marks the boundary between frequentist and Bayesian thinking. Twentieth-century statistical practice focused heavily on direct evidence, on the grounds of superior objectivity. Now, however, new scientific devices such as microarrays routinely produce enormous data sets involving thousands of related situations, where indirect evidence seems too important to ignore. Empirical Bayes methodology offers an attractive direct/indirect compromise. There is already some evidence of a shift toward a less rigid standard of statistical objectivity that allows better use of indirect evidence. This article is basically the text of a recent talk featuring some examples from current practice, with a little bit of futuristic speculation. PMID:21243111

  17. Automated antinuclear immunofluorescence antibody screening: a comparative study of six computer-aided diagnostic systems.

    PubMed

    Bizzaro, Nicola; Antico, Antonio; Platzgummer, Stefan; Tonutti, Elio; Bassetti, Danila; Pesente, Fiorenza; Tozzoli, Renato; Tampoia, Marilina; Villalta, Danilo

    2014-03-01

    Indirect immunofluorescence (IIF) plays an important role in immunological assays for detecting and measuring autoantibodies. However, the method is burdened by some unfavorable features: the need for expert morphologists, the subjectivity of interpretation, and a low degree of standardization and automation. Following the recent statement by the American College of Rheumatology that the IIF technique should be considered as the standard screening method for the detection of anti-nuclear antibodies (ANA), the biomedical industry has developed technological solutions which might significantly improve automation of the procedure, not only in the preparation of substrates and slides, but also in microscope reading. We collected 104 ANA-positive sera from patients with a confirmed clinical diagnosis of autoimmune disease and 40 ANA-negative sera from healthy blood donors. One aliquot of each serum, without information about pattern and titer, was sent to six laboratories of our group, where the sera were tested with the IIF manual method provided by each of the six manufacturers of automatic systems. Assignment of result (pos/neg), of pattern and titer was made by consensus at a meeting attended by all members of the research team. Result was assigned if consensus for pos/neg was reached by at least four of six certifiers, while for the pattern and for the titer, the value observed with higher frequency (mode) was adopted. Seventeen ANA-positive sera and six ANA-negative sera were excluded. Therefore, the study with the following automatic instrumentation was conducted on 92 ANA-positive sera and on 34 ANA-negative sera: Aklides, EUROPattern, G-Sight (I-Sight-IFA), Helios, Image Navigator, and Nova View. Analytical imprecision was measured in five aliquots of the same serum, randomly added to the sample series. Overall sensitivity of the six automated systems was 96.7% and overall specificity was 89.2%. Most false negatives were recorded for cytoplasmic patterns

  18. [Expression profiling and immunofluorescence localization of the major egg antigen p40 of Schistosoma japonicum in the liver of infected New Zealand white rabbits].

    PubMed

    Xia, Dan; Deng, Ganming; Teng, Pingying; Xie, Yu; Li, Yaomin; Wang, Chunmei; Chen, Shujie; Chen, Minfang; Mai, Rongjia; Liao, Haiyan; Shi, Lingyu; Ou, Liyan; Chen, Qiwei; Chen, Xiaoguang; Zhou, Xiaohong

    2015-06-01

    To examine the expression profile and immunofluorescence localization of the major egg antigen p40 of Schistosoma japonicum (Sjp40) during granuloma formation in the liver of infected New Zealand white rabbits. New Zealand white rabbits were infected with S. japonicum cercariae, and the livers were harvested at 29 and 45 days post-infection (dpi). The total RNA of the liver tissues was extracted for expression profiling of Sjp40 by quantitative reverse transcription-PCR (qRT-PCR) with GAPDH of S. japonicum as the endogenous reference gene. The expression of Sjp40 in the liver were detected by Western blotting using anti-Sjp40 monoclonal antibody (mAb) 9G7 or anti-Toxoplasma gondii tSAG1 mAb Y3A8 (control) as the primary antibody. Paraffin sections of the liver were prepared for observing egg granuloma formation using HE staining and for indirect immunofluorescence assay of Sjp40 location in the trapped eggs and egg granulomas. The level of Sjp40 mRNA in the eggs trapped in rabbit livers was significantly higher at 45 dpi than that at 29 dpi (P<0.05), and Western blotting confirmed the presence of Sjp40 protein in the rabbit livers at both 29 and 45 dpi. Immunofluorescence assay demonstrated localized expression of Sjp40 in the immature eggs in the rabbit liver at 29 dpi, but at 45 dpi fluorescence was detected in clusters of mature eggs containing miracidium and in the surrounding egg granulomas. The transcriptional levels of Sjp40 significantly increased with the maturation of eggs trapped in the rabbit livers. Sjp40 protein spread from the eggs to the surrounding egg granuloma at 45 dpi when acute liver granulomatous lesions occur, suggesting that Sjp40 plays a key role in egg granulomas formation in the livers of infected New Zealand white rabbits.

  19. Macrophage colony-stimulating factor induces indirect angiogenesis in vivo.

    PubMed

    Phillips, G D; Aukerman, S L; Whitehead, R A; Knighton, D R

    1993-01-01

    The cytokine macrophage colony-stimulating factor was implanted in the rabbit cornea over a wide dose range (1 ng to 100 microg) to assay its angiogenic activity in vivo. Neovascularization occurred in a dose-dependent manner, and maximum angiogenesis occurred only with 100 microg. Histologic analysis revealed that the corneas were free of inflammation at the lower doses, but had slight inflammation at 50 and 100 microg. Nonspecific esterase staining of frozen sections and transmission electron microscopy revealed that the inflammatory cells were predominantly macrophages, with very few neutrophils present. This association of capillary formation with inflammation suggests an indirect mechanism of angiogenesis. The lack of neutrophils within the inflammatory cell infiltrate demonstrates that indirect angiogenesis can proceed without the local presence of neutrophils. This distinguishes macrophage colony-stimulating factor from other indirect-acting angiogenesis factors that have been identified to date.

  20. Evaluation of culture, immunofluorescence, and serology for the diagnosis of pertussis.

    PubMed Central

    Halperin, S A; Bortolussi, R; Wort, A J

    1989-01-01

    Nasopharyngeal culture, direct immunofluorescence, and serology of acute-phase and paired serum specimens were compared for the laboratory diagnosis of infections due to Bordetella pertussis in a community-based pediatric population with both high vaccine usage and high pertussis incidence. In 77 (37%) of 210 patients evaluated, one or more tests were positive for pertussis. A clinical illness compatible with pertussis was present in 52 (71%) of 73 pertussis test-positive and 42 (35%) of 119 test-negative patients (P less than 0.001). Nasopharyngeal culture was of low sensitivity (20 [26%] of 77 positive tests) but was most commonly confirmed by another positive pertussis test (85%). Direct immunofluorescence was both insensitive and nonspecific; only 6 (30%) of 20 cases positive by culture were positive by immunofluorescence, and only 4 (33%) of 12 of the culture-negative, immunofluorescence-positive cases could be confirmed by another positive pertussis test. Although serology by enzyme immunoassay proved to be the most sensitive of the laboratory tests (87%), this sensitivity could be achieved only by assaying both acute-phase and paired serum specimens and measuring immunoglobulin G (IgG), IgA, and IgM antibodies to two pertussis antigens (pertussis toxin and filamentous hemagglutinin). Loss of sensitivity occurred with any reduction in the number of these serologic assays performed. Optimal laboratory diagnosis of endemic pertussis in a pediatric population requires both nasopharyngeal culture and serology by enzyme immunoassay. PMID:2542366

  1. Detection of Giardia lamblia and Cryptosporidium parvum by direct immunofluorescence assay in stool specimen.

    PubMed

    Rahman, M M; Hossain, M A; Paul, S K; Ahmed, S; Islam, A; Ehsan, M A; Alam, M M; Kabir, M R; Sarkar, S R

    2014-07-01

    Giardia and Cryptosporidium are the pathogens which transmitted through contaminated soil and contaminated water are significant causes of diarrhea and nutritional disorders in institutional and community peoples. Children and immune compromise persons are more vulnerable for these infections. Both Giardiasis and Cryptosporidiosis were included in 2004 as WHO Neglected Disease. So this is a major public health problem in developing countries. The present study was carried out to detect the Giardia and Cryptosporidium from diarrheic or patient having loose stool by Direct Immunofluorescence assay. The study was conducted during July 20012 to February 2013 and the work was done in Mymensingh Medical College in the department of Microbiology and in Bangladesh Agricultural University in the department of Veterinary Medicine. A total of 100 loose stools were collected from school children of different area and hospital under sadar upazilla, Mymensingh. The detection of Giardia lamblia and Cryptosporidium parvum showed the individual prevalence 8% and 4% respectively. The highest cyst/oocyst count was 85,000 and 1,000/gm of stool and the lowest being 100 and 50/gm of stool for Giardiasis and Cryptosporidiosis respectively. The detection rate of Giardia and Cryptosporidium by Immunofluorescence assay was relatively higher than the previous study done in Bangladesh and this was the first report from Bangladesh over human stool specimen using Immunofluorescence assay. So, Immunofluorescence assay could be adapted for rapid and accurate detection of Giardia and Cryptosporidium.

  2. BLIND TRIALS EVALUATING IN VITRO INFECTIVITY OF CRYPTOSPORIDIUM PARVUM OOCYSTS USING CELL CULTURE IMMUNOFLUORESCENCE

    EPA Science Inventory

    An optimized cell culture-immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in 'blind' trials to determine the sensitivity and reproducibility for measuring infectivity of flow cytometry prepared inocula of C. parvum oocysts. In separate trials, suspens...

  3. BLIND TRIALS EVALUATING IN VITRO INFECTIVITY OF CRYPTOSPORIDIUM PARVUM OOCYSTS USING CELL CULTURE IMMUNOFLUORESCENCE

    EPA Science Inventory

    An optimized cell culture-immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in 'blind' trials to determine the sensitivity and reproducibility for measuring infectivity of flow cytometry prepared inocula of C. parvum oocysts. In separate trials, suspens...

  4. Detection of Specific Strains and Variants of Streptococcus cremoris in Mixed Cultures by Immunofluorescence

    PubMed Central

    Hugenholtz, Jeroen; Veldkamp, Hans; Konings, Wil N.

    1987-01-01

    Antisera against four different strains of Streptococcus cremoris were raised by injecting rabbits with washed suspensions of whole cells. These antisera interacted specifically with the corresponding strain in a mixture of up to nine different S. cremoris strains. The antisera could be used for analyzing the composition of mixed cultures containing these strains by immunofluorescence. Competition experiments were performed in batch and continuous cultures under amino acid limitation. A bacteriophage-sensitive variant of S. cremoris SK11 (SK1128) could be distinguished from a bacteriophage-resistant variant (SK1143) by the same immunofluorescence technique. The competition between the two variants and the stability of both variants in pure cultures were followed with the specific antibodies. Antibodies against the purified proteolytic system of S. cremoris Wg2 were used to determine the presence of proteases by immunofluorescence in several S. cremoris strains under different culture conditions. The described immunofluorescence methods can be used to analyze complex mixed starter cultures common in the dairy industry as the strains and variants present in these mixtures can be recognized microscopically. Images PMID:16347256

  5. Comparison of Immunofluorescence and Desmoglein Enzyme-linked Immunosorbent Assay in the Diagnosis of Pemphigus: A Prospective, Cross-sectional Study in a Tertiary Care Hospital

    PubMed Central

    Ravi, Deepthi; Prabhu, S Smitha; Rao, Raghavendra; Balachandran, C; Bairy, Indira

    2017-01-01

    Background: Pemphigus is an acquired immunobullous disorder in which antibodies are directed against epidermal cadherins. Despite the commercial availability and less cost of enzyme-linked immunosorbent assays (ELISAs) to detect antidesmoglein 1 (Dsg1) and anti-Dsg3, immunofluorescence is still widely used for confirmation of diagnosis. Aims: (1) To compare the usefulness of indirect immunofluorescence (IIF) and ELISA tests in the diagnosis of pemphigus. (2) To find the clinical correlation between the tests and severity of the disease. Materials and Methods: Sixty-one patients (27 women and 34 men, age distribution from 20 to 75) were clinically diagnosed as pemphigus (pemphigus foliaceus - 11, pemphigus vulgaris - 50) and were recruited for the study. IIF and Dsg ELISA were performed and the findings were compared with each other and with the pemphigus area activity score. Data were entered in SPSS and were analyzed using Kruskal–Wallis test. Results: There was a moderate positive correlation between the cutaneous score and Dsg1 titer, and mucosal score and Dsg3 titer. The titer of IIF showed statistically significant positive correlation with the cutaneous score but not the mucosal score. Dsg ELISA showed higher sensitivity (90.2%) than IIF (75.4%) in the diagnosis of pemphigus. Conclusions: Dsg ELISA is a more sensitive method than IIF and shows more correlation with the disease severity.

  6. Evaluation of quantum dot immunofluorescence and a digital CMOS imaging system as an alternative to conventional organic fluorescence dyes and laser scanning for quantifying protein microarrays.

    PubMed

    Jain, Aarti; Taghavian, Omid; Vallejo, Derek; Dotsey, Emmanuel; Schwartz, Dan; Bell, Florian G; Greef, Chad; Davies, D Huw; Grudzien, Jennipher; Lee, Abraham P; Felgner, Philip L; Liang, Li

    2016-04-01

    Organic fluorescent dyes are widely used for the visualization of bound antibody in a variety of immunofluorescence assays. However, the detection equipment is often expensive, fragile, and hard to deploy widely. Quantum dots (Qdot) are nanocrystals made of semiconductor materials that emit light at different wavelengths according to the size of the crystal, with increased brightness and stability. Here, we have evaluated a small benchtop "personal" optical imager (ArrayCAM) developed for quantification of protein arrays probed by Qdot-based indirect immunofluorescence. The aim was to determine if the Qdot imager system provides equivalent data to the conventional organic dye-labeled antibody/laser scanner system. To do this, duplicate proteome microarrays of Vaccinia virus, Brucella melitensis and Plasmodium falciparum were probed with identical samples of immune sera, and IgG, IgA, and IgM profiles visualized using biotinylated secondary antibodies followed by a tertiary reagent of streptavidin coupled to either P3 (an organic cyanine dye typically used for microarrays) or Q800 (Qdot). The data show excellent correlation for all samples tested (R > 0.8) with no significant change of antibody reactivity profiles. We conclude that Qdot detection provides data equivalent to that obtained using conventional organic dye detection. The portable imager offers an economical, more robust, and deployable alternative to conventional laser array scanners. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Evaluation of Quantum dot immunofluorescence and a digital CMOS imaging system as an alternative to conventional organic fluorescence dyes and laser scanning for quantifying protein microarrays

    PubMed Central

    Jain, Aarti; Taghavian, Omid; Vallejo, Derek; Dotsey, Emmanuel; Schwartz, Dan; Bell, Florian G.; Greef, Chad; Davies, D. Huw; Grudzien, Jennipher; Lee, Abraham P.; Felgner, Philip; Liang, Li

    2016-01-01

    Organic fluorescent dyes are widely used for the visualization of bound antibody in a variety of immunofluorescence assays. However, the detection equipment is often expensive, fragile and hard to deploy widely. Quantum dots (Qdot®) are nanocrystals made of semiconductor materials that emit light at different wavelengths according to the size of the crystal, with increased brightness and stability. Here we have evaluated a small benchtop ‘personal’ optical imager (ArrayCAM™) developed for quantification of protein arrays probed by Qdot -based indirect immunofluorescence. The aim was to determine if the Qdot imager system provides equivalent data to the conventional organic dye-labelled antibody/laser scanner system. To do this, duplicate proteome microarrays of Vaccinia virus, Brucella melitensis and Plasmodium falciparum were probed with identical samples of immune sera, and IgG, IgA and IgM profiles visualized using biotinylated secondary antibodies followed by a tertiary reagent of streptavidin coupled to either P3 (an organic cyanine dye typically used for microarrays) or Q800 (Qdot). The data show excellent correlation for all samples tested (R>0.8) with no significant change of antibody reactivity profiles. We conclude that Qdot detection provides data equivalent to that obtained using conventional organic dye detection. The portable imager offers an economical, more robust and deployable alternative to conventional laser array scanners. PMID:26842269

  8. A history of urine microscopy.

    PubMed

    Cameron, J Stewart

    2015-11-01

    The naked-eye appearance of the urine must have been studied by shamans and healers since the Stone Age, and an elaborate interpretation of so-called Uroscopy began around 600 AD as a form of divination. A 1000 years later, the first primitive monocular and compound microscopes appeared in the Netherlands, and along with many other objects and liquids, urine was studied from around 1680 onwards as the enlightenment evolved. However, the crude early instruments did not permit fine study because of chromatic and linear/spherical blurring. Only after complex multi-glass lenses which avoided these problems had been made and used in the 1820s in London by Lister, and in Paris by Chevalier and Amici, could urinary microscopy become a practical, clinically useful tool in the 1830s. Clinical urinary microscopy was pioneered by Rayer and his pupils in Paris (especially Vigla), in the late 1830s, and spread to UK and Germany in the 1840s, with detailed descriptions and interpretations of cells and formed elements of the urinary sediment by Nasse, Henle, Robinson and Golding Bird. Classes were held, most notably by Donné in Paris. After another 50 years, optical microscopy had reached its apogee, with magnifications of over 1000 times obtainable free of aberration, using immersion techniques. Atlases of the urinary sediment were published in all major European countries and in the US. Polarised light and phase contrast was used also after 1900 to study urine, and by the early 20th century, photomicroscopy (pioneered by Donné and Daguerre 50 years previously, but then ignored) became usual for teaching and recording. In the 1940s electron microscopy began, followed by detection of specific proteins and cells using immunofluorescent antibodies. All this had been using handheld methodology. Around 1980, machine-assisted observations began, and have dominated progress since.

  9. Demonstration of TGF-β and XIIIα in Endocardial Biopsies of Carcinoid Heart Disease Patients: an Immunofluorescence Study

    PubMed Central

    Diepholz, Dorgrit; Wilke, Andreas; Maisch, Bernhard; Steverding, Dietmar

    2011-01-01

    Background Serotonin and other vasoactive substances play a critical role in the development of carcinoid heart disease, but the exact etiology of the illness is still unknown. Methods By using immunofluorescence microscopy, we investigated the expression of transforming growth factor-β (TGF-β) and the presence of fibrin-stabilizing factor (XIIIα) in endomyocardial biopsy specimens of patients with carcinoid heart disease. In addition, the tissue integrity of the specimens was studied by staining for laminin. Results Both TGF-β and XIIIα co-localized in the endocardium beneath carcinoid plaques: while TGF-β was found within myocytes, XIIIα was detected on the surface of cells in fibrotic lesions stretching out into the tissue. Laminin staining revealed that the integrity of the endocardium was dissolved and that the tissue consisted of hypertrophic and hypotrophic myocytes. Conclusions The results suggest that the presence of TGF-β and XIIIα in carcinoid heart lesions indicates that endocardial damage induced by serotonin and other vasoactive substances gives rise to an overshooting wound healing process.

  10. Immunofluorescence and immunoelectron microscopic localization of medicinal substance, Rb1, in several plant parts of Panax ginseng.

    PubMed

    Yokota, Sadaki; Onohara, Yuko; Shoyama, Yukihiro

    2011-03-01

    It is important to know the localization of medicinal substance, Rb1, of Ginseng, Panax ginseng, in this plant in order to achieve efficient extraction of Rb1 or to culture producing cells. In this report, we describe the localization of Rb1 in various parts of the plant as determined by immunofluorescence (IF) and immunoelectron microscopies (IEM). Using IF, we show that Rb1 is localized to chloroplasts, peroxisomes and cytoplasm but not to vacuoles of leaf parenchymal cells. In the leaf stem, Rb1 is localized to the vascular bundles as well as vacuoles. In the root, vacuoles of parenchymal cells are stained at various intensities. Using IEM, gold particles showing Rb1 antigenic sites are present in the compartments stained by IF technique. In addition, Rb1 is localized in the sieve elements of the phloem and degrading primary cell wall of xylem, and in the root parenchymal cells Rb1 is associated with electron dense polymorphic materials but not in starch granules. Translocation and storage of Rb1 and effective utilization of leaves are discussed.

  11. A bead-based immunofluorescence-assay on a microfluidic dielectrophoresis platform for rapid dengue virus detection.

    PubMed

    Iswardy, Edwar; Tsai, Tien-Chun; Cheng, I-Fang; Ho, Tzu-Chuan; Perng, Guey Chuen; Chang, Hsien-Chang

    2017-09-15

    The proof of concept of utilizing a microfluidic dielectrophoresis (DEP) chip was conducted to rapidly detect a dengue virus (DENV) in vitro based on the fluorescence immunosensing. The mechanism of detection was that the DEP force was employed to capture the modified beads (mouse anti-flavivirus monoclonal antibody-coated beads) in the microfluidic chip and the DENV modified with fluorescence label, as the detection target, can be then captured on the modified beads by immunoreaction. The fluorescent signal was then obtained through fluorescence microscopy, and then quantified by ImageJ freeware. The platform can accelerate an immuno-reaction time, in which the on-chip detection time was 5min, and demonstrating an ability for DENV detection as low as 10(4) PFU/mL. Furthermore, the required volume of DENV samples dramatically reduced, from the commonly used ~50µL to ~15µL, and the chip was reusable (>50x). Overall, this platform provides a rapid detection (5min) of the DENV with a low sample volume, compared to conventional methods. This proof of concept with regard to a microfluidic dielectrophoresis chip thus shows the potential of immunofluorescence based-assay applications to meet diagnostic needs. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Predicting and replacing the pathological Gleason grade with automated gland ring morphometric features from immunofluorescent prostate cancer images.

    PubMed

    Khan, Faisal M; Scott, Richard; Donovan, Michael; Fernandez, Gerardo

    2017-04-01

    The Gleason grade is the most common architectural and morphological assessment of prostate cancer severity and prognosis. There have been numerous algorithms developed to approximate and duplicate the Gleason scoring system, mostly developed in standard H&E brightfield microscopy. Immunofluorescence (IF) image analysis of tissue pathology has recently been proven to be robust in developing prognostic assessments of disease, particularly in prostate cancer. We leverage a method of segmenting gland rings in IF images for predicting the pathological Gleason, both the clinical and the image specific grades, which may not necessarily be the same. We combine these measures with nuclear specific characteristics. In 324 images from 324 patients, our individual features correlate well univariately with the Gleason grades and in a multivariate setting have an accuracy of 85% in predicting the Gleason grade. Additionally, these features correlate strongly with clinical progression outcomes [concordance index (CI) of 0.89], significantly outperforming the clinical Gleason grades (CI of 0.78). Finally, in multivariate models for multiple prostate cancer progression endpoints, replacing the Gleason with these features results in equivalent or improved performances. This work presents the first assessment of morphological gland unit features from IF images for predicting the Gleason grade, and even replacing it in prostate cancer prognostics.

  13. Indirect reciprocity with optional interactions.

    PubMed

    Ghang, Whan; Nowak, Martin A

    2015-01-21

    Indirect reciprocity is a mechanism for the evolution of cooperation that is relevant for prosocial behavior among humans. Indirect reciprocity means that my behavior towards you also depends on what you have done to others. Indirect reciprocity is associated with the evolution of social intelligence and human language. Most approaches to indirect reciprocity assume obligatory interactions, but here we explore optional interactions. In any one round a game between two players is offered. A cooperator accepts a game unless the reputation of the other player indicates a defector. For a game to take place, both players must accept. In a game between a cooperator and a defector, the reputation of the defector is revealed to all players with probability Q. After a sufficiently large number of rounds the identity of all defectors is known and cooperators are no longer exploited. The crucial condition for evolution of cooperation can be written as hQB>1, where h is the average number of rounds per person and B=(b/c)-1 specifies the benefit-to-cost ratio. We analyze both stochastic and deterministic evolutionary game dynamics. We study two extensions that deal with uncertainty: hesitation and malicious gossip. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Modeling Indirect Tunneling in Silicon

    NASA Astrophysics Data System (ADS)

    Chen, Edward

    Indirect tunneling in silicon p-n junctions catches people's attention again in recent years. First, the phenomenon induces a serious leakage problem, so called gate-induced drain leakage (GIDL) effect, in modern metal-oxide-semiconductor field-effect transistors (MOSFETs). Second, it is utilized to develop a novel tunneling transistor with the sharp turn-on ability for continuing ITRS roadmap. Although the indirect tunneling is important for the state-of-the-art transistor-technology, the accuracy of the present tunneling models in technology computer-aided design (TCAD) tools is still vague. In the research work, the theory of indirect tunneling in silicon has been thoroughly studied. The phonon-assisted tunneling model has been developed and compared with the existing ones in the Sentaurus-Synopsys, Medici-Synopsys, and Atlas-Silvaco TCAD tools. Beyond these existing models, ours successfully predicts the indirect tunneling current under the different field direction in silicon. In addition, bandgap narrowing in heavily-doped p-n junctions under the reverse-biased condition is also studied during the model development. At the end of the research work, the application to low standby power (LSTP) transistors is demonstrated to show the capability of our tunneling model in the device level.

  15. Indirect methods in nuclear astrophysics

    NASA Astrophysics Data System (ADS)

    Bertulani, C. A.; Shubhchintak; Mukhamedzhanov, A.; Kadyrov, A. S.; Kruppa, A.; Pang, D. Y.

    2016-04-01

    We discuss recent developments in indirect methods used in nuclear astrophysics to determine the capture cross sections and subsequent rates of various stellar burning processes, when it is difficult to perform the corresponding direct measurements. We discuss in brief, the basic concepts of Asymptotic Normalization Coefficients, the Trojan Horse Method, the Coulomb Dissociation Method, (d,p), and charge-exchange reactions.

  16. Localization of extracellular matrix components in developing mouse salivary glands by confocal microscopy

    NASA Technical Reports Server (NTRS)

    Hardman, P.; Spooner, B. S.

    1992-01-01

    The importance of the extracellular matrix (ECM) in epithelial-mesenchymal interactions in developing organisms is well established. Proteoglycans and interstitial collagens are required for the growth, morphogenesis, and differentiation of epithelial organs and the distribution of these molecules has been described. However, much less is known about other ECM macromolecules in developing epithelial organs. We used confocal microscopy to examine the distribution of laminin, heparan sulfate (BM-1) proteoglycan, fibronectin, and collagen types I, IV, and V, in mouse embryonic salivary glands. Organ rudiments were isolated from gestational day 13 mouse embryos and cultured for 24, 48, or 72 hours. Whole mounts were stained by indirect immunofluorescence and then examined using a Zeiss Laser Scan Microscope. We found that each ECM component examined had a distinct distribution and that the distribution of some molecules varied with culture time. Laminin was mainly restricted to the basement membrane. BM-1 proteoglycan was concentrated in the basement membrane and also formed a fine network throughout the mesenchyme. Type IV collagen was mainly located in the basement membrane of the epithelium, but it was also present throughout the mesenchyme. Type V collagen was distributed throughout the mesenchyme at 24 hours, but at 48 hours was principally located in the basement membrane. Type I collagen was distributed throughout the mesenchyme at all culture times, and accumulated in the clefts and particularly at the epithelial-mesenchymal interface as time in culture increased. Fibronectin was observed throughout the mesenchyme at all times.

  17. Localization of extracellular matrix components in developing mouse salivary glands by confocal microscopy

    NASA Technical Reports Server (NTRS)

    Hardman, P.; Spooner, B. S.

    1992-01-01

    The importance of the extracellular matrix (ECM) in epithelial-mesenchymal interactions in developing organisms is well established. Proteoglycans and interstitial collagens are required for the growth, morphogenesis, and differentiation of epithelial organs and the distribution of these molecules has been described. However, much less is known about other ECM macromolecules in developing epithelial organs. We used confocal microscopy to examine the distribution of laminin, heparan sulfate (BM-1) proteoglycan, fibronectin, and collagen types I, IV, and V, in mouse embryonic salivary glands. Organ rudiments were isolated from gestational day 13 mouse embryos and cultured for 24, 48, or 72 hours. Whole mounts were stained by indirect immunofluorescence and then examined using a Zeiss Laser Scan Microscope. We found that each ECM component examined had a distinct distribution and that the distribution of some molecules varied with culture time. Laminin was mainly restricted to the basement membrane. BM-1 proteoglycan was concentrated in the basement membrane and also formed a fine network throughout the mesenchyme. Type IV collagen was mainly located in the basement membrane of the epithelium, but it was also present throughout the mesenchyme. Type V collagen was distributed throughout the mesenchyme at 24 hours, but at 48 hours was principally located in the basement membrane. Type I collagen was distributed throughout the mesenchyme at all culture times, and accumulated in the clefts and particularly at the epithelial-mesenchymal interface as time in culture increased. Fibronectin was observed throughout the mesenchyme at all times.

  18. 19 CFR 10.879 - Indirect materials.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 19 Customs Duties 1 2012-04-01 2012-04-01 false Indirect materials. 10.879 Section 10.879 Customs... of Origin § 10.879 Indirect materials. Indirect materials are to be disregarded in determining..., except that the cost of such indirect materials may be included in meeting the value-content...

  19. 19 CFR 10.816 - Indirect materials.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 19 Customs Duties 1 2012-04-01 2012-04-01 false Indirect materials. 10.816 Section 10.816 Customs... Rules of Origin § 10.816 Indirect materials. Indirect materials are to be disregarded in determining..., except that the cost of such indirect materials may be included in meeting the value-content...

  20. 19 CFR 10.816 - Indirect materials.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 19 Customs Duties 1 2014-04-01 2014-04-01 false Indirect materials. 10.816 Section 10.816 Customs... Rules of Origin § 10.816 Indirect materials. Indirect materials are to be disregarded in determining..., except that the cost of such indirect materials may be included in meeting the value-content...

  1. 19 CFR 10.879 - Indirect materials.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 1 2011-04-01 2011-04-01 false Indirect materials. 10.879 Section 10.879 Customs... of Origin § 10.879 Indirect materials. Indirect materials are to be disregarded in determining..., except that the cost of such indirect materials may be included in meeting the value-content...

  2. 19 CFR 10.816 - Indirect materials.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 1 2011-04-01 2011-04-01 false Indirect materials. 10.816 Section 10.816 Customs... Rules of Origin § 10.816 Indirect materials. Indirect materials are to be disregarded in determining..., except that the cost of such indirect materials may be included in meeting the value-content...

  3. 19 CFR 10.776 - Indirect materials.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 19 Customs Duties 1 2013-04-01 2013-04-01 false Indirect materials. 10.776 Section 10.776 Customs... Rules of Origin § 10.776 Indirect materials. Indirect materials are to be disregarded in determining..., except that the cost of such indirect materials may be included in meeting the value-content...

  4. 19 CFR 10.879 - Indirect materials.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 19 Customs Duties 1 2014-04-01 2014-04-01 false Indirect materials. 10.879 Section 10.879 Customs... of Origin § 10.879 Indirect materials. Indirect materials are to be disregarded in determining..., except that the cost of such indirect materials may be included in meeting the value-content...

  5. 19 CFR 10.776 - Indirect materials.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 1 2010-04-01 2010-04-01 false Indirect materials. 10.776 Section 10.776 Customs... Rules of Origin § 10.776 Indirect materials. Indirect materials are to be disregarded in determining..., except that the cost of such indirect materials may be included in meeting the value-content...

  6. 19 CFR 10.776 - Indirect materials.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 19 Customs Duties 1 2014-04-01 2014-04-01 false Indirect materials. 10.776 Section 10.776 Customs... Rules of Origin § 10.776 Indirect materials. Indirect materials are to be disregarded in determining..., except that the cost of such indirect materials may be included in meeting the value-content...

  7. 19 CFR 10.776 - Indirect materials.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 1 2011-04-01 2011-04-01 false Indirect materials. 10.776 Section 10.776 Customs... Rules of Origin § 10.776 Indirect materials. Indirect materials are to be disregarded in determining..., except that the cost of such indirect materials may be included in meeting the value-content...

  8. 19 CFR 10.816 - Indirect materials.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 1 2010-04-01 2010-04-01 false Indirect materials. 10.816 Section 10.816 Customs... Rules of Origin § 10.816 Indirect materials. Indirect materials are to be disregarded in determining..., except that the cost of such indirect materials may be included in meeting the value-content...

  9. The immunofluorescent profile of dermatomyositis: a comparative study with lupus erythematosus.

    PubMed

    Magro, C M; Crowson, A N

    1997-10-01

    We have demonstrated a role for microvascular injury mediated by the membrane attack complex of complement (C5b-9) in the genesis of cutaneous lesions of dermatomyositis (DM) (1). The purpose of this study is to revisit the immunofluorescent (IF) profile of DM, to further investigate the role of C5b-9 in the pathogenesis of cutaneous lesions, and to see if any features of the IF profile reliably distinguish DM from LE. Lesional skin biopsies from 24 patients with clinical findings characteristic of DM were received in formalin and in Michel's transport medium. Conventional light microscopy, and IF studies with antibodies monospecific for IgG, IgA, IgM, C3, fibrin and C5b-9 were performed. The control group comprised biopsies from 31 patients with well-documented LE. A positive lupus band test (LBT) correlated highly with a diagnosis of LE, with a sensitivity of 64.5% and a specificity of 95.6% (p=0.001). The LBT was most sensitive in the setting of DLE and SLE and was least sensitive in the setting of SCLE. The finding of vascular C5b-9 deposition correlated with a diagnosis of DM versus LE (p=0.001) although the false positive rate was 21.4%. The false negative rate was reduced when vascular C5b-9 was seen in the absence of antibodies to Ro, La, or RNP. While a negative LBT correlated with a diagnosis of DM (p=0.001), the specificity was only 64.5%. However, when it was seen in concert with C5b-9 along the DEJ, specificity was increased to 80.6% (p=0.001). The presence of C5b-9 in vessels and along the DEJ in concert with a negative LBT was predictive of DM (p=0.001) with a specificity of 93.5%, sensitivity of 78.3%, a false positive rate of 10% and a false negative rate of 14.7%. The combination of a negative LBT, vascular C5b-9 deposition and negative serology for Ro, La, and RNP was a predictor of DM versus LE with a sensitivity of 90.5%, a specificity of 96.8%, a false positive rate of 5% and a false negative rate of 6.2% (p=0.001). The IF profile of DM in

  10. Antinuclear antibody prevalence in a general pediatric cohort from Mexico City: discordance between immunofluorescence and multiplex assays

    PubMed Central

    Somers, Emily C; Monrad, Seetha U; Warren, Jeffrey S; Solano, Maritsa; Schnaas, Lourdes; Hernandez-Avila, Mauricio; Tellez-Rojo, Martha Maria; Hu, Howard

    2017-01-01

    Objective To characterize antinuclear antibody (ANA) prevalence according to distinct assay methodologies in a pediatric cohort from Mexico City, and to further examine associations with age and sex. Methods Serum ANA were measured by indirect immunofluorescence assay (IFA) and multiplex immunoassay in 114 children aged 9–17 years. IFA was considered positive at a cutoff titer of ≥1:80. Agreement between assay methods was assessed by kappa statistic. Sensitivity, specificity, and 95% confidence intervals (CIs) of the multiplex were computed with IFA as the reference standard. Results Of the 114 children (mean age 14.7 [standard deviation 2.1] years; 54 [47%] female), 18 of 114 (15.8%) were ANA positive by IFA, and 11 of 114 (9.6%) by 11-antigen multiplex assay. ANA prevalence was higher in females compared with males by both of the methods (ratios 1.6–1.9 to 1). Agreement between tests was classified as slight by kappa (κ=0.177 [95% CI −0.051, 0.406]). The multiplex immunoassay had sensitivity of 22.2% (95% CI 6.4, 47.6) and specificity of 92.7% (95% CI 85.6, 97.0), and failed to capture 3 of 4 (75%) of the high-titer (≥1:1280) IFA-positives. Conclusion Up to 15% of children in this general population cohort were ANA positive, with a higher rate of positivity among females according to both assay methods. Substantial discordance in ANA results was found between IFA and multiplex methods, even for high-titer IFA positives. These findings underscore the need to sufficiently account for assay characteristics when interpreting ANA test results, and support IFA as the more appropriate assay for studies of subclinical autoimmunity. PMID:28053555

  11. Presence of enolase in the M-band of skeletal muscle and possible indirect interaction with the cytosolic muscle isoform of creatine kinase.

    PubMed Central

    Foucault, G; Vacher, M; Merkulova, T; Keller, A; Arrio-Dupont, M

    1999-01-01

    Glycerol-skinned skeletal muscle fibres retain the defined sarcomeric structure of the myofibrils. We show here that a small fraction of two enzymes important for energy metabolism, the cytosolic muscle isoform of creatine kinase (EC 2.7.3.2), MM-creatine kinase (MM-CK), and enolase (EC 4.2.1.11), remains bound to skinned fibres. CK is slowly exchangeable, whereas enolase is firmly bound. Two-dimensional gel electrophoresis followed by Western blot analyses demonstrates that both alpha (ubiquitous) and beta (muscle-specific) subunits of enolase are present in these preparations. Enolase and CK were co-localized at the M-band of the sarcomeres, as observed by indirect immunofluorescence and confocal microscopy. Cross-linking experiments were performed on skinned fibres with three bifunctional succinimidyl esters of different lengths and yielded a protein complex of 150 kDa that reacted with antibodies directed against either M-CK or beta-enolase. The cross-linking efficiency was greatest for the longest reagent and zero for the shortest one. The length of the cross-linker giving a covalent complex between the two enzymes does not support the notion of a direct interaction between M-CK and enolase. This is the first demonstration of the presence of an enzyme of energy metabolism other than CK at the M-band of myofibres. PMID:9931306

  12. Comparison of two immunochromatographic assays and the indirect immunofluorescence antibody test for diagnosis of Trypanosoma cruzi infection in dogs in south central Louisiana.

    PubMed

    Nieto, Prixia D; Boughton, Roger; Dorn, Patricia L; Steurer, Frank; Raychaudhuri, Syamal; Esfandiari, Javan; Gonçalves, Edson; Diaz, James; Malone, John B

    2009-11-12

    Two rapid tests evaluated in dogs considered to be of high risk of infection with the Chagas parasite Trypanosoma cruzi using two immunochromatographic assays: Trypanosoma Detect for canine, InBios, Seattle, WA and CHAGAS STAT-PAK assay, Chembio Diagnostic Systems, Medford, NY, in south central Louisiana. For this purpose a serological survey was carried out in a total of 122 dogs and a serum bank was created. These 122 animals were first tested by IFAT that was used as the standard test. From the serum bank 50 samples were tested using the two rapid Chagas assays and results compared to the standard test IFAT. The serological survey using IFAT showed a prevalence of T. cruzi infection in 22.1% of the tested dogs. In the immunochromatographic assays, 13 and 11 animals were positive on rapid assay: Trypanosoma Detect for canine, InBios and CHAGAS STAT-PAK, Chembio Diagnostic Systems, respectively compared to 11 positive by IFAT. These two immunochromatographic tests have shown high susceptibility and specificity compared to our standard method IFAT. The rapid, easy and accurate screening assays used in conjunction with confirmatory tests, would be an excellent tool for veterinarians to diagnose T. cruzi infection. Early detection of T. cruzi infection may prevent complications through an effective treatment. Greater awareness by veterinarians of the risk, clinical findings, history along with diagnostic methods will contribute greatly to an understanding of the true prevalence of Chagas disease in dogs in Louisiana.

  13. Evaluation of indirect immunofluorescence antibody test and enzyme-linked immunosorbent assay for the diagnosis of infection by Leishmania infantum in clinically normal and sick cats.

    PubMed

    Chatzis, Manolis K; Leontides, Leonidas; Athanasiou, Labrini V; Papadopoulos, Elias; Kasabalis, Dimitrios; Mylonakis, Mathios; Rallis, Timoleon; Koutinas, Alexandros F; Andreadou, Margarita; Ikonomopoulos, John; Saridomichelakis, Manolis N

    2014-12-01

    Cats that live in areas where canine and human leishmaniosis due to Leishmania infantum is endemic may become infected and may develop anti-Leishmania antibodies. In this study 50 clinically normal and 50 cats with cutaneous and/or systemic signs that lived in an endemic area and had been previously examined for infection by L. infantum using PCR in four different tissues were serologically tested for the presence of anti-Leishmania IgG (IFAT and ELISA) and IgM (IFAT). The aim was to compare the results of IFAT, ELISA and PCR and to investigate the possible associations between seropositivity to Leishmania spp and signalment, living conditions, season of sampling, health status of the cats, and seropositivity to other infectious agents. Low concentrations of anti-Leishmania IgG were detected by IFAT in 10% of the cats and by ELISA in 1%, whereas anti-Leishmania IgM were detected by IFAT in 1%. There was disagreement between the results of IFAT and ELISA for anti-Leishmania IgG (P = 0.039) and between all serological tests and PCR (P < 0.001). The diagnostic sensitivity of all serological tests, using PCR as the gold standard, was very low, but ELISA and IFAT for anti-Leishmania IgM had 100% specificity. The diagnostic sensitivity of all serological tests could not be improved by changing the cut-off values. Seropositivity for Leishmania spp was not associated with signalment, living conditions, season of sampling and health status of the cats or with seropositivity to feline leukemia virus, feline immunodeficiency virus, feline coronavirus, Toxoplasma gondii and Bartonella henselae. In conclusion, because of their low sensitivity and very high specificity two of the evaluated serological tests (ELISA for anti-Leishmania IgG and IFAT for anti-Leishmania IgM) may be useless as population screening tests but valuable for diagnosing feline infection by L. infantum. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. The burden of the variability introduced by the HEp-2 assay kit and the CAD system in ANA indirect immunofluorescence test.

    PubMed

    Infantino, M; Meacci, F; Grossi, V; Manfredi, M; Benucci, M; Merone, M; Soda, P

    2017-02-01

    According to the recent recommendations of the American College of Rheumatology, ANA Task Force, IIF technique should be considered the gold standard in antinuclear antibodies (ANAs) testing. To overcome the lack of standardization, biomedical industries have developed several computer-aided diagnosis (CAD) systems. Two hundred and sixty-one consecutive samples with suspected autoimmune diseases were tested for ANA by means of IIF on routinely HEp-2 assay kit (Euroimmun AG). Assignment of result was made if consensus for positive/negative was reached by at least 2 out of 3 expert physicians. ANA-IIF was also carried out using 3 CAD systems: Zenit G-Sight (n = 84), Helios (n = 85) and NOVA View (n = 92); human evaluation was repeated on the same substrate of each CAD system (Immco, Aesku and Inova HEp-2 cells, respectively). To anonymize the results, we randomly named these three systems as A, B and C. We ran a statistical analysis computing several measures of agreement between the ratings, and we also improved the evaluation by using the Wilcoxon's test for nonparametric data. Agreement between the human readings on routinely HEp-2 assay kit and human readings on CAD HEp-2 assay was substantial for A (k = 0.82) and B (k = 0.72), and almost perfect for C (k = 0.89). Such readings were statistically different only in case A. Comparing experts' readings with the readings of CAD systems, when the samples were prepared using CAD HEp-2 assay kits, we found almost perfect agreement for B and C (k = 0.86; k = 0.82) and substantial agreement for A (k = 0.73). Again, human and CAD readings were statistically different only in A. When we compared the readings of medical experts on routinely HEp-2 assay kit with the output of the CAD systems that worked using their own slides, we found substantial agreement for all the systems (A: k = 0.62; B: k = 0.65; C: k = 0.71). Such readings were not statistically different. The change of the assay kit and/or the introduction of a CAD system affect the laboratory reporting, with an evident impact on the autoimmune laboratory workflow. The CAD systems may represent one of the most important novel elements of harmonization in the autoimmunity field, reducing intra- and inter-laboratory variability in a new vision of the diagnostic autoimmune platform.

  15. Comparison of direct and indirect methods of measuring airborne chrysotile fibre concentration.

    PubMed

    Eypert-Blaison, Celine; Veissiere, Sylvie; Rastoix, Olivier; Kauffer, Edmond

    2010-01-01

    Transmission electron microscopy observations most frequently form a basis for estimating asbestos fibre concentration in the environment and in buildings with asbestos-containing materials. Sampled fibres can be transferred to microscope grids by applying either a direct [ISO (1995) Draft International ISO/DIS 10312. Ambient air. Determination of asbestos fibres. Direct transfer transmission electron microscopy procedure. Geneva, Switzerland: International Standardization Organization] or an indirect [AFNOR (1996) Détermination de la concentration en fibres d'amiante par microscopie électronique à transmission-Méthode indirecte. Cedex, France: AFNOR, p. 42; ISO (1997) Draft International ISO/DIS 13794. Ambient air. Determination of asbestos fibres. Indirect-transfer transmission electron microscopy procedure. Geneva, Switzerland: International Standardization Organization] method. In the latter case, ISO Standard 13794 recommends filtering calcination residues either on a polycarbonate (PC) filter (PC indirect method) or on a cellulose ester (CE) membrane (CE indirect method). The PC indirect method requires that fibres deposited on a PC filter be covered by a carbon layer, whereas in the CE indirect method, the CE membrane has to be directly processed using a method described in ISO Standard 10312. The purpose of this study was to compare results obtained using, on the one hand, direct preparation methods and, on the other hand, PC indirect or CE indirect methods, for counting asbestos fibres deposited on filters as a result of liquid filtration or air sampling. In direct method-based preparation, we observed that an etching time of 6-14 min does not affect the measured densities, except for fibres <1 microm deposited by liquid filtration. Moreover, in all cases, the direct method gives higher densities than the PC indirect method because of possible fibre disappearance when using the carbon evaporator implemented in the PC indirect method. The CE membrane

  16. Immunofluorescence staining for the detection of immunoglobulins and complement (C3) in dogs with renal disease.

    PubMed

    Aresu, L; Pregel, P; Bollo, E; Palmerini, D; Sereno, A; Valenza, F

    2008-12-06

    Renal cortical biopsies from 74 dogs with different degrees of renal failure were studied by immunofluorescence to assess the frequency and extent of the deposition of immunoglobulins G, M and A (IgG, IgM, IgA) and complement C3. The dogs were divided into two groups on the basis of their clinical signs, and standard histological and electron microscopical examinations, according to whether their disease was an immune-mediated nephropathy (IMN) or a non-immune-mediated nephropathy (NIMN). In the dogs with an imn there was strong immunofluorescence due to IgG in the mesangium and the glomerular basement membrane and to IgM in the mesangium. The mechanism of immune complex trapping in the glomerulus also resulted in positive reactions to IgM in the dogs with an NIMN.

  17. Polymyalgia rheumatica (PMR): clinical, laboratory, and immunofluorescence studies in 13 patients.

    PubMed

    Shintani, Shuzo; Shiigai, Tatsuo; Matsui, Yoshiki

    2002-01-01

    Thirteen elderly patients with polymyalgia rheumatica (PMR) are presented. The clinical and laboratory findings suggest that many progressive symptoms are due to the non-specific inflammatory changes in various organs of the body, especially in muscles and joints. An immunofluorescence study of muscle biopsy specimens revealed IgG, IgA, and fibrinogen deposits in the perifascicular area of the perimysium. This finding suggests that immune complexes play a role in the pathogenesis of this condition and that the pathophysiology of PMR is an interstitial inflammatory process. We think that the inflammatory findings affecting the interstitial tissue of muscles in the immunofluorescence study are relatively specific to PMR, and will be affected by steroid treatment.

  18. Microwave oven-based technique for immunofluorescent staining of paraffin-embedded tissues

    PubMed Central

    Buggs, Colleen

    2011-01-01

    Immunohistochemical analysis of formalin-fixed paraffin-embedded tissues can be challenging due to potential modifications of protein structure by exposure to formalin. Heat-induced antigen retrieval techniques can reverse reactions between formalin and proteins that block antibody recognition. Interactions between antibodies and antigens are further enhanced by microwave irradiation, which has simplified immunohistochemical staining protocols. In this report, we modify a technique for antigen retrieval and immunofluorescent staining of formalin-fixed paraffin-embedded tissues by showing that it works well with several antibodies and buffers. This microwave-assisted method for antigen retrieval and immunofluorescent staining eliminates the need for blocking reagents and extended washes, which greatly simplifies the protocol allowing one to complete the analysis in less than 3 h. PMID:17653827

  19. Microwave oven-based technique for immunofluorescent staining of paraffin-embedded tissues.

    PubMed

    Long, Delwin J; Buggs, Colleen

    2008-02-01

    Immunohistochemical analysis of formalin-fixed paraffin-embedded tissues can be challenging due to potential modifications of protein structure by exposure to formalin. Heat-induced antigen retrieval techniques can reverse reactions between formalin and proteins that block antibody recognition. Interactions between antibodies and antigens are further enhanced by microwave irradiation, which has simplified immunohistochemical staining protocols. In this report, we modify a technique for antigen retrieval and immunofluorescent staining of formalin-fixed paraffin-embedded tissues by showing that it works well with several antibodies and buffers. This microwave-assisted method for antigen retrieval and immunofluorescent staining eliminates the need for blocking reagents and extended washes, which greatly simplifies the protocol allowing one to complete the analysis in less than 3 h.

  20. Concerning the localization of steroids in centrioles and basal bodies by immunofluorescence

    PubMed Central

    Nenci, I.; Marchetti, E.

    1978-01-01

    Specific steroid antibodies, by the immunofluorescence technique, regularly reveal fluorescent centrioles and cilia-bearing basal bodies in target and nontarget cells. Although the precise identity of the immunoreactive steroid substance has not yet been established, it seems noteworthy that exogenous steroids can be vitally concentrated by centrioles, perhaps by exchange with steroids already present at this level. This unexpected localization suggests that steroids may affect cell growth and differentiation in some way different from the two-step receptor mechanism. PMID:10605436

  1. [Immunofluorescent-histologic localization of a placenta-antigen with antibodies from pregnancy blood (author's transl)].

    PubMed

    Skrabei, H; Meyer-Falk, E; Mettler, L

    1977-08-01

    Placenta-specific antibodies were isolated by the incubation of washed placental tissue with pregancy serum and then desorption with citric acid pH3. The antigen responsible for the formation of the antibody could be localized via the immunofluorescence technique together with application of the placenta antibody in the syncytiotrophoblast layer. The antibodies are not complement-fixing and belong to the IgG class.

  2. Fibrinogen Demonstration in Oral Lichen Planus: An Immunofluorescence Study on Archival Tissues.

    PubMed

    Shirol, Pallavi D; Naik, Veena; Kale, Alka

    2015-10-01

    Lichen planus is a premalignant condition with minimal diagnostic aids. This study is an attempt to use paraffin embedded sections of lichen planus with immunofluorescein stain and to evaluate the immunofluorescent sections to establish pattern of fibrinogen deposition. Thirty-five paraffin embedded sections of old and new cases of oral lichen planus (study group) and five normal oral mucosa (control group) were chosen. Two sections of each (H & E) case were taken, one was stained with hematoxylin and eosin and another with fluorescein isothiocynate conjugate (FITC) polyclonal rabbit antibody against fibrinogen. Fluorescent findings were examined with a fluorescent microscope. A high statistical significant correlation was found in respect to fluorescence positivity, intensity of fluorescence and distribution of fluorescence each with p < 0.0001 and fluorescence at blood vessel walls (p = 0.0003). This study suggested that paraffin embedded sections can be successfully used in direct immunofluorescence staining in routine set up where only formalin fixed tissues are received. Paraffin embedded sections can be successfully used in direct immunofluorescence staining when only formalin fixed tissues are received.

  3. Analysis on near field scattering spectra around nanoparticles by using parametric indirect microscopic imaging

    NASA Astrophysics Data System (ADS)

    Guoyan, Liu; Kun, Gao; Xuefeng, Liu; Guoqiang, Ni

    2016-10-01

    We report the simulation and measurement results of near field spatial scattering spectra around nanoparticles. Our measurement and simulations results have indicated that Parametric Indirect Microscopic Imaging can image the near field spatial scattering to a much larger distance from the scattering source of the particle under measurement whereas this part of spatial scattering was lost in the conventional microscopy. Both FDTD modeling and measurement provided evidence that parameters of indirect optical wave vector have higher sensitivity to near field scattering.

  4. 7 CFR 2903.4 - Indirect costs.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AGRICULTURE BIODIESEL FUEL EDUCATION PROGRAM General Information § 2903.4 Indirect costs. (a) For the Biodiesel Fuel Education Program, applicants should use the current indirect cost rate negotiated with...

  5. 7 CFR 2903.4 - Indirect costs.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AGRICULTURE BIODIESEL FUEL EDUCATION PROGRAM General Information § 2903.4 Indirect costs. (a) For the Biodiesel Fuel Education Program, applicants should use the current indirect cost rate negotiated with the...

  6. Bosonic cascades of indirect excitons

    NASA Astrophysics Data System (ADS)

    Nalitov, A. V.; De Liberato, S.; Lagoudakis, P.; Savvidis, P. G.; Kavokin, A. V.

    2017-08-01

    Recently, the concept of the terahertz bosonic cascade laser (BCL) based on a parabolic quantum well (PQW) embedded in a microcavity was proposed. We refine this proposal by suggesting transitions between indirect exciton (IX) states as a source of terahertz emission. We explicitly propose a structure containing a narrow-square QW and a wide-parabolic QW for the realisation of a bosonic cascade. Advantages of this type of structures are in large dipole matrix elements for terahertz transitions and in long exciton radiative lifetimes which are crucial for realisation of threshold and quantum efficiency BCLs.

  7. High-resolution imaging by scanning electron microscopy of semithin sections in correlation with light microscopy.

    PubMed

    Koga, Daisuke; Kusumi, Satoshi; Shodo, Ryusuke; Dan, Yukari; Ushiki, Tatsuo

    2015-12-01

    In this study, we introduce scanning electron microscopy (SEM) of semithin resin sections. In this technique, semithin sections were adhered on glass slides, stained with both uranyl acetate and lead citrate, and observed with a backscattered electron detector at a low accelerating voltage. As the specimens are stained in the same manner as conventional transmission electron microscopy (TEM), the contrast of SEM images of semithin sections was similar to TEM images of ultrathin sections. Using this technique, wide areas of semithin sections were also observed by SEM, without the obstruction of grids, which was inevitable for traditional TEM. This study also applied semithin section SEM to correlative light and electron microscopy. Correlative immunofluorescence microscopy and immune-SEM were performed in semithin sections of LR white resin-embedded specimens using a FluoroNanogold-labeled secondary antibody. Because LR white resin is hydrophilic and electron stable, this resin is suitable for immunostaining and SEM observation. Using correlative microscopy, the precise localization of the primary antibody was demonstrated by fluorescence microscopy and SEM. This method has great potential for studies examining the precise localization of molecules, including Golgi- and ER-associated proteins, in correlation with LM and SEM.

  8. 19 CFR 10.460 - Indirect materials.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 19 Customs Duties 1 2012-04-01 2012-04-01 false Indirect materials. 10.460 Section 10.460 Customs... of Origin § 10.460 Indirect materials. An indirect material, as defined in § 10.402(o), will be considered to be an originating material without regard to where it is produced. Example. Chilean Producer...

  9. 19 CFR 10.603 - Indirect materials.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 19 Customs Duties 1 2012-04-01 2012-04-01 false Indirect materials. 10.603 Section 10.603 Customs... States Free Trade Agreement Rules of Origin § 10.603 Indirect materials. An indirect material, as defined in § 10.582(m) of this subpart, will be considered to be an originating material without regard...

  10. 19 CFR 10.460 - Indirect materials.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 19 Customs Duties 1 2013-04-01 2013-04-01 false Indirect materials. 10.460 Section 10.460 Customs... of Origin § 10.460 Indirect materials. An indirect material, as defined in § 10.402(o), will be considered to be an originating material without regard to where it is produced. Example. Chilean Producer...

  11. 19 CFR 10.460 - Indirect materials.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 1 2011-04-01 2011-04-01 false Indirect materials. 10.460 Section 10.460 Customs... of Origin § 10.460 Indirect materials. An indirect material, as defined in § 10.402(o), will be considered to be an originating material without regard to where it is produced. Example. Chilean Producer...

  12. 19 CFR 10.2024 - Indirect materials.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 19 Customs Duties 1 2014-04-01 2014-04-01 false Indirect materials. 10.2024 Section 10.2024... Agreement Rules of Origin § 10.2024 Indirect materials. An indirect material, as defined in § 10.2013(i), will be considered to be an originating material without regard to where it is produced....

  13. 19 CFR 10.541 - Indirect materials.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 1 2010-04-01 2010-04-01 false Indirect materials. 10.541 Section 10.541 Customs... Rules of Origin § 10.541 Indirect materials. An indirect material, as defined in § 10.502(j) of this subpart, will be considered to be an originating material without regard to where it is produced, and...

  14. 19 CFR 10.541 - Indirect materials.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 1 2011-04-01 2011-04-01 false Indirect materials. 10.541 Section 10.541 Customs... Rules of Origin § 10.541 Indirect materials. An indirect material, as defined in § 10.502(j) of this subpart, will be considered to be an originating material without regard to where it is produced, and...

  15. 19 CFR 10.541 - Indirect materials.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 19 Customs Duties 1 2014-04-01 2014-04-01 false Indirect materials. 10.541 Section 10.541 Customs... Rules of Origin § 10.541 Indirect materials. An indirect material, as defined in § 10.502(j) of this subpart, will be considered to be an originating material without regard to where it is produced, and...

  16. 19 CFR 10.541 - Indirect materials.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 19 Customs Duties 1 2013-04-01 2013-04-01 false Indirect materials. 10.541 Section 10.541 Customs... Rules of Origin § 10.541 Indirect materials. An indirect material, as defined in § 10.502(j) of this subpart, will be considered to be an originating material without regard to where it is produced, and...

  17. 19 CFR 10.1024 - Indirect materials.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 19 Customs Duties 1 2014-04-01 2014-04-01 false Indirect materials. 10.1024 Section 10.1024... Agreement Rules of Origin § 10.1024 Indirect materials. An indirect material, as defined in § 10.1002(n) of.... Korean Producer A produces good C using non-originating material B. Producer A imports...

  18. 19 CFR 10.603 - Indirect materials.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 19 Customs Duties 1 2013-04-01 2013-04-01 false Indirect materials. 10.603 Section 10.603 Customs... States Free Trade Agreement Rules of Origin § 10.603 Indirect materials. An indirect material, as defined in § 10.582(m) of this subpart, will be considered to be an originating material without regard...

  19. 19 CFR 10.924 - Indirect materials.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 19 Customs Duties 1 2014-04-01 2014-04-01 false Indirect materials. 10.924 Section 10.924 Customs... Rules of Origin § 10.924 Indirect materials. An indirect material, as defined in § 10.902(m) of this subpart, will be considered to be an originating material without regard to where it is produced....

  20. 19 CFR 10.3024 - Indirect materials.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 19 Customs Duties 1 2014-04-01 2014-04-01 false Indirect materials. 10.3024 Section 10.3024... Promotion Agreement Rules of Origin § 10.3024 Indirect materials. An indirect material, as defined in § 10.3013(h), will be considered to be an originating material without regard to where it is...

  1. 19 CFR 10.603 - Indirect materials.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 1 2010-04-01 2010-04-01 false Indirect materials. 10.603 Section 10.603 Customs... States Free Trade Agreement Rules of Origin § 10.603 Indirect materials. An indirect material, as defined in § 10.582(m) of this subpart, will be considered to be an originating material without regard...

  2. 19 CFR 10.603 - Indirect materials.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 19 Customs Duties 1 2014-04-01 2014-04-01 false Indirect materials. 10.603 Section 10.603 Customs... States Free Trade Agreement Rules of Origin § 10.603 Indirect materials. An indirect material, as defined in § 10.582(m) of this subpart, will be considered to be an originating material without regard...

  3. 19 CFR 10.460 - Indirect materials.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 19 Customs Duties 1 2014-04-01 2014-04-01 false Indirect materials. 10.460 Section 10.460 Customs... of Origin § 10.460 Indirect materials. An indirect material, as defined in § 10.402(o), will be considered to be an originating material without regard to where it is produced. Example. Chilean Producer...

  4. 19 CFR 10.603 - Indirect materials.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 1 2011-04-01 2011-04-01 false Indirect materials. 10.603 Section 10.603 Customs... States Free Trade Agreement Rules of Origin § 10.603 Indirect materials. An indirect material, as defined in § 10.582(m) of this subpart, will be considered to be an originating material without regard...

  5. 46 CFR 154.1720 - Indirect refrigeration.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 5 2011-10-01 2011-10-01 false Indirect refrigeration. 154.1720 Section 154.1720... § 154.1720 Indirect refrigeration. A refrigeration system that is used to cool acetaldehyde, ethylene oxide, or methyl bromide, must be an indirect refrigeration system that does not use vapor compression....

  6. 46 CFR 154.1720 - Indirect refrigeration.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 5 2010-10-01 2010-10-01 false Indirect refrigeration. 154.1720 Section 154.1720... § 154.1720 Indirect refrigeration. A refrigeration system that is used to cool acetaldehyde, ethylene oxide, or methyl bromide, must be an indirect refrigeration system that does not use vapor compression....

  7. 46 CFR 154.1720 - Indirect refrigeration.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 5 2013-10-01 2013-10-01 false Indirect refrigeration. 154.1720 Section 154.1720... § 154.1720 Indirect refrigeration. A refrigeration system that is used to cool acetaldehyde, ethylene oxide, or methyl bromide, must be an indirect refrigeration system that does not use vapor compression....

  8. 46 CFR 154.1720 - Indirect refrigeration.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 5 2012-10-01 2012-10-01 false Indirect refrigeration. 154.1720 Section 154.1720... § 154.1720 Indirect refrigeration. A refrigeration system that is used to cool acetaldehyde, ethylene oxide, or methyl bromide, must be an indirect refrigeration system that does not use vapor compression....

  9. 46 CFR 154.1720 - Indirect refrigeration.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 5 2014-10-01 2014-10-01 false Indirect refrigeration. 154.1720 Section 154.1720... § 154.1720 Indirect refrigeration. A refrigeration system that is used to cool acetaldehyde, ethylene oxide, or methyl bromide, must be an indirect refrigeration system that does not use vapor compression....

  10. 7 CFR 3430.54 - Indirect costs.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 15 2011-01-01 2011-01-01 false Indirect costs. 3430.54 Section 3430.54 Agriculture... Post-Award and Closeout § 3430.54 Indirect costs. Indirect cost rates for grants and cooperative agreements shall be determined in accordance with the applicable assistance regulations and cost...

  11. 48 CFR 31.203 - Indirect costs.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 1 2011-10-01 2011-10-01 false Indirect costs. 31.203... REQUIREMENTS CONTRACT COST PRINCIPLES AND PROCEDURES Contracts With Commercial Organizations 31.203 Indirect costs. (a) For contracts subject to full CAS coverage, allocation of indirect costs shall be based...

  12. 7 CFR 2903.4 - Indirect costs.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AGRICULTURE BIODIESEL FUEL EDUCATION PROGRAM General Information § 2903.4 Indirect costs. (a) For the Biodiesel Fuel Education Program, applicants should use the current indirect cost rate negotiated with the... funds. Grantees electing this alternative will not be allowed to charge, as direct costs, indirect...

  13. 7 CFR 2903.4 - Indirect costs.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AGRICULTURE BIODIESEL FUEL EDUCATION PROGRAM General Information § 2903.4 Indirect costs. (a) For the Biodiesel Fuel Education Program, applicants should use the current indirect cost rate negotiated with the... funds. Grantees electing this alternative will not be allowed to charge, as direct costs, indirect...

  14. 7 CFR 2903.4 - Indirect costs.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AGRICULTURE BIODIESEL FUEL EDUCATION PROGRAM General Information § 2903.4 Indirect costs. (a) For the Biodiesel Fuel Education Program, applicants should use the current indirect cost rate negotiated with the... funds. Grantees electing this alternative will not be allowed to charge, as direct costs, indirect...

  15. Multilabel immunofluorescence and antigen reprobing on formalin-fixed paraffin-embedded sections: novel applications for precision pathology diagnosis.

    PubMed

    Pan, Jie; Thoeni, Cornelia; Muise, Aleixo; Yeger, Herman; Cutz, Ernest

    2016-06-01

    We report new methods for multilabel immunofluorescence (MIF) and reprobing of antigen epitopes on the same formalin-fixed paraffin-embedded (FFPE) sections. The MIF method includes an antigen-retrieval step followed by multilabel immunostaining and examination by confocal microscopy. As examples, we illustrate epitopes localized to the apical and basolateral membranes, and the cytoplasm of enterocytes of normal small intestine and in cases of congenital enteropathies (microvillous inclusion disease and congenital tufting enteropathy). We also demonstrate localization of the bile salt excretion pump protein (BSEP) in bile canalicular membrane of normal hepatocytes and in cases of primary sclerosing cholangitis. To demonstrate colocalization of cytoplasmic and nuclear epitopes we analyzed normal control and hyperplastic pulmonary neuroendocrine cells (PNEC) and neuroepithelial bodies (NEBs), presumed airway sensors in the lungs of infants with bronchopulmonary dysplasia (BPD). As cytoplasmic markers we used anti-bombesin or anti-synaptic vesicle protein 2 (SV2) antibody, respectively, and for nuclear localization, antibodies against neurogenic genes mammalian achaete-scute homolog (Mash1) and prospero homeobox 1 (Prox1), essential for NEB cells differentiation and maturation, hypoxia-inducible factor 1α (HIF1α) a downstream modulator of hypoxia response and a proliferation marker Ki67. The reprobing method consisted of removal of the previously immunolabeled target and immunostaining with different antibodies, facilitating colocalization of enterocyte brush border epitopes as well as HIF1α, Mash1 and Prox1 in PNEC/NEB PNEC and NEBs. As these methods are suitable for routine FFPE pathology samples from various tissues, allowing visualization of multiple epitopes in the same cells/sections with superior contrast and resolution, they are suitable for a wide range of applications in diagnostic pathology and may be particularly well suited for precision medicine

  16. Direct immunofluorescence of the outer root sheath in anagen and telogen hair in pemphigus vulgaris and pemphigus foliaceus.

    PubMed

    Tanasilovic, Srdjan; Medenica, Ljiljana; Popadic, Svetlana

    2014-11-01

    Direct immunofluorescence of peri-lesional skin is the gold standard in the diagnosis of pemphigus. A specific immunofluorescence pattern may also be demonstrated in the outer root sheath of anagen and telogen hair. We demonstrated an intercellular reticular deposition of immunoglobulin G in the outer root sheath of plucked anagen and telogen hair in all pemphigus vulgaris patients with active disease and for the first time in all patients with active pemphigus foliaceus. Moreover, we demonstrated for the first time that plucked hair samples may be kept at -20°C for at least 2 weeks before immunofluorescent staining and analysis.

  17. Indirect reciprocity with trinary reputations.

    PubMed

    Tanabe, Shoma; Suzuki, Hideyuki; Masuda, Naoki

    2013-01-21

    Indirect reciprocity is a reputation-based mechanism for cooperation in social dilemma situations when individuals do not repeatedly meet. The conditions under which cooperation based on indirect reciprocity occurs have been examined in great details. Most previous theoretical analysis assumed for mathematical tractability that an individual possesses a binary reputation value, i.e., good or bad, which depends on their past actions and other factors. However, in real situations, reputations of individuals may be multiple valued. Another puzzling discrepancy between the theory and experiments is the status of the so-called image scoring, in which cooperation and defection are judged to be good and bad, respectively, independent of other factors. Such an assessment rule is found in behavioral experiments, whereas it is known to be unstable in theory. In the present study, we fill both gaps by analyzing a trinary reputation model. By an exhaustive search, we identify all the cooperative and stable equilibria composed of a homogeneous population or a heterogeneous population containing two types of players. Some results derived for the trinary reputation model are direct extensions of those for the binary model. However, we find that the trinary model allows cooperation under image scoring under some mild conditions.

  18. Indirect costs of rheumatoid arthritis

    PubMed Central

    Raciborski, Filip; Kwiatkowska, Brygida

    2015-01-01

    It is estimated that in Poland about 400,000 persons in general suffer from inflammatory joint diseases, including rheumatoid arthritis (RA). Epidemiological surveys documenting the frequency and disturbance of musculoskeletal disorders in the Polish population are few in number. Most of the estimations are based on epidemiological data from other countries (prevalence of 0.5–1%). According to the data of the National Health Fund in Poland 135,000–157,000 persons in total are treated because of rheumatoid arthritis per year [ICD10 (International Statistical Classification of Diseases and Related Health Problems): M05, M06]. In the case of this group of diseases indirect costs significantly outweigh the direct costs. Indirect costs increase together with activity level of the disease. The cost analysis of productivity loss of RA patients indicates that sickness absenteeism and informal care are the most burdensome. At the national level it amounts in total from 1.2 billion to 2.8 billion PLN per year, depending on the method of analysis. These costs could be significantly reduced through early diagnosis and introduction of effective treatment. PMID:27407258

  19. Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence.

    PubMed

    Neubauer, Heidi A; Pitson, Stuart M

    2016-01-01

    Sphingosine kinase 2 (SK2) is a ubiquitously expressed lipid kinase that has important, albeit complex and poorly understood, roles in regulating cell survival and cell death. In addition to being able to promote cell cycle arrest and apoptosis under certain conditions, it has recently been shown that SK2 can promote neoplastic transformation and tumorigenesis in vivo. Therefore, well validated and reliable tools are required to study and better understand the true functions of SK2. Here, we compare two commercially available SK2 antibodies: a rabbit polyclonal antibody from Proteintech that recognizes amino acids 266-618 of human SK2a, and a rabbit polyclonal antibody from ECM Biosciences that recognizes amino acids 36-52 of human SK2a. We examine the performance of these antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence staining of endogenous SK2, using human HEK293 and HeLa cell lines, as well as mouse embryonic fibroblasts (MEFs). Furthermore, we assess the specificity of these antibodies to the target protein through the use of siRNA-mediated SK2 knockdown and SK2 knockout ( Sphk2(-/-)) MEFs. Our results demonstrate that the Proteintech anti-SK2 antibody reproducibly displayed superior sensitivity and selectivity towards SK2 in immunoblot analyses, while the ECM Biosciences anti-SK2 antibody was reproducibly superior for SK2 immunoprecipitation and detection by immunofluorescence staining. Notably, both antibodies produced non-specific bands and staining in the MEFs, which was not observed with the human cell lines. Therefore, we conclude that the Proteintech SK2 antibody is a valuable reagent for use in immunoblot analyses, and the ECM Biosciences SK2 antibody is a useful tool for SK2 immunoprecipitation and immunofluorescence staining, at least in the human cell lines employed in this study.

  20. Detection of Pituitary Antibodies by Immunofluorescence: Approach and Results in Patients With Pituitary Diseases

    PubMed Central

    Ricciuti, Adriana; De Remigis, Alessandra; Landek-Salgado, Melissa A.; De Vincentiis, Ludovica; Guaraldi, Federica; Lupi, Isabella; Iwama, Shintaro; Wand, Gary S.; Salvatori, Roberto

    2014-01-01

    Context: Pituitary antibodies have been measured mainly to identify patients whose disease is caused or sustained by pituitary-specific autoimmunity. Although reported in over 100 publications, they have yielded variable results and are thus considered of limited clinical utility. Objectives: Our objectives were to analyze all publications reporting pituitary antibodies by immunofluorescence for detecting the major sources of variability, to experimentally test these sources and devise an optimized immunofluorescence protocol, and to assess prevalence and significance of pituitary antibodies in patients with pituitary diseases. Study Design and Outcome Measures: We first evaluated the effect of pituitary gland species, section fixation, autofluorescence quenching, blockade of unwanted antibody binding, and use of purified IgG on the performance of this antibody assay. We then measured cross-sectionally the prevalence of pituitary antibodies in 390 pituitary cases and 60 healthy controls, expressing results as present or absent and according to the (granular, diffuse, perinuclear, or mixed) staining pattern. Results: Human pituitary was the best substrate to detect pituitary antibodies and yielded an optimal signal-to-noise ratio when treated with Sudan black B to reduce autofluorescence. Pituitary antibodies were more common in cases (95 of 390, 24%) than controls (3 of 60, 5%, P = .001) but did not discriminate among pituitary diseases when reported dichotomously. However, when expressed according to their cytosolic staining, a granular pattern was highly predictive of pituitary autoimmunity (P < .0001). Conclusion: We report a comprehensive study of pituitary antibodies by immunofluorescence and provide a method and an interpretation scheme that should be useful for identifying and monitoring patients with pituitary autoimmunity. PMID:24606106

  1. Optimized Protocol of Methanol Treatment for Immunofluorescent Staining in Fixed Brain Slices

    PubMed Central

    Yuan, Feng; Cohen, Noam A.; Cohen, Akiva S.

    2017-01-01

    We optimized methanol treatment in paraformaldehyde-fixed slices for immunofluorescent staining of ependymal basal bodies in brain ventricles. As 100% methanol induced severe deformations to the slices (including rolling and folding over), we tried to decrease methanol concentration. We found that 33.3% to 75% methanol could result in ideal immunostaining of basal bodies without inducing obvious deformations. Instead of treating slices at −20°C (without proper cryoprotection measurements) as suggested in previous studies, we carried out methanol treatment at room temperature. Our modified protocol can not only raise immunostaining efficiency in tissue slices, it may also prevent potential freezing damages to the samples. PMID:26509907

  2. Optimized Protocol of Methanol Treatment for Immunofluorescent Staining in Fixed Brain Slices.

    PubMed

    Yuan, Feng; Xiong, Guoxiang; Cohen, Noam A; Cohen, Akiva S

    2017-03-01

    We optimized methanol treatment in paraformaldehyde-fixed slices for immunofluorescent staining of ependymal basal bodies in brain ventricles. As 100% methanol induced severe deformations to the slices (including rolling and folding over), we tried to decrease methanol concentration. We found that 33.3% to 75% methanol could result in ideal immunostaining of basal bodies without inducing obvious deformations. Instead of treating slices at -20°C (without proper cryoprotection measurements) as suggested in previous studies, we carried out methanol treatment at room temperature. Our modified protocol can not only raise immunostaining efficiency in tissue slices, it may also prevent potential freezing damages to the samples.

  3. Diploid and tetraploid precursors of megakaryocytes in normal human bone marrow detected by immunofluorescence.

    PubMed

    Renner, D; Propp, H; Queisser, W

    1987-11-01

    A sequential preparation method is described which allows immunological identification, morphological characterization, cytophotometric determination of relative DNA content of the megakaryocyte lineage as well as quantitation of megakaryocyte precursors in human bone marrow aspirates. We compared several monoclonal (anti-GP IIIa and HD 19) and polyclonal (A225, RAHPS) antiplatelet antibodies for immunofluorescent staining. Among the identified cells, a small number of cells showing a diploid and tetraploid DNA content were found which must be regarded as promegakaryoblasts, representing 2.5-4.7% of all megakaryocytes. The heterogenous morphology of these precursors in panoptically stained smears is described.

  4. Validation of a monoclonal antibody-based immunofluorescent assay to detect Burkholderia pseudomallei in blood cultures

    PubMed Central

    Dulsuk, Adul; Paksanont, Suporn; Sangchankoom, Adisak; Ekchariyawat, Peeraya; Phunpang, Rungnapa; Jutrakul, Yaowaruk; West, T. Eoin

    2016-01-01

    Abstract Background Identification of Burkholderia pseudomallei, the cause of melioidosis, using routine methods takes several days. Use of a monoclonal antibody-based immunofluorescent assay (IFA) on positive blood cultures may speed diagnosis. Methods We tested the diagnostic accuracy of the IFA on 545 blood cultures positive for Gram-negative organisms at Udon Thani Hospital, Thailand, between June 2015 and August 2016. Results Sensitivity of the IFA was 100% and specificity was 99.6%. The median decrease in time to pathogen identification between the IFA result and routine methods was 28 h (IQR 25–51), p<0.0001. Conclusions The IFA accurately expedites the diagnosis of melioidosis. PMID:28115683

  5. Direct immunofluorescence of skin using formalin-fixed paraffin-embedded sections.

    PubMed Central

    Mera, S L; Young, E W; Bradfield, J W

    1980-01-01

    The technique of direct immunofluorescence has been applied to skin biopsy specimens fixed in formalin and embedded in paraffin wax. The results have been compared with those obtained by using snap-frozen biopsy specimens from the same patients. Trypsinisation of the dewaxed material allowed subsequent detection of immunoglobulins, complement, and fibrinogen. When compared to the fluorescence in the snap-frozen specimens, the staining in the paraffin sections was less bright and there was a higher rate of negatives. Even so, it was possible to establish the diagnosis in most cases of pemphigus, pemphigoid, and lupus erythematosus. Images Fig. 1 Fig. 2 Fig. 3 PMID:6995489

  6. Localization of mouse isoantigens on teh cell surface as revealed by immunofluorescence

    PubMed Central

    Cerottini, J.-C.; Brunner, K. T.

    1967-01-01

    An immunofluorescent technique is described which permits the localization and the semi-quantitative evaluation of isoantigens. Studies of normal and tumour cells have demonstrated that H-2 isoantigens are located in discrete areas on the cell surface. The thymus of 20–25-day-old mice was found to contain approximately 85 per cent lymphoid cells with a very low isoantigen content. These cells are considered to represent thymus cortical lymphoid cells. Cortisol treatment of mice reduced the relative number of cells with low isoantigen content to 10 per cent. ImagesFIG. 1FIG. 2-4FIG. 5-7 PMID:4168095

  7. Whole-slide, quadruple immunofluorescence labeling of routinely processed paraffin sections.

    PubMed

    Buscone, Serena; Argentieri, Maria C; Pilla, Daniela; Cattoretti, Giorgio

    2014-04-01

    Whole-slide images (WSI) have acquired a stable place in diagnostic histopathology and immunohistochemistry. Immunofluorescence (IF) techniques hold a limited and selective role in diagnostics (eg, renal and cutaneous pathology) and so far remain excluded from the digital pathology evolution, with notable exceptions, such as quantitative immunopathology. We explored the ability of a commercial fluorescent slide scanner to provide 4-color IF WSI from routinely processed tissues. With minor modifications and a careful match between filters and fluorochromes, we show that 4-color IF WSI can be obtained from routine material with negligible autofluorescence, good sensitivity, and diagnostic power.

  8. ISOLATION AND DETECTION OF GIARDIA CYSTS FROM WATER USING DIRECT IMMUNOFLUORESCENCE.

    USGS Publications Warehouse

    Sorenson, Stephen K.; Riggs, John L.; Dileanis, Peter D.; Suk, Thomas J.

    1986-01-01

    A water-sampling apparatus used for the isolation and detection of Giardia cysts in water has been designed and tested. The sampling apparatus uses one of a variety of pumps or waterline pressure to move water through a filter. Two of the optional pumps are lightweight enough to make the apparatus portable and thus suitable for sampling in remote areas. This technique of sample processing produces good cyst recovery in much less time than is required with previously established methods. Giardia cysts are identified using direct immunofluorescence.

  9. Simultaneous detection and semiquantification of DNA damage in normal and apoptotic cells: triple-immunofluorescent labeling using DAPI, antibodies, and TUNEL.

    PubMed

    Agrawal, Anant; Godar, Dianne E

    2012-07-01

    We developed a triple-labeling immunofluorescence technique that simultaneously identifies total DNA (DAPI), DNA damage (antibodies), and dead cells [terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells] and a method that semiquantifies DNA damage in paraffin-embedded tissues. Using this technique in combination with our analysis method, scientists can now simultaneously detect and compare the relative amounts of DNA damage of almost any kind (except single-strand and double-strand breaks), using indirect fluorescent antibody labeling, in both normal and dying cells of different tissues. Simultaneous labeling of DNA damage and dead or TUNEL-positive cells can reduce processing costs and analysis time, and can lead to discoveries concerning how cells die from different DNA damages. We used increasing doses of UV (290 to 400 nm) radiation to create DNA damage in the form of cyclobutane pyrimidine dimers and 6-4 photoproducts that kill some of the cells in 3-dimensional tissue-engineered skin and vaginal samples. We describe a protocol that reliably detects and semiquantifies DNA damage in both normal and apoptotic cells. We show this triple-labeling immunofluorescence technique and analysis method yields linear UV dose response curves for damage to DNA bases that allows semiquantification of cyclobutane pyrimidine dimers and calculation of its repair rate (T=1 and 24 h), whereas TUNEL allows quantification of the number of apoptotic cells. Scientists can now create beautiful fluorescent pictures that simultaneously detect DNA damage in both normal and apoptotic cells to assess and semiquantify the damage to understand better how different insults lead to the cell's demise.

  10. [Antinuclear antibodies, patterns and characteristics obtained by immunofluorescence. The importance of the IgA, IgM and IgG isotypes].

    PubMed

    Arcavi, Miriam; Dadone, Jorge

    2009-01-01

    The indirect immunofluorescence with epitelial cell line from human laryngeal carcinoma as substrate (IIF-HEp2) and anti-IgG or anti-total Ig as antisera, is the technique currently used for the detection of antinuclear antibodies. The most important antibodies for the diagnosis and follow-up of connective tissue diseases (CTD) are the IgG-ANA, while the IgM-ANA have no clinical relevance. However the IgA-ANA have not been thoroughly investigated so far. The aim of this work was to study the prevalence of different ANA isotypes of Ig antibodies in CTD patients and to evaluate the convenience of the use of monovalent or polyvalent conjugate. We examined the sera of 100 patients with different CTD by IIF-HEp2 and detected a prevalence of 38% IgA-ANA (titles > or = 1:80) and 12% IgM-ANA (titles < or = 1:160). In twenty nine cases we detected IgA-ANA in absence of IgM-ANA, and in 3 cases IgM-ANA in absence of IgA-ANA. In all the cases IgG-ANA were present. In 6 sera a change in the immunofluorescence pattern was observed while using anti-IgA conjugate, whereas in 3 the change was observed with the use of anti-IgM conjugate. Because of the high prevalence of ANA-IgA detected by IIF-HEp2, we emphasize the convenience of employing anti-total Ig in spite of anti-IgG conjugated until the role of ANA-IgA is dilucidated in CTD patients, in order to establish its relevance in the diagnosis, prognosis and follow-up of systemic rheumatic diseases.

  11. Separate spheres and indirect benefits

    PubMed Central

    Brock, Dan W

    2003-01-01

    On any plausible account of the basis for health care resource prioritization, the benefits and costs of different alternative resource uses are relevant considerations in the prioritization process. Consequentialists hold that the maximization of benefits with available resources is the only relevant consideration. Non-consequentialists do not reject the relevance of consequences of benefits and costs, but insist that other considerations, and in particular the distribution of benefits and costs, are morally important as well. Whatever one's particular account of morally justified standards for the prioritization of different health interventions, we must be able to measure those interventions' benefits and costs. There are many theoretical and practical difficulties in that measurement, such as how to weigh extending life against improving health and quality of life as well as how different quality of life improvements should be valued, but they are not my concern here. This paper addresses two related issues in assessing benefits and costs for health resource prioritization. First, should benefits be restricted only to health benefits, or include as well other non health benefits such as economic benefits to employers from reducing the lost work time due to illness of their employees? I shall call this the Separate Spheres problem. Second, should only the direct benefits, such as extending life or reducing disability, and direct costs, such as costs of medical personnel and supplies, of health interventions be counted, or should other indirect benefits and costs be counted as well? I shall call this the Indirect Benefits problem. These two issues can have great importance for a ranking of different health interventions by either a cost/benefit or cost effectiveness analysis (CEA) standard. PMID:12773217

  12. Quantitative Immunofluorescent Blotting of the Multidrug Resistance-associated Protein 2 (MRP2)

    PubMed Central

    Gerk, Phillip M.

    2011-01-01

    Introduction Quantitation of the expression levels of proteins involved in drug transport and disposition is needed to overcome limitations of film-based detection of chemiluminescent immunoblots. Purpose The purpose was to describe and validate a quantitative immunofluorescent blotting method for detection of ATP-Binding Cassette Transporter Isoform C2/Multidrug Resistance-associated Protein 2 (ABCC2/MRP2). Methods Western blotting was performed by electrophoresis of membrane vesicle protein isolated from Sf9 cells overexpressing MRP2 subsequently blotting with infrared labeled secondary antibody. The bound complex was detected using the Odyssey Infrared Imaging System (Li-Cor; Lincoln, NE). The images were analyzed using the Odyssey Application Software to obtain the integrated intensities, followed by linear regression of the intensity data. Results The limits of quantitation for the time-insensitive technique described here were from 0.001μg to 0.5μg of total membrane protein, the coefficient of variation of the slope was 8.9%; r2 values were 0.986 ± 0.012. The utility and sensitivity of this technique was demonstrated in quantitating expression of MRP2 in human placental tissue samples, in which MRP2 was present in low abundance. Discussion The immunofluorescent blotting technique described provides sensitive, reproducible, and quantitative determinations of large, integral membrane proteins such as MRP2, all with potential long-term cost savings. PMID:21277982

  13. Immunofluorescent localization of intermediate filaments (IFs) in helminths using anti-mammalian IFs monoclonal antibody.

    PubMed

    Sato, H; Kamiya, H

    2000-08-01

    Intermediate filaments (IFs) make up the cytoskeleton of most eukaryotic cells. In vertebrates, a number of IF proteins have been identified, showing distributions unique to tissue or cell type. Information on helminth IFs is limited to some nematode species. To observe immunofluorescent localization of IFs in helminth tissues, we selected a murine hybridoma clone producing IgM antibody to multiple types of mammalian IF proteins and examined cross-reactivity to helminth proteins. The selected monoclonal antibody (HUSM-9) cross-reacted well with IFs from nematode species such as Toxocara canis, Dirofilaria immitis, Anisakis simplex, and Trichinella britovi; strong immunofluorescence on cryostat sections was detected in the hypodermis, cords, body muscle, smooth muscle of the uterus, and other epithelial structures. In platyhelminths, i.e., adult Schistosoma mansoni, larval Taenia taeniaeformis, adult Taenia crassiceps, and Echinococcus multilocularis protoscolex, the reactivity was weaker than in nematodes, and localized in the body wall muscle and subtegumental tissue. Western blotting of 8 M urea extracts of parasites with the antibody detected a pair of clear bands in nematodes but not in S. mansoni or the cestodes. These results might be explained by sparse distribution of IFs in platyhelminths, or low affinity of the used antibody to platyhelminth IF proteins, or both.

  14. Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

    PubMed Central

    Kishen, Ria E. B.; Kluth, David C.; Bellamy, Christopher O. C.

    2016-01-01

    The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family). Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705), Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches. PMID:27632367

  15. Classification of the intrafusal muscle fibres in the frog muscle spindle: histochemical and immunofluorescent studies.

    PubMed Central

    Yoshimura, A; Fujitsuka, N; Sokabe, M; Naruse, K; Nomura, K; Diwan, F H; Ito, F

    1990-01-01

    Intrafusal muscle fibres from bull-frog semitendinosus, iliofibularis and sartorius muscles were classified into three types using the histochemical, immunofluorescent and morphological characteristics, with reference to the extrafusal muscle fibres, which were classified into five types in accordance with Rowlerson & Spurway (1988). Immunofluorescent reactions with antibodies against slow or fast myosins obtained from anterior or posterior latissimus dorsi muscles (ALD or PLD), respectively, of chicken were used as the primary criterion. Histochemical profiles of muscle fibres were classified into nine types of myosin ATPase activity as the secondary criterion. Anti-PLD intrafusal fibres (polar zone) with ATPase profiles of moderate to high acid and alkaline stabilities correspond to large nuclear bag fibres in the classification of Diwan & Ito (1989), whereas anti-ALD fibres (polar zone) with alkaline-labile ATPase profiles correspond to medium nuclear bag fibres. On the basis of diameter, anti-PLD fibres (polar zone) with ATPase profiles of moderate to low acid stability and moderate to high alkaline stability seem to correspond to two types of small nuclear chain fibre. Variations between muscles, between intra- and extrafusal fibres and also between zones along intrafusal fibres are discussed. Images Fig. 1 Fig. 3 PMID:2148748

  16. Imaging and cell count in cleared intact cochlea in the Mongolian gerbil using laser scanning confocal microscopy.

    PubMed

    Risoud, M; Sircoglou, J; Dedieu, G; Tardivel, M; Vincent, C; Bonne, N-X

    2017-09-01

    To draw up a clearing protocol for Mongolian gerbil cochlea, and to assess the feasibility of quantifying and analyzing 3D cell architecture in the transparent cochleae. Freshly dissected inner ears were prepared on a 13-day protocol: fixation, microdissection, post-fixation, decalcification, pretreatment (signal enhancement, permeabilization and blocking), fluorescent labeling (indirect immunolabeling and direct labeling), dehydration, clearing in Spalteholz solution (MSBB: methyl salicylate and benzyl benzoate) and mounting. Image acquisition used laser scanning confocal microscopy. ImageJ software was used to measure the length of the organ of Corti thus available for analysis and to count inner and outer hair cells. Four cochleas underwent imaging. 3D reconstruction enabled organ of Corti length to be measured, at a mean 1269±346μm. Mean inner and outer hair-cell count per organ of Corti length was 142±44 and 400±122, respectively. Cochlear clearing by MSBB was feasible in Mongolian gerbils and provided high-resolution immunofluorescence-labeled inner-ear images. To our knowledge, this was the first application of the technique in this species. Cell count could thus be performed along the organ of Corti length without traumatic dissection. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  17. Delay, change and bifurcation of the immunofluorescence distribution attractors in health statuses diagnostics and in medical treatment

    NASA Astrophysics Data System (ADS)

    Galich, Nikolay E.; Filatov, Michael V.

    2008-07-01

    Communication contains the description of the immunology experiments and the experimental data treatment. New nonlinear methods of immunofluorescence statistical analysis of peripheral blood neutrophils have been developed. We used technology of respiratory burst reaction of DNA fluorescence in the neutrophils cells nuclei due to oxidative activity. The histograms of photon count statistics the radiant neutrophils populations' in flow cytometry experiments are considered. Distributions of the fluorescence flashes frequency as functions of the fluorescence intensity are analyzed. Statistic peculiarities of histograms set for healthy and unhealthy donors allow dividing all histograms on the three classes. The classification is based on three different types of smoothing and long-range scale averaged immunofluorescence distributions and their bifurcation. Heterogeneity peculiarities of long-range scale immunofluorescence distributions allow dividing all histograms on three groups. First histograms group belongs to healthy donors. Two other groups belong to donors with autoimmune and inflammatory diseases. Some of the illnesses are not diagnosed by standards biochemical methods. Medical standards and statistical data of the immunofluorescence histograms for identifications of health and illnesses are interconnected. Possibilities and alterations of immunofluorescence statistics in registration, diagnostics and monitoring of different diseases in various medical treatments have been demonstrated. Health or illness criteria are connected with statistics features of immunofluorescence histograms. Neutrophils populations' fluorescence presents the sensitive clear indicator of health status.

  18. Bridging fluorescence microscopy and electron microscopy

    PubMed Central

    2008-01-01

    Development of new fluorescent probes and fluorescence microscopes has led to new ways to study cell biology. With the emergence of specialized microscopy units at most universities and research centers, the use of these techniques is well within reach for a broad research community. A major breakthrough in fluorescence microscopy in biology is the ability to follow specific targets on or in living cells, revealing dynamic localization and/or function of target molecules. One of the inherent limitations of fluorescence microscopy is the resolution. Several efforts are undertaken to overcome this limit. The traditional and most well-known way to achieve higher resolution imaging is by electron microscopy. Moreover, electron microscopy reveals organelles, membranes, macromolecules, and thus aids in the understanding of cellular complexity and localization of molecules of interest in relation to other structures. With the new probe development, a solid bridge between fluorescence microscopy and electron microscopy is being built, even leading to correlative imaging. This connection provides several benefits, both scientifically as well as practically. Here, I summarize recent developments in bridging microscopy. PMID:18575880

  19. Indirect Lightning Safety Assessment Methodology

    SciTech Connect

    Ong, M M; Perkins, M P; Brown, C G; Crull, E W; Streit, R D

    2009-04-24

    Lightning is a safety hazard for high-explosives (HE) and their detonators. In the However, the current flowing from the strike point through the rebar of the building The methodology for estimating the risk from indirect lighting effects will be presented. It has two parts: a method to determine the likelihood of a detonation given a lightning strike, and an approach for estimating the likelihood of a strike. The results of these two parts produce an overall probability of a detonation. The probability calculations are complex for five reasons: (1) lightning strikes are stochastic and relatively rare, (2) the quality of the Faraday cage varies from one facility to the next, (3) RF coupling is inherently a complex subject, (4) performance data for abnormally stressed detonators is scarce, and (5) the arc plasma physics is not well understood. Therefore, a rigorous mathematical analysis would be too complex. Instead, our methodology takes a more practical approach combining rigorous mathematical calculations where possible with empirical data when necessary. Where there is uncertainty, we compensate with conservative approximations. The goal is to determine a conservative estimate of the odds of a detonation. In Section 2, the methodology will be explained. This report will discuss topics at a high-level. The reasons for selecting an approach will be justified. For those interested in technical details, references will be provided. In Section 3, a simple hypothetical example will be given to reinforce the concepts. While the methodology will touch on all the items shown in Figure 1, the focus of this report is the indirect effect, i.e., determining the odds of a detonation from given EM fields. Professor Martin Uman from the University of Florida has been characterizing and defining extreme lightning strikes. Using Professor Uman's research, Dr. Kimball Merewether at Sandia National Laboratory in Albuquerque calculated the EM fields inside a Faraday-cage type

  20. Indirect discrimination and breast screening.

    PubMed

    Botha, J L; Manku-Scott, T K; Moledina, F; Williams, A

    1993-01-01

    Uptake of screening services in inner-city communities has been low, particularly in older age groups, lower social classes, and ethnic minorities. In Leicester City, where up to 25% of the population belong to ethnic minorities, this may have important implications for breast screening. We randomly sampled 701 inner-city women aged 45 to 64 years, stratified by neighborhood and by women's "likely home language." Trained interviewers succeeded in interviewing 79% of those eligible, and we report here a preliminary analysis of 413 respondents. Knowledge of breast cancer and screening varied markedly and significantly by actual language: 60.4% of English-speaking and 12.5% of non-English-speaking women correctly answered 10 or more questions (of 14) about breast cancer and screening (chi 2(1) = 89.884; P = .000). Despite that, 80% or more women stated their intention to attend for screening and assessment if necessary, irrespective of neighborhood, language, age, or social class. We suggest that the difference in knowledge between language groups arose from indirect discrimination in the way in which health-related information is disseminated in British society. However, after providing appropriate screening information, we report similarly high intended acceptance rates in the two language groups.

  1. Lifespan based indirect response models

    PubMed Central

    Ruixo, Juan Jose Perez

    2012-01-01

    In the field of hematology, several mechanism-based pharmacokinetic-pharmacodynamic models have been developed to understand the dynamics of several blood cell populations under different clinical conditions while accounting for the essential underlying principles of pharmacology, physiology and pathology. In general, a population of blood cells is basically controlled by two processes: the cell production and cell loss. The assumption that each cell exits the population when its lifespan expires implies that the cell loss rate is equal to the cell production rate delayed by the lifespan and justifies the use of delayed differential equations for compartmental modeling. This review is focused on lifespan models based on delayed differential equations and presents the structure and properties of the basic lifespan indirect response (LIDR) models for drugs affecting cell production or cell lifespan distribution. The LIDR models for drugs affecting the precursor cell production or decreasing the precursor cell population are also presented and their properties are discussed. The interpretation of transit compartment models as LIDR models is reviewed as the basis for introducing a new LIDR for drugs affecting the cell lifespan distribution. Finally, the applications and limitations of the LIDR models are discussed. PMID:22212685

  2. Value of electron microscopy in the diagnosis of glomerular diseases.

    PubMed

    Darouich, Sihem; Goucha, Rym Louzir; Jaafoura, Mohamed Habib; Moussa, Fatma Ben; Zekri, Semy; Maiz, Hédi Ben

    2010-04-01

    To evaluate the contribution of electron microscopy to the final diagnosis of glomerulopathies, the authors established a prospective study during the first semester of 2006. A total of 52 kidney biopsies were performed with 3 samples for light microscopy, immunofluorescence, and electron microscopy. Among these renal biopsies, only 20 were examined with electron microscopy because the diagnosis made on the basis of conventional methods had remained unclear or doubtful. In 18 cases, electron microscopy was undertaken for the investigation of primary kidney disease. The 2 remaining cases were transplant biopsies. In this series of 20 patients, there were 3 children with an average age of 9 years and 17 adults with an average age of 35.5 years. Fifteen patients (75%) were nephrotic. The study revealed that electron microscopy was essential for diagnosis in 8 cases (40%) and was helpful in 12 cases (60%). In conclusion, the results showed that the ultrastructural study provides essential or helpful information in many cases of glomerular diseases, and therefore electron microscopy should be considered an important tool of diagnostic renal pathology. As was recommended, it is important to reserve renal tissue for ultrastructural study unless electron microscopy can be routinely used in all biopsies. Thus, this technique could be performed wherever a renal biopsy has to be ultrastructurally evaluated.

  3. RELATION BETWEEN EPSTEIN-BARR VIRAL AND CELL MEMBRANE IMMUNOFLUORESCENCE OF BURKITT TUMOR CELLS

    PubMed Central

    Klein, G.; Pearson, G.; Nadkarni, J. S.; Nadkarni, J. J.; Klein, E.; Henle, G.; Henle, W.; Clifford, P.

    1968-01-01

    A comparison was made of the immunofluorescence tests for detection of cell membrane and Epstein-Barr virus antigens in cells from Burkitt tumor biopsies or continuous cultures derived therefrom. On the whole, cell membrane fluorescence in established lines appeared to depend not only upon the presence of EBV but to a considerable degree also upon the extent of the persistent viral infection. There was no constant relationship, however, between the results of the two tests and exceptions to the rule were noted. These observations indicate that different antigens are involved in the two tests. Biopsy cells in general and young cultures may reveal strong MIF activity but few, if any, EBV-positive cells. The reverse, the presence of relatively large numbers of EBV antigen-containing cells in the absence of significant MIF reactions, was also noted on occasion in a few established cultures. The possible interpretations of these findings have been discussed. PMID:4878906

  4. Comparison of immunofluorescence and immunoperoxidase for demonstration of antinuclear antibodies on HEp-2 substrate.

    PubMed

    Tipping, P G; Dimech, W J; Littlejohn, G O; Holdsworth, S R

    1987-06-01

    A comparison of antinuclear antibody (ANA) detection on HEp-2 cell substrate, using immunofluorescence (IF) and immunoperoxidase (IP) techniques, was performed on 183 sera from 178 individuals, with and without connective tissue diseases. All sera were number coded and scored blindly by two independent observers. ANA was detected in 61% of sera by IF and 62% of sera by IP. Positive and negative results corresponded between the two techniques in 97% of sera. Patterns and titres corresponded in 85% of positive sera. The two independent observers scored ANA more consistently with IP than with IF. Both methods were technically simple to perform and produced consistent results with control sera. These data show that IP provides results equivalent to the traditional IF technique for demonstrating ANA on HEp-2 substrate. Fewer discordant results between two independent observers using IP suggests that this technique has technical advantages for interpretation of ANA on HEp-2 substrate.

  5. DETECTION OF SPECIFIC ANTIGEN IN SV40-TRANSFORMED CELLS BY IMMUNOFLUORESCENCE

    PubMed Central

    Pope, John H.; Rowe, Wallace P.

    1964-01-01

    With an immunofluorescent technique involving the use of serum of hamsters with SV40 tumors, nuclear fluorescence was detected in each of five cell lines, derived from four mammalian species, transformed by SV40 virus. Essentially all nuclei, including those of multinuclear cells, were fluorescent-stainable. Serum of hamsters bearing SV40 tumors was also found to give nuclear fluorescence in susceptible cells (AGMK or BSC-1) acutely infected with SV40 virus. These findings provide further evidence that cellular incorporation of the SV40 viral genome, with partial expression of the genome by synthesis of at least one virus-specific antigen, is an integral property of all SV40 transformed cells. PMID:14206435

  6. Skeletal Muscle Tissue Clearing for LacZ and Fluorescent Reporters, and Immunofluorescence Staining

    PubMed Central

    Verma, Mayank; Murkonda, Bhavani SR; Asakura, Yoko; Asakura, Atsushi

    2016-01-01

    Skeletal muscle is a highly ordered yet complex tissue containing several cell types that interact with each other in order to maintain structure and homeostasis. It is also a highly regenerative tissue that responds to damage in a highly intricate but stereotypic manner, with distinct spatial and temporal kinetics. Proper examination of this process requires one to look at the three-dimensional orientation of the cellular and subcellular components, which can be accomplished through tissue clearing. While there has been a recent surge of protocols to study biology in whole tissue, it has primarily focused on the nervous system. This chapter describes the workflow for whole mount analysis of murine skeletal muscle for LacZ reporters, fluorescent reporters and immunofluorescence staining. Using this technique, we are able to visualize LacZ reporters more effectively in deep tissue samples, and to perform fluorescent imaging with a depth greater than 1700 μm. PMID:27492170

  7. Immunofluorescent localization of ubiquitin and proteasomes in nucleolar vacuoles of soybean root meristematic cells.

    PubMed

    Stępiński, D

    2012-05-30

    In this study, using the immunofluorescent method, the immunopositive signals to ubiquitin and proteasomes in nucleoli of root meristematic cells of soybean seedlings have been observed. In fact, those signals were present exclusively in nucleolar vacuoles. No signals were observed in the nucleolar territory out of the nucleolar vacuoles or in the nucleoli without vacuoles. The ubiquitin-proteasome system (UPS) may act within the nucleoli of plants with high metabolic activities and may provide an additional level of regulation of intracellular proteolysis via compartment-specific activities of their components. It is suggested that the presence of the UPS solely in vacuolated nucleoli serves as a mechanism that enhances the speed of ribosome subunit production in very actively transcribing nucleoli. On the other hand, nucleolar vacuoles in a cell/nucleus could play additional roles associated with temporary sequestration or storage of some cellular factors, including components of the ubiquitin-proteasome system.

  8. Dual Immunofluorescence Study of Citrullinated Proteins in Alzheimer Diseased Frontal Cortex

    PubMed Central

    Nicholas, Anthony P.

    2013-01-01

    Deimination is a post-translational modification of proteins in which selected arginine amino acids are enzymatically converted to citrullines. Using dual-color immunofluorescence, the present study is the first to examine the frontal cortex of patients with Alzheimer's disease (AD) versus age-matched controls with an established monoclonal antibody (F95) against peptidyl-citrulline moieties. In AD specimens, a number of new findings were discovered, including evidence for deiminated proteins in extracellular plaques, the walls of large blood vessels, the nuclei of selective neurons immunoreactive for phosphorylated tau and numerous reactive astrocytes concentrated around extracellular plaques, ventricular surfaces and at the interface between the gray and white matter of the cortex. Although the identities of these citrullinated proteins remain largely unknown, the present study adds to the growing number of locations in which deiminated proteins may be found in the brains of patients with AD. PMID:23648390

  9. Muscle Histology Characterization Using H&E Staining and Muscle Fiber Type Classification Using Immunofluorescence Staining

    PubMed Central

    Wang, Chao; Yue, Feng; Kuang, Shihuan

    2017-01-01

    Muscle function is determined by its structure and fiber type composition. Here we describe a protocol to examine muscle histology and myofiber types using hematoxylin and eosin (H&E) and immunofluorescence staining, respectively. H&E stain nucleus in blue and cytoplasm in red, therefore allowing for morphological analyses, such as myofiber diameter, the presence of degenerated and regenerated myofibers, and adipocytes and fibrotic cells. Muscle fibers in adult skeletal muscles of rodents are classified into 4 subtypes based on the expression of myosin heavy chain proteins: Myh7 (type I fiber), Myh2 (type IIA fiber), Myh1 (type IIX fiber), Myh4 (type IIB fiber). A panel of monoclonal antibodies can be used to specifically label these muscle fiber subtypes. These protocols are commonly used in the study of muscle development, growth and regeneration (for example: Wang et al., 2015; Nie et al., 2016; Yue et al., 2016; Wang et al., 2017). PMID:28752107

  10. Validation of a monoclonal antibody-based immunofluorescent assay to detect Burkholderia pseudomallei in blood cultures.

    PubMed

    Dulsuk, Adul; Paksanont, Suporn; Sangchankoom, Adisak; Ekchariyawat, Peeraya; Phunpang, Rungnapa; Jutrakul, Yaowaruk; Chantratita, Narisara; West, T Eoin

    2017-01-22

    Identification of Burkholderia pseudomallei, the cause of melioidosis, using routine methods takes several days. Use of a monoclonal antibody-based immunofluorescent assay (IFA) on positive blood cultures may speed diagnosis. We tested the diagnostic accuracy of the IFA on 545 blood cultures positive for Gram-negative organisms at Udon Thani Hospital, Thailand, between June 2015 and August 2016. Sensitivity of the IFA was 100% and specificity was 99.6%. The median decrease in time to pathogen identification between the IFA result and routine methods was 28 h (IQR 25-51), p<0.0001. The IFA accurately expedites the diagnosis of melioidosis. © The Author 2017. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.

  11. Dual immunofluorescence study of citrullinated proteins in Alzheimer diseased frontal cortex.

    PubMed

    Nicholas, Anthony P

    2013-06-17

    Deimination is a post-translational modification of proteins in which selected arginine amino acids are enzymatically converted to citrullines. Using dual-color immunofluorescence, the present study is the first to examine the frontal cortex of patients with Alzheimer's disease (AD) versus age-matched controls with an established monoclonal antibody (F95) against peptidyl-citrulline moieties. In AD specimens, a number of new findings were discovered, including evidence for deiminated proteins in extracellular plaques, the walls of large blood vessels, the nuclei of selective neurons immunoreactive for phosphorylated tau and numerous reactive astrocytes concentrated around extracellular plaques, ventricular surfaces and at the interface between the gray and white matter of the cortex. Although the identities of these citrullinated proteins remain largely unknown, the present study adds to the growing number of locations in which deiminated proteins may be found in the brains of patients with AD. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  12. Demonstration of antimyelin antibodies by immunofluorescence in Guillain-Barré syndrome

    PubMed Central

    Tse, K. S.; Arbesman, C. E.; Tomasi, T. B.; Tourville, D.

    1971-01-01

    IgG, IgA and IgM antibodies reacting with myelin sheaths were found in the sera but not in the cerebrospinal fluid of four out of six patients with Guillain-Barré syndrome. These antibodies had organ specificity for myelin of the central and peripheral nervous systems but did not demonstrate species specificity. Similar antibodies, but in much lower titres, were found infrequently in the sera of patients with liver disease and rarely in normal individuals. The organ specificity and the close correlation between antibody titres and disease activity suggest the possibility that these antibodies may play a role in the pathogenesis of Guillain-Barré syndrome. The detection of antimyelin antibodies by immunofluorescence may provide a relatively simple ancillary laboratory aid in the diagnosis of the neurological entity. ImagesFIG. 1 PMID:4997072

  13. Two immunofluorescent methods compared with a radial immunodiffusion method for measurement of serum immunoglobulins.

    PubMed

    Blanchard, G C; Gardner, R

    1978-05-01

    We found high correlations when the values for immunoglobulins G, A, and M, obtained from the analysis of a group of 50 or more human sera by a measured-time radial immunodiffusion method, were compared to values found by two new commercial immunofluorescent methods. The Fiax and the Immuno-Fluor systems make use of fluorescently labeled antibodies and solid-phase immunoadsorbents and offer simple, precise, and objective methods for immunoglobulin quantitation. The Fiax procedure is less time consuming than is the Immuno-Fluor and gives IgG and IgA concentrations that are in close agreement with those found by radial immunodiffusion and IgM values that are significantly lower than the radial immunodiffusion values. The IgG results determined by the Immuno-Fluor method compare well with the radial immunodiffusion results, uhile the IgA values are significantly higher and IgM values significantly lower than those obtained by radial immunodiffusion.

  14. A rare case of signet ring cell lymphoma: Diagnosis aided by immunofluorescent staining

    PubMed Central

    Gore, Charusheela R; Panicker, N K; Kumar, Harsh; Buch, Archana; Chandanwale, S S

    2017-01-01

    Signet ring cell lymphomas are the proliferations of malignant lymphoid cells containing cytoplasmic vacuoles or globules which displace the nuclei, imparting it a signet ring appearance. This rare tumor is a variant of non-Hodgkin lymphoma. Signet ring appearance is due to cytoplasmic accumulation of immunoglobulin or vacuoles derived from multivesicular bodies. These cells, particularly with cytoplasmic vacuoles, may be mistaken for adenocarcinoma cells. We are presenting one such case where immunofluorescence helped us to demonstrate the immunoglobulins on fine needle aspiration smears. This is an innovative technique and has not been reported earlier. Our aim of presenting this case is to review the awareness of this rare lymphoma among pathologists to give due consideration for avoiding inappropriate investigations and treatment. PMID:28182089

  15. A rare case of signet ring cell lymphoma: Diagnosis aided by immunofluorescent staining.

    PubMed

    Gore, Charusheela R; Panicker, N K; Kumar, Harsh; Buch, Archana; Chandanwale, S S

    2017-01-01

    Signet ring cell lymphomas are the proliferations of malignant lymphoid cells containing cytoplasmic vacuoles or globules which displace the nuclei, imparting it a signet ring appearance. This rare tumor is a variant of non-Hodgkin lymphoma. Signet ring appearance is due to cytoplasmic accumulation of immunoglobulin or vacuoles derived from multivesicular bodies. These cells, particularly with cytoplasmic vacuoles, may be mistaken for adenocarcinoma cells. We are presenting one such case where immunofluorescence helped us to demonstrate the immunoglobulins on fine needle aspiration smears. This is an innovative technique and has not been reported earlier. Our aim of presenting this case is to review the awareness of this rare lymphoma among pathologists to give due consideration for avoiding inappropriate investigations and treatment.

  16. Ultrastructural and immunofluorescent detection of herpes simplex virus after embalming and burial.

    PubMed

    Sperry, K; Sweeney, E S

    1987-09-01

    The practice of embalming preserves body tissues, and embalmed bodies may resist decay processes for many decades with relatively little change. As the chemicals used for embalming are poisonous to microorganisms, bacterial and viral cultures are futile after such funerary procedures are performed. However, embalming may act as a virtual tissue fixative, especially with arterial perfusion, and identification methods other than culture may be used to detect and identify pathogenic organisms. In the case presented here, a death from a fulminating, but unidentified, illness in a young girl was successfully diagnosed as herpes hepatitis by immunofluorescent and electron-microscopic studies of tissue obtained 3 weeks after she was embalmed and interred. Routine embalming and burial should not eliminate these diagnostic procedures from consideration in specific situations where potentially useful information may be realized.

  17. Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts.

    PubMed

    Kumar, Manish; Tanwar, Pradeep

    2017-01-13

    Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

  18. Immunofluorescence staining of paraffin sections: creating DAB staining like virtual digital images using CMYK color conversion.

    PubMed

    Buchynska, L; Kashuba, E; Szekely, L

    2008-12-01

    Crystal violet treatment of formalin fixed paraffin embedded tissue slides greatly reduces the endogenous autofluorescence, and allows immunofluorescence (IF) staining with FITC or Alexa488 conjugated antibodies. Using cold CCD camera to capture the fluorescence images makes this staining method very sensitive. Here we show that combination of IF with the simultaneous recording of crystal violet induced red and Hoechst 33258 induced blue fluorescence permits the localization of the IF signal over a cytoplasmic: nuclear red:blue stain that visualizes the microscopic anatomy of the underlying tissue. To make the visual interpretation of the IF staining easier for microscopists, who are used to DAB staining over weak hematoxilin-eosin background, we created a simple color conversion procedure that turns the captured three-color fluorescence RGB (red, green, blue) images over a black background into four color CMYK (cyan, magenta, yellow, key color (black)) images.

  19. Indirect Self-Destructiveness and Emotional Intelligence.

    PubMed

    Tsirigotis, Konstantinos

    2016-06-01

    While emotional intelligence may have a favourable influence on the life and psychological and social functioning of the individual, indirect self-destructiveness exerts a rather negative influence. The aim of this study has been to explore possible relations between indirect self-destructiveness and emotional intelligence. A population of 260 individuals (130 females and 130 males) aged 20-30 (mean age of 24.5) was studied by using the Polish version of the chronic self-destructiveness scale and INTE, i.e., the Polish version of the assessing emotions scale. Indirect self-destructiveness has significant correlations with all variables of INTE (overall score, factor I, factor II), and these correlations are negative. The intensity of indirect self-destructiveness differentiates significantly the height of the emotional intelligence and vice versa: the height of the emotional intelligence differentiates significantly the intensity of indirect self-destructiveness. Indirect self-destructiveness has negative correlations with emotional intelligence as well as its components: the ability to recognize emotions and the ability to utilize emotions. The height of emotional intelligence differentiates the intensity of indirect self-destructiveness, and vice versa: the intensity of indirect self-destructiveness differentiates the height of emotional intelligence. It seems advisable to use emotional intelligence in the prophylactic and therapeutic work with persons with various types of disorders, especially with the syndrome of indirect self-destructiveness.

  20. Two stage indirect evaporative cooling system

    SciTech Connect

    Bourne, Richard C.; Lee, Brian E.; Callaway, Duncan

    2005-08-23

    A two stage indirect evaporative cooler that moves air from a blower mounted above the unit, vertically downward into dry air passages in an indirect stage and turns the air flow horizontally before leaving the indirect stage. After leaving the dry passages, a major air portion travels into the direct stage and the remainder of the air is induced by a pressure drop in the direct stage to turn 180.degree. and returns horizontally through wet passages in the indirect stage and out of the unit as exhaust air.

  1. Immunofluorescence Analysis and Diagnosis of Primary Ciliary Dyskinesia with Radial Spoke Defects.

    PubMed

    Frommer, Adrien; Hjeij, Rim; Loges, Niki T; Edelbusch, Christine; Jahnke, Charlotte; Raidt, Johanna; Werner, Claudius; Wallmeier, Julia; Große-Onnebrink, Jörg; Olbrich, Heike; Cindrić, Sandra; Jaspers, Martine; Boon, Mieke; Memari, Yasin; Durbin, Richard; Kolb-Kokocinski, Anja; Sauer, Sascha; Marthin, June K; Nielsen, Kim G; Amirav, Israel; Elias, Nael; Kerem, Eitan; Shoseyov, David; Haeffner, Karsten; Omran, Heymut

    2015-10-01

    Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder caused by several distinct defects in genes responsible for ciliary beating, leading to defective mucociliary clearance often associated with randomization of left/right body asymmetry. Individuals with PCD caused by defective radial spoke (RS) heads are difficult to diagnose owing to lack of gross ultrastructural defects and absence of situs inversus. Thus far, most mutations identified in human radial spoke genes (RSPH) are loss-of-function mutations, and missense variants have been rarely described. We studied the consequences of different RSPH9, RSPH4A, and RSPH1 mutations on the assembly of the RS complex to improve diagnostics in PCD. We report 21 individuals with PCD (16 families) with biallelic mutations in RSPH9, RSPH4A, and RSPH1, including seven novel mutations comprising missense variants, and performed high-resolution immunofluorescence analysis of human respiratory cilia. Missense variants are frequent genetic defects in PCD with RS defects. Absence of RSPH4A due to mutations in RSPH4A results in deficient axonemal assembly of the RS head components RSPH1 and RSPH9. RSPH1 mutant cilia, lacking RSPH1, fail to assemble RSPH9, whereas RSPH9 mutations result in axonemal absence of RSPH9, but do not affect the assembly of the other head proteins, RSPH1 and RSPH4A. Interestingly, our results were identical in individuals carrying loss-of-function mutations, missense variants, or one amino acid deletion. Immunofluorescence analysis can improve diagnosis of PCD in patients with loss-of-function mutations as well as missense variants. RSPH4A is the core protein of the RS head.

  2. Immunofluorescence Analysis and Diagnosis of Primary Ciliary Dyskinesia with Radial Spoke Defects

    PubMed Central

    Frommer, Adrien; Hjeij, Rim; Loges, Niki T.; Edelbusch, Christine; Jahnke, Charlotte; Raidt, Johanna; Werner, Claudius; Wallmeier, Julia; Große-Onnebrink, Jörg; Olbrich, Heike; Cindrić, Sandra; Jaspers, Martine; Boon, Mieke; Memari, Yasin; Durbin, Richard; Kolb-Kokocinski, Anja; Sauer, Sascha; Marthin, June K.; Nielsen, Kim G.; Amirav, Israel; Elias, Nael; Kerem, Eitan; Shoseyov, David; Haeffner, Karsten

    2015-01-01

    Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder caused by several distinct defects in genes responsible for ciliary beating, leading to defective mucociliary clearance often associated with randomization of left/right body asymmetry. Individuals with PCD caused by defective radial spoke (RS) heads are difficult to diagnose owing to lack of gross ultrastructural defects and absence of situs inversus. Thus far, most mutations identified in human radial spoke genes (RSPH) are loss-of-function mutations, and missense variants have been rarely described. We studied the consequences of different RSPH9, RSPH4A, and RSPH1 mutations on the assembly of the RS complex to improve diagnostics in PCD. We report 21 individuals with PCD (16 families) with biallelic mutations in RSPH9, RSPH4A, and RSPH1, including seven novel mutations comprising missense variants, and performed high-resolution immunofluorescence analysis of human respiratory cilia. Missense variants are frequent genetic defects in PCD with RS defects. Absence of RSPH4A due to mutations in RSPH4A results in deficient axonemal assembly of the RS head components RSPH1 and RSPH9. RSPH1 mutant cilia, lacking RSPH1, fail to assemble RSPH9, whereas RSPH9 mutations result in axonemal absence of RSPH9, but do not affect the assembly of the other head proteins, RSPH1 and RSPH4A. Interestingly, our results were identical in individuals carrying loss-of-function mutations, missense variants, or one amino acid deletion. Immunofluorescence analysis can improve diagnosis of PCD in patients with loss-of-function mutations as well as missense variants. RSPH4A is the core protein of the RS head. PMID:25789548

  3. Novel multicolor immunofluorescence technique using primary antibodies raised in the same host species.

    PubMed

    Frisch, Jillian; Houchins, J P; Grahek, Michael; Schoephoerster, Jordan; Hagen, Jodi; Sweet, Joseph; Mendoza, Leopoldo; Schwartz, David; Kalyuzhny, Alexander E

    2011-01-01

    Simultaneous detection of multiple tissue antigens is one of the most frequently used immunohistochemical (IHC) techniques. In order to avoid cross-reactivity of each secondary antibody with multiple primary antibodies when doing either dual- or triple-labeling immunofluorescence, it is necessary to use primary antibodies raised in different host species such as mouse, rabbit, and goat. However, in many cases, suitable primary antibodies raised in different species are unavailable. We have developed a novel technique for triple-labeling immunofluorescence that can be used with primary antibodies derived from a single host source. This technique includes modification of one primary antibody with biotin (ChromaLink™ Biotin) and a second primary antibody with DIG (ChromaLink™ Digoxigenin). For IHC staining, cells or tissue sections are incubated first with unconjugated primary antibody against the first target protein followed by detection with antiprimary secondary antibody conjugated to NorthernLights™ NL-637 tag (fluorescence in the far-red spectral region). Subsequently, the same tissue sections are incubated with a mixture of same species biotin-labeled primary antibody (against the second target protein) and DIG-labeled primary antibody (against the third target protein) followed by detection using a mixture of Streptavidin NorthernLights™ NL-493 tag (green fluorescence) and anti-DIG secondary antibody conjugated to a Rhodamine Red X™ tag (red fluorescence). This technique provides good spectral separation of colors depicting different antigens of interest while avoiding cross-reactivity between irrelevant primary and secondary antibodies. In addition, this multiplexed IHC technique provides significant convenience to researchers who have only primary antibodies raised in the same host species at their disposal.

  4. Confocal reference free traction force microscopy

    PubMed Central

    Bergert, Martin; Lendenmann, Tobias; Zündel, Manuel; Ehret, Alexander E.; Panozzo, Daniele; Richner, Patrizia; Kim, David K.; Kress, Stephan J. P.; Norris, David J.; Sorkine-Hornung, Olga; Mazza, Edoardo; Poulikakos, Dimos; Ferrari, Aldo

    2016-01-01

    The mechanical wiring between cells and their surroundings is fundamental to the regulation of complex biological processes during tissue development, repair or pathology. Traction force microscopy (TFM) enables determination of the actuating forces. Despite progress, important limitations with intrusion effects in low resolution 2D pillar-based methods or disruptive intermediate steps of cell removal and substrate relaxation in high-resolution continuum TFM methods need to be overcome. Here we introduce a novel method allowing a one-shot (live) acquisition of continuous in- and out-of-plane traction fields with high sensitivity. The method is based on electrohydrodynamic nanodrip-printing of quantum dots into confocal monocrystalline arrays, rendering individually identifiable point light sources on compliant substrates. We demonstrate the undisrupted reference-free acquisition and quantification of high-resolution continuous force fields, and the simultaneous capability of this method to correlatively overlap traction forces with spatial localization of proteins revealed using immunofluorescence methods. PMID:27681958

  5. Confocal reference free traction force microscopy.

    PubMed

    Bergert, Martin; Lendenmann, Tobias; Zündel, Manuel; Ehret, Alexander E; Panozzo, Daniele; Richner, Patrizia; Kim, David K; Kress, Stephan J P; Norris, David J; Sorkine-Hornung, Olga; Mazza, Edoardo; Poulikakos, Dimos; Ferrari, Aldo

    2016-09-29

    The mechanical wiring between cells and their surroundings is fundamental to the regulation of complex biological processes during tissue development, repair or pathology. Traction force microscopy (TFM) enables determination of the actuating forces. Despite progress, important limitations with intrusion effects in low resolution 2D pillar-based methods or disruptive intermediate steps of cell removal and substrate relaxation in high-resolution continuum TFM methods need to be overcome. Here we introduce a novel method allowing a one-shot (live) acquisition of continuous in- and out-of-plane traction fields with high sensitivity. The method is based on electrohydrodynamic nanodrip-printing of quantum dots into confocal monocrystalline arrays, rendering individually identifiable point light sources on compliant substrates. We demonstrate the undisrupted reference-free acquisition and quantification of high-resolution continuous force fields, and the simultaneous capability of this method to correlatively overlap traction forces with spatial localization of proteins revealed using immunofluorescence methods.

  6. Optical inversions based on polarization parameters indirect microscopic imaging

    NASA Astrophysics Data System (ADS)

    Liu, Guoyan; Gao, Kun; Liu, Xuefeng; Huang, Zicheng; Ni, Guoqiang

    2016-10-01

    The resolution of conventional optical microscope is intrinsically limited by the optical diffraction, therefore it cannot be used in the measurement of sub-100nm shape and structural detection. Non-optical imaging techniques are not limited by the optical diffraction. For example, scanning tunneling microscopy (STM) and atomic force microscopy (AFM), but both of them have the weakness of narrow view field, low efficiency, and excessive cost. To detect nanoscale material, a new microscopic imaging technique is introduced in this paper, i.e. the polarization parameter indirect microscopic imaging technique. A conventional reflection microscopic system is used as the basic optical system, with polarization-modulation mechanics being inserted into it. The near-field structural characteristics can be delivered by optical wave and material coupling. According to coupling and conduction physics, changes of the optical wave parameters can be calculated, and then curves of the image intensity can be obtained. By analyzing the near-field polarization parameters in nanoscale, indirect polarization parameter imaging can be established.

  7. Immunofluorescent examination of the skin of rabies-infected animals as a means of early detection of rabies virus antigen.

    PubMed Central

    Blenden, D C; Bell, J F; Tsao, A T; Umoh, J U

    1983-01-01

    Correlations were made on immunofluorescence positivity to antirabies conjugate between cranium-derived nerve fibers in skin and traditional samplings of brain tissue from several species and illness categories of animals with naturally acquired rabies. The overall correlation of results from all categories was about 98% (n, 104) for those that were brain positive and 100% (n, 99) for those that were brain negative. Some animals that ultimately developed rabies were found to have immunofluorescence-positive results 2 or more days before the onset of clinical signs in both natural and experimental infections. The percentage of those with positive skin immunofluorescence results increased as the onset of symptoms approached. From the midcourse period of illness to death, the correlation between skin and brain approached 100%. Different vaccines, commonly given to prevent rabies and other diseases of dogs and cats, were administered to groups of mice and were found to not produce false-positive results when their skin was examined by immunofluorescence for rabies virus antigen. These data suggest that examination of surgical biopsy specimens by immunofluorescence for rabies virus antigen is a useful and reliable diagnostic tool to evaluate the rabies status of biting dogs or cats, or to confirm a clinical diagnosis of rabies in the species tested. The biopsy evaluation of any other species as a means of assessing bite risk is not suggested by these data. PMID:6355152

  8. Dictionary of Microscopy

    NASA Astrophysics Data System (ADS)

    Heath, Julian

    2005-10-01

    The past decade has seen huge advances in the application of microscopy in all areas of science. This welcome development in microscopy has been paralleled by an expansion of the vocabulary of technical terms used in microscopy: terms have been coined for new instruments and techniques and, as microscopes reach even higher resolution, the use of terms that relate to the optical and physical principles underpinning microscopy is now commonplace. The Dictionary of Microscopy was compiled to meet this challenge and provides concise definitions of over 2,500 terms used in the fields of light microscopy, electron microscopy, scanning probe microscopy, x-ray microscopy and related techniques. Written by Dr Julian P. Heath, Editor of Microscopy and Analysis, the dictionary is intended to provide easy navigation through the microscopy terminology and to be a first point of reference for definitions of new and established terms. The Dictionary of Microscopy is an essential, accessible resource for: students who are new to the field and are learning about microscopes equipment purchasers who want an explanation of the terms used in manufacturers' literature scientists who are considering using a new microscopical technique experienced microscopists as an aide mémoire or quick source of reference librarians, the press and marketing personnel who require definitions for technical reports.

  9. Basic confocal microscopy.

    PubMed

    Smith, Carolyn L

    2011-07-01

    This unit introduces the reader to the basic principles of confocal microscopy and the design and capabilities of current confocal microscopes. The advantages and disadvantages of confocal microscopy compared to other techniques for fluorescence imaging are described. There are also practical guidelines for sample preparation and optimization of imaging parameters, as well as examples of some of the applications of confocal microscopy.

  10. Use of Peptide-Based Enzyme-Linked Immunosorbent Assay followed by Immunofluorescence Assay To Document Ehrlichia chaffeensis as a Cause of Febrile Illness in Nicaragua

    PubMed Central

    Chikeka, Ijeuru; Matute, Armando J.; Woods, Christopher W.; Mayorga, Orlando

    2016-01-01

    Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis (HME), has been extensively studied as a cause of acute febrile illness and an emerging tick-borne zoonosis in the United States. Limited data suggest its presence in other regions, including Central and South America but not Nicaragua to date. Diagnosis of E. chaffeensis infection by indirect immunofluorescence assay (IFA) is the reference standard due to its presumed high sensitivity and specificity, but IFA is impractical, variably reproducible, and cumbersome for large epidemiologic studies and for clinical diagnosis in resource-poor regions. We evaluated a high-throughput, objective peptide-based enzyme-linked immunosorbent assay (ELISA) for use alone or in combination with IFA. We found that it performed best as a screening test (sensitivity, 100%; specificity, 84%) to reduce the proportion of serum samples that were required by the more cumbersome and subjective IFA testing to <20%. Using a two-step diagnostic approach (IFA is performed if the ELISA is positive), we identified E. chaffeensis or a serologically and antigenically similar organism as a heretofore unrecognized cause of acute febrile illness in humans in Nicaragua and demonstrated the utility of the peptide ELISA as a screening tool for large-scale clinical studies. PMID:27053675

  11. Detection of Bacteroides fragilis, Bacteroides thetaiotaomicron, and Bacteroides ovatus in clinical specimens by immunofluorescence with a monoclonal antibody to B. fragilis lipopolysaccharide.

    PubMed Central

    Viljanen, M K; Linko, L; Lehtonen, O P

    1988-01-01

    A total of 1,897 clinical specimens (1,019 aspirates and 876 swabs) were studied by indirect immunofluorescence (IF) with a mouse monoclonal antibody (MAb) against a D-galactose oligomer of Bacteroides fragilis lipopolysaccharide. The MAb has been shown to react with 96% of clinical B. fragilis isolates and with about 50% of Bacteroides ovatus and Bacteroides thetaiotaomicron isolates but not with other aerobic or anaerobic organisms tested. The sensitivity of IF in comparison with culturing was 78.9% for all three species. Of the 32 strains originating from culture-positive, IF-negative specimens, 13 lacked the target determinant for the MAb. Sensitivity was highest with specimens taken from the perineal area (87.1%) and lowest with those taken from undefined sites (56.6%). Sensitivity was better with aspirates (86.8%) than with swabs (72.6%). The specificity of IF was 95.6% for all of the material. Positive and negative predictive values were 51.1 and 98.0%, respectively. Neither long transportation times of specimens nor antimicrobial therapy seemed to correlate with the occurrence of IF-positive, culture-negative specimens. This study shows that a single MAb can be used to establish an IF assay that can complement isolation in the detection of these three members of the B. fragilis group. Images PMID:3281973

  12. Indirect techniques in nuclear astrophysics: a review.

    PubMed

    Tribble, R E; Bertulani, C A; Cognata, M La; Mukhamedzhanov, A M; Spitaleri, C

    2014-10-01

    In this review, we discuss the present status of three indirect techniques that are used to determine reaction rates for stellar burning processes, asymptotic normalization coefficients, the Trojan Horse method and Coulomb dissociation. A comprehensive review of the theory behind each of these techniques is presented. This is followed by an overview of the experiments that have been carried out using these indirect approaches.

  13. 48 CFR 31.203 - Indirect costs.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... contract or other work, indirect costs are those remaining to be allocated to intermediate or two or more... by removing individual elements. All items properly includable in an indirect cost base shall bear a... unallowable, and these items shall bear their pro rata share of G&A costs. (e) The method of allocating...

  14. 29 CFR 452.119 - Indirect elections.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 2 2010-07-01 2010-07-01 false Indirect elections. 452.119 Section 452.119 Labor... STANDARDS GENERAL STATEMENT CONCERNING THE ELECTION PROVISIONS OF THE LABOR-MANAGEMENT REPORTING AND DISCLOSURE ACT OF 1959 Election Procedures; Rights of Members § 452.119 Indirect elections. National...

  15. Indirect Costs in Universities. ACE Special Report.

    ERIC Educational Resources Information Center

    Woodrow, Raymond J.

    Indirect costs of sponsored research projects and educational programs are as necessary as are the direct costs. This report demonstrates that they are real costs and that sponsors such as the Federal Government receive more than equitable treatment in the computation and application of indirect costs. The areas discussed include: the computation…

  16. 27 CFR 6.26 - Indirect interest.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Indirect interest. 6.26 Section 6.26 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS âTIED-HOUSEâ Unlawful Inducements Interest in Retail License § 6.26 Indirect interest. Industry member interest in...

  17. 27 CFR 6.32 - Indirect interest.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Indirect interest. 6.32 Section 6.32 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS âTIED-HOUSEâ Unlawful Inducements Interest in Retail Property § 6.32 Indirect interest. Industry member interest in...

  18. Sliding indirect hernia containing both ovaries.

    PubMed

    Fowler, Carol L

    2005-09-01

    Although sliding indirect inguinal hernias containing the ipsilateral ovary and fallopian tube are not uncommon in infant girls, sliding hernias containing both ovaries are rare. This report describes a large indirect inguinal hernia in a 1-year-old infant girl that contained the left uterine fundus, left bladder ear, as well as both ovaries and fallopian tubes.

  19. Photovoltaic efficiency of an indirect bandgap material

    NASA Astrophysics Data System (ADS)

    Tomasik, Michelle; Mangan, Niall; Grossman, Jeffrey

    2015-03-01

    Photovoltaic materials with direct band gap transitions absorb light more readily than those with indirect gaps, allowing for thinner devices. However, direct bands also suffer faster rates of radiative recombination than indirect bandgap materials. Some novel photovoltaic absorber materials, such as tin sulfide, have both direct and indirect gaps. Such materials raise the question of whether the multiple energy states benefit or harm device efficiency. We develop a model for current in a device with direct and indirect band gaps using detailed balance, similar to the Shockley-Quiesser model for direct band photovoltaics. We explore the effects of the following on device performance: transition probability of carriers between the direct and indirect state, and relative transport rate in each band.

  20. Gliding Motility of Babesia bovis Merozoites Visualized by Time-Lapse Video Microscopy

    PubMed Central

    Asada, Masahito; Goto, Yasuyuki; Yahata, Kazuhide; Yokoyama, Naoaki; Kawai, Satoru; Inoue, Noboru; Kaneko, Osamu; Kawazu, Shin-ichiro

    2012-01-01

    Background Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite that induces babesiosis in cattle after transmission by ticks. During specific stages of the apicomplexan parasite lifecycle, such as the sporozoites of Plasmodium falciparum and tachyzoites of Toxoplasma gondii, host cells are targeted for invasion using a unique, active process termed “gliding motility”. However, it is not thoroughly understood how the merozoites of B. bovis target and invade host red blood cells (RBCs), and gliding motility has so far not been observed in the parasite. Methodology/Principal Findings Gliding motility of B. bovis merozoites was revealed by time-lapse video microscopy. The recorded images revealed that the process included egress of the merozoites from the infected RBC, gliding motility, and subsequent invasion into new RBCs. The gliding motility of B. bovis merozoites was similar to the helical gliding of Toxoplasma tachyzoites. The trails left by the merozoites were detected by indirect immunofluorescence assay using antiserum against B. bovis merozoite surface antigen 1. Inhibition of gliding motility by actin filament polymerization or depolymerization indicated that the gliding motility was driven by actomyosin dependent process. In addition, we revealed the timing of breakdown of the parasitophorous vacuole. Time-lapse image analysis of membrane-stained bovine RBCs showed formation and breakdown of the parasitophorous vacuole within ten minutes of invasion. Conclusions/Significance This is the first report of the gliding motility of B. bovis. Since merozoites of Plasmodium parasites do not glide on a substrate, the gliding motility of B. bovis merozoites is a notable finding. PMID:22506073

  1. Diagnostic value of eccrine glands and hair follicles in direct immunofluorescent analysis of pemphigus vulgaris and bullous pemphigoid.

    PubMed

    Zhou, Cheng; Yu, Yan; Elston, Dirk M

    2016-04-01

    The immunofluorescence pattern in adnexal structures may be of value, especially when the epidermis is not well represented in diagnostic sections. We studied a total of 88 cases of pemphigus vulgaris (PV) and bullous pemphigoid (BP) accessioned between 2010 and 2015 (40 cases of PV and 48 cases of BP). Immunofluorescence patterns and sensitivity in adnexal structures were similar to those observed in the epidermis. One case of PV and three cases of BP showed weak or absent fluorescence in the epidermis, while the eccrine glands were strongly positive, suggesting that careful examination of adnexal structures can be of value.

  2. Immunofluorescence in skin specimens from three different biopsy sites in patients with scleroderma.

    PubMed

    Chen, Z Y; Dobson, R L; Ainsworth, S K; Silver, R M; Rust, P F; Maricq, H R

    1985-01-01

    Immunofluorescence (IF) data from three different biopsy sites (nailfold, forearm, buttock) were studied in 18 patients with scleroderma (SD, systemic sclerosis) and the results compared with those obtained from 10 normal controls (NC) and 7 patients with systemic lupus erythematosus (SLE). Immunoglobulin (Ig) deposits were detected by direct IF technique at the dermo-epidermal junction (DEJ) in 8/14 nailfolds, 6/15 forearms and in none of the buttock specimens of SD patients. Epidermal nuclear staining was present in 6/14 nailfolds, and in 6/15 forearms and buttocks. The most prominent finding was the observation of multiple Ig deposits in the cuticle of 9/14 patients with SD. NC group was negative in all sites for epidermal nuclear staining and the only DEJ deposit occurred in the forearm of one subject. In conclusion, this study demonstrates that Ig deposits in SD, both at the DEJ and in the epidermal nuclei, occur more often than previously reported and are especially frequent in the nailfold & cuticle area.

  3. Segment and fit thresholding: a new method for image analysis applied to microarray and immunofluorescence data.

    PubMed

    Ensink, Elliot; Sinha, Jessica; Sinha, Arkadeep; Tang, Huiyuan; Calderone, Heather M; Hostetter, Galen; Winter, Jordan; Cherba, David; Brand, Randall E; Allen, Peter J; Sempere, Lorenzo F; Haab, Brian B

    2015-10-06

    Experiments involving the high-throughput quantification of image data require algorithms for automation. A challenge in the development of such algorithms is to properly interpret signals over a broad range of image characteristics, without the need for manual adjustment of parameters. Here we present a new approach for locating signals in image data, called Segment and Fit Thresholding (SFT). The method assesses statistical characteristics of small segments of the image and determines the best-fit trends between the statistics. Based on the relationships, SFT identifies segments belonging to background regions; analyzes the background to determine optimal thresholds; and analyzes all segments to identify signal pixels. We optimized the initial settings for locating background and signal in antibody microarray and immunofluorescence data and found that SFT performed well over multiple, diverse image characteristics without readjustment of settings. When used for the automated analysis of multicolor, tissue-microarray images, SFT correctly found the overlap of markers with known subcellular localization, and it performed better than a fixed threshold and Otsu's method for selected images. SFT promises to advance the goal of full automation in image analysis.

  4. New insights on ctenophore neural anatomy: immunofluorescence study in Pleurobrachia pileus (Müller, 1776).

    PubMed

    Jager, Muriel; Chiori, Roxane; Alié, Alexandre; Dayraud, Cyrielle; Quéinnec, Eric; Manuel, Michaël

    2011-05-15

    Ctenophores are non-bilaterian animals sharing with cnidarians and bilaterians the presence of sensory receptors, nerve cells, and synapses, absent in placozoans and sponges. Although recent immunofluorescence studies have renewed our knowledge of cnidarian neuro-anatomy, ctenophores have been much less investigated despite their importance to understanding the origin and early evolution of the nervous system. In this study, the neuro-anatomy of the ctenophore Pleurobrachia pileus (Müller, 1776) was explored by whole-mount fluorescent antibody staining using antibodies against tyrosylated -tubulin, FMRFamide, and vasopressin. We describe the morphology of nerve nets and their local specializations, and the organization of the aboral neuro-sensory complex comprising the apical organ and polar fields. Two distinct nerve nets are distinguished: a mesogleal nerve net, loosely organized throughout body mesoglea, and a much more compact “nerve net” with polygonal meshes in the ectodermal epithelium. The latter is organized as a plexus of short nerve cords. This epithelial nervous system contains distinct sub-populations of dispersed FMRFamide and vasopressin immunoreactive nerve cells. In the aboral neuro-sensory complex, our most significant observations include specialized nerve nets underlying the apical organ and polar fields, a tangential bundle of actin-rich fibers (interpreted as a muscle) within the polar fields, and distinct groups of neurons labeled by anti-FMRFamide and anti-vasopressin antibodies, within the apical organ floor. These results are discussed in a comparative perspective. Copyright © 2011 Wiley-Liss, Inc., A Wiley Company.

  5. Clinicopathological Significance among Patients with In vivo Epidermal Nuclear Staining by Direct Immunofluorescence Study

    PubMed Central

    Sirikudta, Wararat; Tuchinda, Papapit; Chularojanamontri, Leena; Kulthanan, Kanokvalai; Pinkaew, Samruay

    2017-01-01

    Context: In vivo epidermal nuclear staining (ENS) can be found in patients with autoimmune connective tissue diseases (CNTDs) and other diseases. Aims: The aim of this study was to reveal the underlying diseases and direct immunofluorescence (DIF) characters of patients with in vivo ENS and association of in vivo ENS with circulating autoantibodies. Settings and Design: A retrospective analysis was conducted involving skin biopsy specimens submitted for DIF study at the Dermatoimmunology Laboratory at Siriraj Hospital between 2002 and 2012. Subjects and Methods: The findings of DIF study, clinical manifestations, and diagnosis of patients who had positive ENS were investigated. Statistical Analysis Used: The SPSS software version 18.0. Descriptive statistics were used to report demographic data, clinical characteristics, and laboratory investigation results. Moreover, Chi-squared test or Fisher's exact test were used to compare the categorical variables. Results: One hundred and thirty-eight out of 3735 submitted specimens (3.7%) showed positive ENS. The most common diagnosis was CNTD (79%) followed by vasculitis (10.1%). Lupus erythematosus was the most common diagnosis among CNTD (78%). No association between levels of serum antinuclear antibodies (ANA) titer and intensity of ENS (P = 0.660). However, we found that patients with positive in vivo ANA had lower prevalence of systemic involvement. Conclusions: Although lupus erythematosus was the most common diagnosis among patients with in vivo ENS, the presence of ENS does not indicate any specific diagnosis. However, patients with ENS tend to have less systemic involvement. PMID:28400653

  6. Regeneration of axotomized olfactory neurons in young and adult locusts quantified by fasciclin I immunofluorescence.

    PubMed

    Wasser, Hannah; Biller, Alexandra; Antonopoulos, Georgios; Meyer, Heiko; Bicker, Gerd; Stern, Michael

    2017-04-01

    The olfactory pathway of the locust Locusta migratoria is characterized by a multiglomerular innervation of the antennal lobe (AL) by olfactory receptor neurons (ORNs). After crushing the antenna and thereby severing ORN axons, changes in the AL were monitored. First, volume changes were measured at different times post-crush with scanning laser optical tomography in 5th instar nymphs. AL volume decreased significantly to a minimum volume at 4 days post-crush, followed by an increase. Second, anterograde labeling was used to visualize details in the AL and antennal nerve (AN) during de- and regeneration. Within 24 h post-crush (hpc) the ORN fragments distal to the lesion degenerated. After 48 hpc, regenerating fibers grew through the crush site. In the AL, labeled ORN projections disappeared completely and reappeared after a few days. A weak topographic match between ORN origin on the antenna and the position of innervated glomeruli that was present in untreated controls did not reappear after regeneration. Third, the cell surface marker fasciclin I that is expressed in ORNs was used for quantifying purposes. Immunofluorescence was measured in the AL during de- and regeneration in adults and 5th instar nymphs: after a rapid but transient, decrease, it reappeared. Both processes happen faster in 5th instar nymphs than in adults.

  7. Is psoriasis an autoimmune disease? Interpretations from an immunofluorescence-based study.

    PubMed

    Anand, Shashi; Gupta, Parikshaa; Bhardwaj, Ranjeet; Narang, Tarun; Dogra, Sunil; Minz, Ranjana W; Saikia, Biman; Chhabra, Seema

    2017-04-01

    Psoriasis is a multisystem, immune-mediated inflammatory disease. Some authors have proposed an autoimmune basis for psoriasis; however, till date, it has not been definitely established. This study was conducted to explore the autoimmune nature of psoriasis. This was a prospective study in which 43 psoriasis patients were assessed for detailed clinical, histopathological and immunopathological features to explore the diagnostic utility of subtypes, intensity and number of immunoreactants in lesional and non-lesional skin in these patients. In addition, the sera of these patients were analyzed for the presence of various autoantibodies. The patients' age ranged from 14 to 75 years with a male-to-female ratio of 1.52:1. Nine patients (20.93%) were positive for antinuclear and 2 (4.65%) for antismooth muscle antibodies. Direct immunofluorescence (DIF) was positive in 31 (72%) biopsies from the lesional and 27 (63%) biopsies from non-lesional skin. In all these DIF positive cases, granular deposits of C5b-9 were detected at the dermoepidermal junction. No significant difference was observed on comparing the type and pattern of immunoreactant positivity, among lesional and non-lesional skin biopsies (P > .05). No significant association between psoriasis and immunoreactant deposition as well as autoantibody seroprevalence was observed, thereby refuting a definite autoimmune basis for psoriasis. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Vimentin in cultured chromaffin cells: an immunofluorescent, biochemical and functional study.

    PubMed

    Quintanar, J L

    2000-01-01

    In tile present study we seek the presence and possible function of the intermediate filament protein vimentin in adrenomedullary chromaffin cells. Vimentin which is not present in the adrenal medulla was clearly showed up after collagenase digestion of the gland in the cultured chromaffin cells by using an immunofluorescent analysis with double cell labeling with monoclonal antibodies against vimentin and dopamine-beta-hydroxylase. Vimentin was also shown to be phosphorylated in a calcium-dependent manner by acetylcholine. The specific protein phosphatase inhibitor calyculin-A, that has been previously shown to increase vimentin phosphorylation, caused a change in the distribution of vimentin which moved from the Triton X-100 insoluble cytoskeletal preparation to the detergent soluble fraction probably as a result of modifications in filament integrity. The possible role of vimentin in secretion was in addition investigated using digitonin-permeabilized cells, in which the specific antibody for vimentin partially inhibited calcium-induced catecholamine release. These results demonstrate the induction of vimentin expression after collagenase digestion in cultured chromaffin cells and suggest that in these conditions this protein is possibly implicated in the regulation of the secretory process through a phosphorylation-dependent mechanism.

  9. Immunofluorescent studies of the local immune response in the mammary glands of rats.

    PubMed Central

    Lee, C G; Ladds, P W; Watson, D L; Goddard, M E

    1979-01-01

    Two irritants, phosphate-buffered saline and alcohol, and antigens including killed Brucella abortus, live B. abortus, Staphylococcus aureus, and rat parvovirus were separately infused into rat mammary glands during pregnancy, and by using immunofluorescent techniques, the numbers of immunoglobulin-containing cells in glands during lactation and involution were determined. The study provided basic information on the local immune response of the mammary gland to antigens of various types. In all experiments, the number of immunoglobulin M (IgM) cells present was small and no trends were apparent. IgA cells were always more prevalent than IgG cells. Fewer IgA cells were in the glands of rats infused with phosphate-buffered saline and alcohol than in normal rats. IgA cell prevalence was greatest in response to infusion of live B. abortus. Responses to live S. aureus and parvovirus were less pronounced, and infusion of killed B. abortus did not induce an elevation in IgA cell prevalence. IgG cell prevalence was greatest in response to infusion of live B. abortus or S. aureus and was decreasingly less pronounced in response to killed B. abortus and rat parvovirus. With the exception of parvovirus infusion, in regard to IgA cells, all glands locally infused with antigen had elevated IgA and IgG cell numbers when compared with noninfused glands in the same animal. Images PMID:106012

  10. Analysis of chromosome segregation during mammalian meiosis using combined immunofluorescence and fluorescence in situ hubridization

    SciTech Connect

    Hunt, P.A.; Embury, P.B.; Mroz, K.M.

    1994-09-01

    Meiotic non-disjunction is thought to occur in 10-20% of all human oocytes, making this the most common genetic abnormality in our species. Aberrant recombination has been implicated in the genesis of these errors; however, direct studies of the meiotic process have been hampered by the lack of material and appropriate technology. We have developed a technique for the evaluation of meiosis in intact mammalian oocytes that combines immunofluorescence and fluorescence in situ hybridization (FISH). This allows for simultaneous, 3-dimensional visualization of the meiotic spindle, the alignment of the chromosomes on the spindle, and the placement of specific chromosomes. We have used this technology to follow meiotic progression in oocytes from XO female mice to evaluate the behavior of an unsynapsed chromosome during mammalian meiosis. Perturbations in chromosome behavior are evident early in meiosis: during the formation of the first meiotic spindle, the univalent X chromosome is properly positioned. With the onset of anaphase, the single X chromosome most commonly segregates as an intact chromosome, although equational segregation of the X chromatids is seen in a significant minority (approximately 20%) of oocytes. These observations demonstrate that failure of pairing/recombination can result in segregation of sister chromatids at meiosis I. This has obvious implications for human non-disjunction, much of which is thought to be due to recombination deficiencies; accordingly, we are now extending our studies to include analyses of human oocytes.

  11. Distribution of crossing over on mouse synaptonemal complexes using immunofluorescent localization of MLH1 protein.

    PubMed Central

    Anderson, L K; Reeves, A; Webb, L M; Ashley, T

    1999-01-01

    We have used immunofluorescent localization to examine the distribution of MLH1 (MutL homolog) foci on synaptonemal complexes (SCs) from juvenile male mice. MLH1 is a mismatch repair protein necessary for meiotic recombination in mice, and MLH1 foci have been proposed to mark crossover sites. We present evidence that the number and distribution of MLH1 foci on SCs closely correspond to the number and distribution of chiasmata on diplotene-metaphase I chromosomes. MLH1 foci were typically excluded from SC in centromeric heterochromatin. For SCs with one MLH1 focus, most foci were located near the middle of long SCs, but near the distal end of short SCs. For SCs with two MLH1 foci, the distribution of foci was bimodal regardless of SC length, with most foci located near the proximal and distal ends. The distribution of MLH1 foci indicated interference between foci. We observed a consistent relative distance (percent of SC length in euchromatin) between two foci on SCs of different lengths, suggesting that positive interference between MLH1 foci is a function of relative SC length. The extended length of pachytene SCs, as compared to more condensed diplotene-metaphase I bivalents, makes mapping crossover events and interference distances using MLH1 foci more accurate than using chiasmata. PMID:10101178

  12. Identification of mouse mammary epithelial cells by immunofluorescence with rabbit and guinea pig antikeratin antisera.

    PubMed Central

    Asch, B B; Burstein, N A; Vidrich, A; Sun, T T

    1981-01-01

    Few markers are available to identify the three types of mammary epithelial cells--ductal, alveolar, and myoepithelial--especially in pathological conditions and in cell cultures. We have used antisera to human keratins in immunofluorescence to facilitate the identification of the three mouse mammary epithelial cell types. In frozen tissue sections and primary cell cultures, a rabbit antikeratin antiserum specifically stained cytoplasmic filaments in all three types of epithelial cells. A guinea pig antiserum against the same keratin preparation, however, reacted preferentially with filaments in myoepithelial cells and readily detected this cell type in normal, dysplastic, and malignant mammary tissues and cell cultures. Neither antisera reacted with fibroblasts or any other mesenchymal cells. The combined use of the two antikeratin antisera thereby permits rapid surveys of tissue sections and cultures for the localization of not only all epithelial cells but also the subpopulation of myoepithelial cells. Moreover, when mammary cultures established from late-pregnant or lactating mice were stained simultaneously with guinea pig antikeratin and rabbit anticasein antisera, three populations of epithelial cells were mutually exclusive: those stained by anticasein antiserum, those stained by guinea pig antikeratin antiserum, and those stained by neither, consistent with properties of alveolar, myoepithelial, and ductal cells, respectively. These antisera thus offer a tool for studying different epithelial cell types during mammary development, tumorigenesis, and malignant progression. Images PMID:6170984

  13. Immunofluorescent characterization of meiotic recombination in human males with variable spermatogenesis.

    PubMed

    de Vries, M; Ramos, L; de Boer, P

    2013-03-01

    Homologous recombination is the key to meiotic functioning. The basis of this process is provided by numerous SPO11-induced DNA double-strand breaks. Repair of these breaks occurs via the crossover (CO) and non-crossover (NCO) pathways. By means of immunofluorescence staining of Replication protein A (RPA) and MutL homolog 1 (MLH1) in combination with the DNA damage marker γH2AX, we studied transitional (CO and NCO) and late (CO) recombination nodules, respectively. Testicular samples were from non-obstructive azoospermic probands (testicular spermatozoa were found) and probands that had a history of normal fertility prior to a vasectomy. All probands were ICSI candidates. γH2AX foci mostly colocalized with delayed transitional nodules (RPA) for which variation was found among probands. Highest incidences of colocalization were found in patients. The level of MLH1 signal intensity was lower in probands who showed more frequent γH2AX RPA colocalization in late pachytene, suggesting communication between the CO and NCO pathways. Our results suggest the presence of a genetic risk pathway for children conceived from non-obstructive azoospermic probands and urge for follow-up studies investigating the level of recombination involved de novo mutations in these children.

  14. Dual immunofluorescence study of citrullinated proteins in Parkinson diseased substantia nigra.

    PubMed

    Nicholas, Anthony P

    2011-05-09

    Deimination is a post-translational modification of proteins in which selected arginine amino acids are enzymatically converted to citrullines. Using dual-color immunofluorescence and an established monoclonal antibody (F95) against peptidyl-citrulline moieties, the present study is the first to compare immunohistochemical staining patterns for deiminated proteins in human substantia nigra (SN) from patients with Parkinson disease (PD) versus similar control specimens supplied by the Harvard Brain Bank. In control SN sections, many tyrosine hydroxylase (TH)-immunoreactive dopamine neurons were seen surrounded either by small fibers immunoreactive for deiminated proteins, or large reactive astrocytes, co-localized with glial fibrillary acidic protein (GFAP). However, in SN specimens from PD patients, immunoreactivity for deiminated proteins was also demonstrated within the cytoplasm of many surviving dopamine neurons that were also immunoreactive for TH, but this staining was not specifically restricted to Lewy bodies. Although the identity of neuronal deiminated proteins in these SN dopamine neurons is unknown, the present study provides evidence that the anatomical expression of these proteins in PD is altered and thus suggests that deimination may be involved in the pathophysiology of this disease. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  15. Fatal viscerotropic Rocky Mountain spotted fever. Report of a case diagnosed by immunofluorescence.

    PubMed

    Green, W R; Walker, D H; Cain, B G

    1978-03-01

    A case of fatal viscerotropic Rocky Mountain spotted fever with virtual absence of cutaneous lesions was diagnosed at autopsy by specific immunofluorescent demonstration of Rickettsia rickettsii in spleen, kidney, epididymis and skin. The clinical presentation was that of insidious onset of fever, renal failure, hypotension, hyponatremia and obtundation over a 10 day period. The patient had respiratory insufficiency, hypocalcemia, increases in creatinine phosphokinase (CPK), serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), lactic dehydrogenase (LDH), alkaline phosphatase, billirubin and serum phosphate, grand mal seizure, myalgia and unremitting shock with death occurring on day 12 of illness. Postmortem examination revealed severe vasculitis with interstitial nephritis and multifocal tubular necrosis, pericholangitis with bile stasis, glial nodules in the brain, multifocal rhabdomyonecrosis, interstitial pneumonitis and mild interstitial myocarditis. Risk factors which this patient shared with other patients with fatal Rocky Mountain spotted fever were failure to recognize a rash, failure to obtain a tick bite history, male sex, black race and age greater than 30 years.

  16. Immunofluorescence localization of the receptor Mas in cardiovascular-related areas of the rat brain.

    PubMed

    Becker, Lenice K; Etelvino, Gisele M; Walther, Thomas; Santos, Robson A S; Campagnole-Santos, Maria J

    2007-09-01

    The G protein-coupled receptor Mas was recently described as an angiotensin-(1-7) [ANG-(1-7)] receptor. In the present study we evaluated the anatomical localization of Mas using immunofluorescence in the central nervous system of adult male Wistar rats. An abundant labeling was found in the hippocampus, amigdala, anterodorsal thalamic nucleus, cortex, and hypoglossal nucleus. More importantly, a dense ANG-(1-7) receptor Mas immunoreactivity was observed in cardiovascular-related areas of the medulla and forebrain, shown in several previous studies as sites for the action of ANG-(1-7) in the brain. A strong staining was found in the nucleus of the solitary tract, caudal and rostral ventrolateral medulla, inferior olive, parvo and magnocellular portions of the paraventricular hypothalamic nucleus, supraoptic nucleus, and lateral preoptic area. Furthermore, Mas staining was predominantly present in neurons. At the medullary sites, a specific and high-intensity binding for rhodamine-ANG-(1-7) was also shown. The specific ANG-(1-7) binding was completely displaced by the anti-Mas antibody or by the ANG-(1-7) antagonist, A-779. The data presented provide the first anatomical basis for the physiological role of ANG-(1-7)/Mas axis in the modulation of different cardiovascular functions and give new insights for clarifying the role of ANG-(1-7) in the central nervous system.

  17. Segment and Fit Thresholding: A New Method for Image Analysis Applied to Microarray and Immunofluorescence Data

    PubMed Central

    Ensink, Elliot; Sinha, Jessica; Sinha, Arkadeep; Tang, Huiyuan; Calderone, Heather M.; Hostetter, Galen; Winter, Jordan; Cherba, David; Brand, Randall E.; Allen, Peter J.; Sempere, Lorenzo F.; Haab, Brian B.

    2016-01-01

    Certain experiments involve the high-throughput quantification of image data, thus requiring algorithms for automation. A challenge in the development of such algorithms is to properly interpret signals over a broad range of image characteristics, without the need for manual adjustment of parameters. Here we present a new approach for locating signals in image data, called Segment and Fit Thresholding (SFT). The method assesses statistical characteristics of small segments of the image and determines the best-fit trends between the statistics. Based on the relationships, SFT identifies segments belonging to background regions; analyzes the background to determine optimal thresholds; and analyzes all segments to identify signal pixels. We optimized the initial settings for locating background and signal in antibody microarray and immunofluorescence data and found that SFT performed well over multiple, diverse image characteristics without readjustment of settings. When used for the automated analysis of multi-color, tissue-microarray images, SFT correctly found the overlap of markers with known subcellular localization, and it performed better than a fixed threshold and Otsu’s method for selected images. SFT promises to advance the goal of full automation in image analysis. PMID:26339978

  18. Quantitative Immunofluorescence of Regulated eps Gene Expression in Single Cells of Ralstonia solanacearum

    PubMed Central

    Kang, Yaowei; Saile, Elke; Schell, Mark A.; Denny, Timothy P.

    1999-01-01

    Ralstonia solanacearum, a phytopathogenic bacterium, uses an environmentally sensitive and complex regulatory network to control expression of multiple virulence genes. Part of this network is an unusual autoregulatory system that produces and senses 3-hydroxypalmitic acid methyl ester. In culture, this autoregulatory system ensures that expression of virulence genes, such as those of the eps operon encoding biosynthesis of the acidic extracellular polysaccharide, occurs only at high cell density (>107 cells/ml). To determine if regulation follows a similar pattern within tomato plants, we first developed a quantitative immunofluorescence (QIF) method that measures the relative amount of a target protein within individual bacterial cells. For R. solanacearum, QIF was used to determine the amount of β-galactosidase protein within wild-type cells containing a stable eps-lacZ reporter allele. When cultured cells were examined to test the method, QIF accurately detected both low and high levels of eps gene expression. QIF analysis of R. solanacearum cells recovered from stems of infected tomato plants showed that expression of eps during pathogenesis was similar to that in culture. These results suggest that there are no special signals or conditions within plants that override or short-circuit the regulatory processes observed in R. solanacearum in culture. Because QIF is a robust, relatively simple procedure that uses generally accessible equipment, it should be useful in many situations where gene expression in single bacterial cells must be determined. PMID:10347013

  19. Serological detection of Helicobacter pylori by a flow microsphere immunofluorescence assay.

    PubMed Central

    Best, L M; Veldhuyzen van Zanten, S J; Bezanson, G S; Haldane, D J; Malatjalian, D A

    1992-01-01

    A flow cytometric immunofluorescence assay (FMIA) for the detection of immunoglobulin G antibodies to Helicobacter pylori was developed. A multicomponent antigen was prepared and used to coat carboxylated polystyrene microspheres for reaction with patient sera followed by fluorescein isothiocyanate-labelled goat anti-human immunoglobulin G. The reacted microspheres were collected with a flow cytometer, and fluorescence was quantitated relative to the cutoff value provided by pooled sera from patients in whom H. pylori could not be demonstrated by culture or histology. Serum samples from 28 H. pylori-positive patients and 27 H. pylori-negative patients were tested by FMIA. Additionally, an in-house enzyme-linked immunosorbent assay (ELISA) employing the same antigen preparation and a commercially available ELISA were used to assay the patient population. Both the FMIA and in-house ELISA were 100% sensitive and 89% specific with positive and negative predictive values of 90 and 100% and no equivocal results. The commercial ELISA was 96% sensitive and 89% specific with positive and negative predictive values of 90 and 96% and five equivocal results. FMIA provides a rapid, inexpensive, and easily performed means for serodiagnosis of H. pylori. Images PMID:1400995

  20. Immunofluorescent localization of enteroglucagon cells in the gastrointestinal tract of the dog

    PubMed Central

    Polak, Julia M.; Bloom, S.; Coulling, I.; Pearse, A. G. E.

    1971-01-01

    Localization of the endocrine polypeptide cells responsible for `glucagon-like immunoreactivity' in the gastrointestinal tract of the dog has been achieved with an immunofluorescent technique using antibodies raised against porcine pancreatic glucagon. The cells, for which we prefer the term `enteroglucagon', could only be demonstrated by this technique in tissues fixed in carbodiimide. The enteroglucagon cells possess cytological, cytochemical, and ultrastructural characteristics in common with those of the pancreatic α2 cell and they are equivalent in the stomach to the A cell and in the intestine to the L cell of the Wiesbaden terminology. Their distribution, predominantly in fundus and jejunum, correlates precisely with the distribution of glucagon-like immunoreactivity by radioimmunoassay and bioassay. The storage form of enteroglucagon differs in many respects from that of pancreatic glucagon although there are some close resemblances between the two forms of specific hormone-containing granule. Elucidation of the role of enteroglucagon should be assisted by the ability to demonstrate enteroglucagon cells. ImagesFig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7 PMID:4930155

  1. Immunofluorescence assay method to detect dengue virus in Paniai-Papua

    NASA Astrophysics Data System (ADS)

    Sucipto, Teguh Hari; Ahwanah, Nur Laila Fitriati; Churrotin, Siti; Matake, Norifumi; Kotaki, Tomohiro; Soegijanto, Soegeng

    2016-03-01

    The dengue viruses (DENV), which include in the family Flaviviridae and the genus Flavivirus, was endemic in tropical areas and had been transmitted to humans by Aedes aegypti. An increasing number of immigrants from endemic areas to the non-endemic areas have emphasized the need for a simple and reliable test for the diagnosis of dengue virus infection. The purpose of this study was to detect the dengue virus by immunofluorescence assay (IFA) in the general population at Paniai-Papua. The results obtained from this study had showed a significantly better discrimination for DENV specific IgG antibodies. A total of 158 samples, 116 samples were IgG antibodies positive and 42 samples were negative. The conclusion of this study, Papua is not only a malaria endemic area, but also dengue virus infections were detected by IFA method. Therefore, the IFA can be used as an important diagnostic tool, which is a quick and an easy way to test samples from immigrants who come to the non-endemic areas.

  2. Collective phenomena in cold indirect excitons

    SciTech Connect

    Butov, L. V.

    2016-03-15

    Due to their long lifetimes, indirect excitons can cool to below the temperature of quantum degeneracy. This gives an opportunity to experimentally study cold composite bosons. Both theoretically predicted phenomena and phenomena that have not been anticipated were observed in a cold gas of indirect excitons. In this contribution, we overview our studies of cold indirect excitons over the past decade, presenting spontaneous coherence and condensation of excitons, spatially modulated exciton state, long-range spin currents and spin textures, and exciton localization–delocalization transitions.

  3. [Introduction to the indirect meta-analyses].

    PubMed

    Bolaños Díaz, Rafael; Calderón Cahua, María

    2014-04-01

    Meta-analyses are studies that aim to compile all available information, grouping them according to an specific theme and evaluating it through methodological quality tools. When there are two specific comparisons of treatments based on randomized clinical trials, standard meta-analyses are the best option, but there are scenarios in which there is no available literature for those direct comparisons. In these cases, an alternative method to consider is indirect comparison or indirect meta-analyses. The aim of this review is to understand the conceptual foundations, the need, applications and limitations of indirect comparisons for further understanding of network meta-analyses.

  4. Capillary electrophoresis with indirect amperometric detection.

    PubMed

    Olefirowicz, T M; Ewing, A G

    1990-01-19

    The use of indirect amperometric detection with capillary electrophoresis is demonstrated. The system consists of a porous glass coupler which allows amperometric detection at a carbon fiber electrode placed in the end of the capillary. 3,4-Dihydroxybenzylamine is added to the buffer system as a continuously eluting electrophore. Indirect amperometric detection in 9-mumol I.D. capillaries provides detection limits as low as 380 attomole for the amino acid arginine. Finally, both direct and indirect amperometric detection can be accomplished simultaneously.

  5. In vivo antinuclear antibodies of the skin: A rare phenomenon on direct immunofluorescence.

    PubMed

    Chhabra, Seema; Arora, Sandeep Kumar; Minz, Ranjana Walker; Dogra, Suneel

    2013-04-15

    Detection of immunoglobulins in epidermal cell nuclei or in vivo antinuclear antibodies on direct immunoflorescence microscopy of skin biopsies is an easily detectable immunopathologic feature. It is an unusual, but not totally rare, occurrence in systemic connective tissue disorders. This positive epidermal nuclear reaction is found to be commonly associated with immunoglobulin G.

  6. Basic confocal microscopy.

    PubMed

    Smith, Carolyn L

    2008-01-01

    This unit introduces the reader to the basic principles of confocal microscopy and the design and capabilities of current confocal microscopes. The advantages and disadvantages of confocal microscopy compared to other techniques for fluorescence imaging are described. There are also practical guidelines for sample preparation and optimization of imaging parameters, as well as examples of some of the applications of confocal microscopy. (c) 2008 by John Wiley & Sons, Inc.

  7. Fluorescence (Multiwave) Confocal Microscopy.

    PubMed

    Welzel, J; Kästle, Raphaela; Sattler, Elke C

    2016-10-01

    In addition to reflectance confocal microscopy, multiwave confocal microscopes with different laser wavelengths in combination with exogenous fluorophores allow fluorescence mode confocal microscopy in vivo and ex vivo. Fluorescence mode confocal microscopy improves the contrast between the epithelium and the surrounding soft tissue and allows the depiction of certain structures, like epithelial tumors, nerves, and glands. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Correlation mapping microscopy

    NASA Astrophysics Data System (ADS)

    McGrath, James; Alexandrov, Sergey; Owens, Peter; Subhash, Hrebesh M.; Leahy, Martin J.

    2015-03-01

    Changes in the microcirculation are associated with conditions such as Raynauds disease. Current modalities used to assess the microcirculation such as nailfold capillaroscopy are limited due to their depth ambiguity. A correlation mapping technique was recently developed to extend the capabilities of Optical Coherence Tomography to generate depth resolved images of the microcirculation. Here we present the extension of this technique to microscopy modalities, including confocal microscopy. It is shown that this correlation mapping microscopy technique can extend the capabilities of conventional microscopy to enable mapping of vascular networks in vivo with high spatial resolution.

  9. Light sheet microscopy.

    PubMed

    Weber, Michael; Mickoleit, Michaela; Huisken, Jan

    2014-01-01

    This chapter introduces the concept of light sheet microscopy along with practical advice on how to design and build such an instrument. Selective plane illumination microscopy is presented as an alternative to confocal microscopy due to several superior features such as high-speed full-frame acquisition, minimal phototoxicity, and multiview sample rotation. Based on our experience over the last 10 years, we summarize the key concepts in light sheet microscopy, typical implementations, and successful applications. In particular, sample mounting for long time-lapse imaging and the resulting challenges in data processing are discussed in detail.

  10. Axial Plane Optical Microscopy

    PubMed Central

    Li, Tongcang; Ota, Sadao; Kim, Jeongmin; Wong, Zi Jing; Wang, Yuan; Yin, Xiaobo; Zhang, Xiang

    2014-01-01

    We present axial plane optical microscopy (APOM) that can, in contrast to conventional microscopy, directly image a sample's cross-section parallel to the optical axis of an objective lens without scanning. APOM combined with conventional microscopy simultaneously provides two orthogonal images of a 3D sample. More importantly, APOM uses only a single lens near the sample to achieve selective-plane illumination microscopy, as we demonstrated by three-dimensional (3D) imaging of fluorescent pollens and brain slices. This technique allows fast, high-contrast, and convenient 3D imaging of structures that are hundreds of microns beneath the surfaces of large biological tissues. PMID:25434770

  11. Detection of Bartonella quintana by Direct Immunofluorescence Examination of Blood Smears of a Patient with Acute Trench Fever

    PubMed Central

    Foucault, C.; Rolain, J. M.; Raoult, D.; Brouqui, P.

    2004-01-01

    We report a case of Bartonella quintana acute symptomatic infection in a homeless man, presenting as a typical trench fever. B. quintana has been retrieved in erythrocytes in large clusters and in erythroblasts. Direct immunofluorescence of blood smears allows a rapid diagnosis. PMID:15472378

  12. Indirect Lighting--a Matter of Economics

    ERIC Educational Resources Information Center

    Modern Schools, 1977

    1977-01-01

    Recent developments in the field of indirect lighting and the use of high intensity discharge light sources reveal that the most efficient lighting system can also be the most economical. (Author/MLF)

  13. Outcome indicators for direct and indirect caregiving.

    PubMed

    Schoenfelder, D P; Swanson, E A; Specht, J K; Maas, M; Johnson, M

    2000-02-01

    Informal caregiving and outcomes for caregiving are an important part of health care and of particular importance in nursing. The purpose of this research is to report the results of a survey mailed to nursing experts for validation of the outcome labels Caregiver Role Performance: Direct Care and Caregiver Role Performance: Indirect Care and their accompanying indicators. Experts were asked to rate how important the identified indicators were for assessing those two outcomes. In addition, the respondents were asked to what extent nursing interventions influence the achievement of each identified indicator for Caregiver Role Performance: Direct Care and Caregiver Role Performance: Indirect Care. In general, the validity of the concept analysis work by the caregiver focus group was supported. Ten indicators for Caregiver Performance: Direct Care were retained, 1 was dropped that was considered most appropriate for indirect care, and 3 new indicators were added to reflect the nurse experts surveyed. For Caregiver Performance: Indirect Care, all of the indicators were retained.

  14. Indirect punishment and generosity toward strangers.

    PubMed

    Ule, Aljaz; Schram, Arthur; Riedl, Arno; Cason, Timothy N

    2009-12-18

    Many people incur costs to reward strangers who have been kind to others. Theoretical and experimental evidence suggests that such "indirect rewarding" sustains cooperation between unrelated humans. Its emergence is surprising, because rewarders incur costs but receive no immediate benefits. It can prevail in the long run only if rewarders earn higher payoffs than "defectors" who ignore strangers' kindness. We provide experimental evidence regarding the payoffs received by individuals who employ these and other strategies, such as "indirect punishment," by imposing costs on unkind strangers. We find that if unkind strangers cannot be punished, defection earns most. If they can be punished, however, then indirect rewarding earns most. Indirect punishment plays this important role, even if it gives a low payoff and is rarely implemented.

  15. 19 CFR 18.43 - Indirect exportation.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Under Cover of A Tir Carnet § 18.43 Indirect exportation. (a) When merchandise is to move from one U.S... loading, sealing or labeling, and affixing of TIR plates. He shall remove one voucher from the carnet...

  16. 19 CFR 18.43 - Indirect exportation.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Under Cover of A Tir Carnet § 18.43 Indirect exportation. (a) When merchandise is to move from one U.S... loading, sealing or labeling, and affixing of TIR plates. He shall remove one voucher from the carnet...

  17. 19 CFR 18.43 - Indirect exportation.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Under Cover of A Tir Carnet § 18.43 Indirect exportation. (a) When merchandise is to move from one U.S... loading, sealing or labeling, and affixing of TIR plates. He shall remove one voucher from the carnet...

  18. 19 CFR 18.43 - Indirect exportation.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Under Cover of A Tir Carnet § 18.43 Indirect exportation. (a) When merchandise is to move from one U.S... loading, sealing or labeling, and affixing of TIR plates. He shall remove one voucher from the carnet...

  19. Indirect Ultraviolet-Reactivation of Phage λ

    PubMed Central

    George, Jacqueline; Devoret, Raymond; Radman, Miroslav

    1974-01-01

    When an F- recipient Escherichia coli K12 bacterium receives Hfr or F-lac+ DNA from an ultraviolet-irradiated donor, its capacity to promote DNA repair and mutagenesis of ultraviolet-damaged phage λ is substantially increased. We call this phenomenon indirect ultraviolet-reactivation, since its features are essentially the same as those of ultraviolet-reactivation; this repair process occurs in pyrimidine dimer excision-deficient strains and produces clear plaque mutations of the restored phage. Moreover, this process is similar to indirect ultraviolet-induction of prophage λ, since it is promoted by conjugation. However, contrarily to indirect induction, it is produced by Hfr donors and occurs in recipients restricting the incoming ultraviolet-damaged donor DNA. The occurrence of indirect ultraviolet-reactivation provides evidence for the existence in E. coli of an inducible error-prone mechanism for the repair of DNA. PMID:4589889

  20. Direct and indirect effects in causal networks

    NASA Astrophysics Data System (ADS)

    Krämer, Andreas

    2013-03-01

    Literature-derived networks of biomolecular interactions representing cause-effect relationships generally contain many indirect relationships where the actually observed causal effect results from a sequence of events represented in the same network. A statistical method is developed, based on an Ising-like spin model operating on the edges of the network, to distinguish between direct and indirect effects using only the network structure itself. This allows to identify paths representing likely causation mechanisms.

  1. Quantification and localization of HLA-DR and intercellular adhesion molecule-1 (ICAM-1) molecules on bronchial epithelial cells of asthmatics using confocal microscopy.

    PubMed Central

    Vignola, A M; Chanez, P; Campbell, A M; Pinel, A M; Bousquet, J; Michel, F B; Godard, P

    1994-01-01

    An increased expression of HLA-DR and ICAM-1 molecules on bronchial epithelial cells has been observed in asthmatic patients. The aim of this study was to evaluate the localization and to quantify the spontaneous expression of HLA-DR and ICAM-1 on bronchial epithelial cells recovered by bronchial brushing of nine asthmatics and nine controls. Epithelial cells constituted over 95% of cells recovered as shown using an anti-cytokeratin MoAb. Expression of HLA-DR and ICAM-1 was studied using indirect immunofluorescence and confocal microscopy. The intensity of fluorescence of epithelial cells expressing HLA-DR and ICAM-1 was significantly (P < 0.003) increased in asthmatics. In asthmatics, but not in controls, the expression of both molecules was localized in the cytoplasm on the apicolateral portions of the cells. This study shows an up-regulation in the expression of HLA-DR and ICAM-1 molecules by bronchial epithelial cells from asthmatics and a localization of these molecules within the cell. Images Fig. 1 Fig. 1 PMID:7908615

  2. An Indirect Route for Ethanol Production

    SciTech Connect

    Eggeman, T.; Verser, D.; Weber, E.

    2005-04-29

    The ZeaChem indirect method is a radically new approach to producing fuel ethanol from renewable resources. Sugar and syngas processing platforms are combined in a novel way that allows all fractions of biomass feedstocks (e.g. carbohydrates, lignins, etc.) to contribute their energy directly into the ethanol product via fermentation and hydrogen based chemical process technologies. The goals of this project were: (1) Collect engineering data necessary for scale-up of the indirect route for ethanol production, and (2) Produce process and economic models to guide the development effort. Both goals were successfully accomplished. The projected economics of the Base Case developed in this work are comparable to today's corn based ethanol technology. Sensitivity analysis shows that significant improvements in economics for the indirect route would result if a biomass feedstock rather that starch hydrolyzate were used as the carbohydrate source. The energy ratio, defined as the ratio of green energy produced divided by the amount of fossil energy consumed, is projected to be 3.11 to 12.32 for the indirect route depending upon the details of implementation. Conventional technology has an energy ratio of 1.34, thus the indirect route will have a significant environmental advantage over today's technology. Energy savings of 7.48 trillion Btu/yr will result when 100 MMgal/yr (neat) of ethanol capacity via the indirect route is placed on-line by the year 2010.

  3. 48 CFR 742.770 - Negotiated indirect cost rate agreement.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... DEVELOPMENT CONTRACT MANAGEMENT CONTRACT ADMINISTRATION Indirect Cost Rates 742.770 Negotiated indirect cost... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false Negotiated indirect cost... Negotiated Indirect Cost Rate Agreement, executed by both parties. The Negotiated Indirect Cost...

  4. 14 CFR 296.3 - Indirect cargo air carrier.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Indirect cargo air carrier. 296.3 Section... PROCEEDINGS) ECONOMIC REGULATIONS INDIRECT AIR TRANSPORTATION OF PROPERTY General § 296.3 Indirect cargo air carrier. An indirect cargo air carrier is any U.S. citizen who undertakes to engage indirectly in...

  5. 14 CFR 296.3 - Indirect cargo air carrier.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 4 2014-01-01 2014-01-01 false Indirect cargo air carrier. 296.3 Section... PROCEEDINGS) ECONOMIC REGULATIONS INDIRECT AIR TRANSPORTATION OF PROPERTY General § 296.3 Indirect cargo air carrier. An indirect cargo air carrier is any U.S. citizen who undertakes to engage indirectly in...

  6. 14 CFR 296.3 - Indirect cargo air carrier.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Indirect cargo air carrier. 296.3 Section... PROCEEDINGS) ECONOMIC REGULATIONS INDIRECT AIR TRANSPORTATION OF PROPERTY General § 296.3 Indirect cargo air carrier. An indirect cargo air carrier is any U.S. citizen who undertakes to engage indirectly in...

  7. 14 CFR 296.3 - Indirect cargo air carrier.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Indirect cargo air carrier. 296.3 Section... PROCEEDINGS) ECONOMIC REGULATIONS INDIRECT AIR TRANSPORTATION OF PROPERTY General § 296.3 Indirect cargo air carrier. An indirect cargo air carrier is any U.S. citizen who undertakes to engage indirectly in...

  8. 14 CFR 296.3 - Indirect cargo air carrier.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 4 2012-01-01 2012-01-01 false Indirect cargo air carrier. 296.3 Section... PROCEEDINGS) ECONOMIC REGULATIONS INDIRECT AIR TRANSPORTATION OF PROPERTY General § 296.3 Indirect cargo air carrier. An indirect cargo air carrier is any U.S. citizen who undertakes to engage indirectly in air...

  9. Specific detection of prostate cancer cells in urine by multiplex immunofluorescence cytology.

    PubMed

    Fujita, Kazutoshi; Pavlovich, Christian P; Netto, George J; Konishi, Yuko; Isaacs, William B; Ali, Syed; De Marzo, Angelo; Meeker, Alan K

    2009-07-01

    Prostate cancer biomarkers are enriched in urine after prostatic manipulation, suggesting that whole cells might also be detectable for diagnosis. We tested multiplex staining of urinary sediments as a minimally invasive method to detect prostate cancer. Urine samples were collected from 35 men who had prostatic massage (attentive digital rectal examination) in a urology clinic and from 15 control men without urologic disease and without massage, for a total of 50 specimens (27 cancer-positive cases and 23 cancer-negative cases). LNCaP prostate cancer cells spiked into urine were used for initial marker optimization. Urine sediments were cytospun onto glass slides and stained. Multiplex urine cytology was compared with conventional urine cytology for cancer detection; anti-alpha-methylacyl-CoA racemase antibody was used as a marker of prostate cancer cells, anti-Nkx3.1 as a marker of prostate epithelial cells, anti-nucleolin as a marker of nucleoli, and 4'-6-diamidino-2-phenylindole to highlight nuclei. Prostate cancer cells were successfully visualized by combined staining for alpha-methylacyl-CoA racemase, Nkx3.1, and nucleolin. Of the 25 informative cases with biopsy-proven prostate cancer, 9 were diagnosed as suspicious or positive by multiplex immunofluorescence urine cytology, but only 4 were similarly judged by conventional cytology. All cases without cancer were read as negative by both methods. The multiplex cytology sensitivity for cancer detection in informative cases was 36% (9/25), and specificity was 100% (8/8). In conclusion, we have successfully achieved multiple staining for alpha-methylacyl-CoA racemase, Nkx3.1, nucleolin, and 4'-6-diamidino-2-phenylindole to detect prostate cancer cells in urine. Further refinements in marker selection and technique may increase sensitivity and applicability for prostate cancer diagnosis.

  10. Detection of receptor-specific murine leukemia virus binding to cells by immunofluorescence analysis.

    PubMed Central

    Kadan, M J; Sturm, S; Anderson, W F; Eglitis, M A

    1992-01-01

    Four classes of murine leukemia virus (MuLV) which display distinct cellular tropisms and bind to different retrovirus receptors to initiate virus infection have been described. In the present study, we describe a rapid, sensitive immunofluorescence assay useful for characterizing the initial binding of MuLV to cells. By using the rat monoclonal antibody 83A25 (L. H. Evans, R. P. Morrison, F. G. Malik, J. Portis, and W. J. Britt, J. Virol. 64:6176-6183, 1990), which recognizes an epitope of the envelope gp70 molecule common to the different classes of MuLV, it is possible to analyse the binding of ecotropic, amphotropic, or xenotropic MuLV by using only a single combination of primary and secondary antibodies. The MuLV binding detected by this assay is envelope receptor specific and matches the susceptibility to infection determined for cells from a variety of species. The binding of amphotropic MuLV to NIH 3T3 cells was shown to be rapid, saturable, and temperature dependent. Chinese hamster ovary (CHO-K1) cells normally lack the ability to bind ecotropic virus and are not infectible by ecotropic vectors. Expression of the cloned ecotropic retrovirus receptor gene (Rec) in CHO-K1 cells confers high levels of ecotropic virus-specific binding and confers susceptibility to infection. Characterization of MuLV binding to primary cells may provide insight into the infectibility of cells by retroviruses and aid in the selection of appropriate vectors for gene transfer experiments. PMID:1312632

  11. Determination of Cutoff of ELISA and Immunofluorescence Assay for Scrub Typhus

    PubMed Central

    Gupta, Nitin; Chaudhry, Rama; Thakur, Chandan Kumar

    2016-01-01

    Background: The most common method employed for diagnosis of scrub typhus is serology. It is widely known that demonstration of ≥4-fold rise in titers of antibody in paired sera is required for diagnosis. However, for guidance of initial treatment, there is a need for rapid diagnosis at the time of admission. Therefore, there is a need for standardized region specific cutoff titers at the time of admission. Materials and Methods: A total of 258 patients of all age groups with clinically suspected scrub typhus over a period of 24 months (October 2013-October 2015) were enrolled. Serum samples of these patients were subjected to immunofluorescent antibody (IFA) for immunoglobulin M (IgM) (Fuller Labs, USA) with dilutions of 1:64, 1:128, 1:256, and 1:512. Serum samples of all 258 patients were subjected to IgM ELISA (Inbios Inc., USA). Any patient with response to antibiotics within 48 h accompanied by either presence of an eschar or positivity by polymerase chain reaction was taken as positive. Receiver operating characteristic (ROC) curve was drawn to generate cutoff for these tests. Results: A total of 20 patients were diagnosed as cases of scrub typhus. The ROC curve analysis revealed a cutoff optical density value of 0.87 with sensitivity and specificity of 100% and 94.12%, respectively. ROC curve analysis of IFA revealed sensitivity and specificity of 100% and 93.5%, respectively at 1:64 dilution. Conclusion: Considering cost constraints, centers in and around New Delhi region can use the cutoffs we determined for the diagnosis of scrub typhus. PMID:27621559

  12. Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies.

    PubMed

    Burghi, Valeria; Fernández, Natalia Cristina; Gándola, Yamila Belén; Piazza, Verónica Gabriela; Quiroga, Diego Tomás; Guilhen Mario, Érica; Felix Braga, Janaína; Bader, Michael; Santos, Robson Augusto Souza; Dominici, Fernando Pablo; Muñoz, Marina Cecilia

    2017-01-01

    Mas receptor (MasR) is a G protein-coupled receptor proposed as a candidate for mediating the angiotensin (Ang)-converting enzyme 2-Ang (1-7) protective axis of renin-angiotensin system. Because the role of this receptor is not definitively clarified, determination of MasR tissue distribution and expression levels constitutes a critical knowledge to fully understanding its function. Commercially available antibodies have been widely employed for MasR protein localization and quantification, but they have not been adequately validated. In this study, we carried on an exhaustive evaluation of four commercial MasR antibodies, following previously established criteria. Western Blotting (WB) and immunohistochemistry studies starting from hearts and kidneys from wild type (WT) mice revealed that antibodies raised against different MasR domains yielded different patterns of reactivity. Furthermore, staining patterns appeared identical in samples from MasR knockout (MasR-KO) mice. We verified by polymerase chain reaction analysis that the MasR-KO mice used were truly deficient in this receptor as MAS transcripts were undetectable in either heart or kidney from this animal model. In addition, we evaluated the ability of the antibodies to detect the human c-myc-tagged MasR overexpressed in human embryonic kidney cells. Three antibodies were capable of detecting the MasR either by WB or by immunofluorescence, reproducing the patterns obtained with an anti c-myc antibody. In conclusion, although three of the selected antibodies were able to detect MasR protein at high expression levels observed in a transfected cell line, they failed to detect this receptor in mice tissues at physiological expression levels. As a consequence, validated antibodies that can recognize and detect the MasR at physiological levels are still lacking.

  13. Immunofluorescent staining reveals hypermethylation of microchromosomes in the central bearded dragon, Pogona vitticeps.

    PubMed

    Domaschenz, Renae; Livernois, Alexandra M; Rao, Sudha; Ezaz, Tariq; Deakin, Janine E

    2015-01-01

    Studies of model organisms have demonstrated that DNA cytosine methylation and histone modifications are key regulators of gene expression in biological processes. Comparatively little is known about the presence and distribution of epigenetic marks in non-model amniotes such as non-avian reptiles whose genomes are typically packaged into chromosomes of distinct size classes. Studies of chicken karyotypes have associated the gene-richness and high GC content of microchromosomes with a distinct epigenetic landscape. To determine whether this is likely to be a common feature of amniote microchromosomes, we have analysed the distribution of epigenetic marks using immunofluorescence on metaphase chromosomes of the central bearded dragon (Pogona vitticeps). This study is the first to study the distribution of epigenetic marks on non-avian reptile chromosomes. We observed an enrichment of DNA cytosine methylation, active modifications H3K4me2 and H3K4me3, as well as the repressive mark H3K27me3 in telomeric regions on macro and microchromosomes. Microchromosomes were hypermethylated compared to macrochromosomes, as they are in chicken. However, differences between macro- and microchromosomes for histone modifications associated with actively transcribed or repressed DNA were either less distinct or not detectable. Hypermethylation of microchromosomes compared to macrochromosomes is a shared feature between P. vitticeps and avian species. The lack of the clear distinction between macro- and microchromosome staining patterns for active and repressive histone modifications makes it difficult to determine at this stage whether microchrosome hypermethylation is correlated with greater gene density as it is in aves, or associated with the greater GC content of P. vitticeps microchromosomes compared to macrochromosomes.

  14. [Efficiency of bacteriological culture and the immunofluorescent assay to detect Campylobacter fetus in bovine genital fluids].

    PubMed

    Marcellino, Romanela B; Morsella, Claudia G; Cano, Dora; Paolicchi, Fernando A

    2015-01-01

    Bovine genital campylobacteriosis is a reproductive disease that affects cattle production. It is caused by Campylobacter fetus subspecies, C. fetus fetus (Cff) and C. fetus venerealis (Cfv). The aim of this study was to identify the presence of C. fetus in genital fluids by bacteriological culture and direct immunofluorescence (DIF) and to compare the results. Two groups of 6 heifers and 5 bulls, one infected with Cff (Cff group) and the other with Cfv (Cfv group) were formed. Two heifers and 2 bulls, all of them uninfected, made up the control group. Samples of cervicovaginal mucus and preputial fluid were processed by culture and DIF. In the Cff group, 100% of the heifers and 80% of the bulls were infected, while in the Cfv group, 50% of the heifers and 60% of the bulls were infected. The degree of agreement (Kappa values) from benchmarking diagnostic techniques were 0.57 for heifers in the Cff group and 0.52 for heifers in the Cfv group, whereas the values for bulls were 0.17 and 0.27, respectively. Heifers yielded more positive results in the DIF assay than in the culture, exhibiting 5.6% increase in the Cff group and 7.4% in the Cfv group. The lowest percentage of positive results for DIF in bulls, 40% less for the Cff group and 5.2% for the Cfv group, could be due to improper sampling. Kappa values showed moderate agreement for the heifers and low for the bulls. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  15. IMMUNOFLUORESCENT STUDIES OF THE POTENTIATION OF AN ADENOVIRUS-ASSOCIATED VIRUS BY ADENOVIRUS 7

    PubMed Central

    Blacklow, Neil R.; Hoggan, M. David; Rowe, Wallace P.

    1967-01-01

    A quantitative immunofluorescent procedure for detection of viral antigen was used to study the potentiation of AAV-1 by Ad.7. AAV viral antigen formed only when the cells were also infected with adenovirus, and only in cell culture systems in which the adenovirus infection proceeded to completion. Ad. 7 infection of AGMK. cell cultures did not potentiate AAV unless the Ad. 7 infection was itself potentiated by SV40. Dose-response studies indicated that a single AAV particle and a single infectious Ad. 7 particle sufficed to initiate AAV antigen synthesis. Sequential inoculation studies showed that AAV antigen formed simultaneously with Ad. 7 viral antigen when the AAV was inoculated any time between 15 hr before to 10 hr after the Ad. 7, both antigens appearing about 15 hr after inoculation of Ad. 7. The AAV-1 antigen formation had a minimum latent period of 5 hr, as seen with Ad. 7 preinfection of 10 hr or more. When UV-irradiated Ad. 7 was used as helper, the AAV antigen still appeared simultaneously with the Ad. 7 viral antigen, even though the latter was delayed by 23 hr compared to nonirradiated virus. When the early replicative events of both viruses were allowed to proceed in FUDR-inhibited cells, and then the FUDR inhibition was reversed, AAV antigen formed within 2 hr, which was 3 hr before the Ad. 7 viral antigen appeared. It was inferred that the event in the adenovirus cycle that renders a cell competent to synthesize AAV occurs after the 10th hr and may be temporally associated with replication of the adenovirus DNA. PMID:4225814

  16. Lasers for nonlinear microscopy.

    PubMed

    Wise, Frank

    2013-03-01

    Various versions of nonlinear microscopy are revolutionizing the life sciences, almost all of which are made possible because of the development of ultrafast lasers. In this article, the main properties and technical features of short-pulse lasers used in nonlinear microscopy are summarized. Recent research results on fiber lasers that will impact future instruments are also discussed.

  17. Influence of Different Antioxidants on X-Ray Induced DNA Double-Strand Breaks (DSBs) Using γ-H2AX Immunofluorescence Microscopy in a Preliminary Study

    PubMed Central

    Brand, Michael; Sommer, Matthias; Ellmann, Stephan; Wuest, Wolfgang; May, Matthias S.; Eller, Achim; Vogt, Sabine; Lell, Michael M.; Kuefner, Michael A.; Uder, Michael

    2015-01-01

    Background Radiation exposure occurs in X-ray guided interventional procedures or computed tomography (CT) and γ-H2AX-foci are recognized to represent DNA double-strand breaks (DSBs) as a biomarker for radiation induced damage. Antioxidants may reduce the induction of γ-H2AX-foci by binding free radicals. The aim of this study was to establish a dose-effect relationship and a time-effect relationship for the individual antioxidants on DSBs in human blood lymphocytes. Materials and Methods Blood samples from volunteers were irradiated with 10 mGy before and after pre-incubation with different antioxidants (zinc, trolox, lipoic acid, ß-carotene, selenium, vitamin E, vitamin C, N-acetyl-L-cysteine (NAC) and Q 10). Thereby, different pre-incubation times, concentrations and combinations of drugs were evaluated. For assessment of DSBs, lymphocytes were stained against the phosphorylated histone variant γ-H2AX. Results For zinc, trolox and lipoic acid regardless of concentration or pre-incubation time, no significant decrease of γ-H2AX-foci was found. However, ß-carotene (15%), selenium (14%), vitamin E (12%), vitamin C (25%), NAC (43%) and Q 10 (18%) led to a significant reduction of γ-H2AX-foci at a pre-incubation time of 1 hour. The combination of different antioxidants did not have an additive effect. Conclusion Antioxidants administered prior to irradiation demonstrated the potential to reduce γ-H2AX-foci in blood lymphocytes. PMID:25996998

  18. Influence of Different Antioxidants on X-Ray Induced DNA Double-Strand Breaks (DSBs) Using γ-H2AX Immunofluorescence Microscopy in a Preliminary Study.

    PubMed

    Brand, Michael; Sommer, Matthias; Ellmann, Stephan; Wuest, Wolfgang; May, Matthias S; Eller, Achim; Vogt, Sabine; Lell, Michael M; Kuefner, Michael A; Uder, Michael

    2015-01-01

    Radiation exposure occurs in X-ray guided interventional procedures or computed tomography (CT) and γ-H2AX-foci are recognized to represent DNA double-strand breaks (DSBs) as a biomarker for radiation induced damage. Antioxidants may reduce the induction of γ-H2AX-foci by binding free radicals. The aim of this study was to establish a dose-effect relationship and a time-effect relationship for the individual antioxidants on DSBs in human blood lymphocytes. Blood samples from volunteers were irradiated with 10 mGy before and after pre-incubation with different antioxidants (zinc, trolox, lipoic acid, ß-carotene, selenium, vitamin E, vitamin C, N-acetyl-L-cysteine (NAC) and Q 10). Thereby, different pre-incubation times, concentrations and combinations of drugs were evaluated. For assessment of DSBs, lymphocytes were stained against the phosphorylated histone variant γ-H2AX. For zinc, trolox and lipoic acid regardless of concentration or pre-incubation time, no significant decrease of γ-H2AX-foci was found. However, ß-carotene (15%), selenium (14%), vitamin E (12%), vitamin C (25%), NAC (43%) and Q 10 (18%) led to a significant reduction of γ-H2AX-foci at a pre-incubation time of 1 hour. The combination of different antioxidants did not have an additive effect. Antioxidants administered prior to irradiation demonstrated the potential to reduce γ-H2AX-foci in blood lymphocytes.

  19. Comparative immunofluorescence and ultrastructural analysis of microtubule organization in Uronema sp., Klebsormidium flaccidum, K. subtilissimum, Stichococcus bacillaris and S. chloranthus (Chlorophyta).

    PubMed

    Katsaros, Christos I; Varvarigos, Vasilios; Gachon, Claire M M; Brand, Jerry; Motomura, Taizo; Nagasato, Chikako; Küpper, Frithjof C

    2011-04-01

    A detailed comparative examination of microtubule (MT) organization in interphase and dividing cells of Uronema sp., Klebsormidium flaccidum, K. subtilissimum, Stichococcus bacillaris and S. chloranthus was made using tubulin immunofluorescence and transmission electron microscopy (TEM). During interphase all the species bear a well-organized cortical MT system, consisting of parallel bundles with different orientations. In Uronema sp. the cortical MT bundles are longitudinally oriented, whereas in the other species they are in transverse orientation to the axis of the cells. Considerable differences in MT organization were also observed during stages of mitosis, mainly preprophase, as well as cytokinesis. In Uronema sp., a particular radial MT assembly is organized during preprophase-early prophase, which was not observed in the other species. In Stichococcus a fine MT ring surrounded the nucleus during preprophase and prophase. An MT ring, together with single cytoplasmic MTs, was also found associated with the developing diaphragm during cytokinesis in Stichococcus. A phycoplast participates in cytokinesis in Uronema sp., but not in the other species. In Uronema sp. the centrosome functions as a microtubule organizing center (MTOC) during mitosis, but not during interphase and cytokinesis. The phylogenetic significance of these differences is discussed in combination with SSU/ITS sequencing and other, existing molecular data.

  20. Serological cross-reaction between Legionella pneumophila and campylobacter in the indirect fluorescent antibody test.

    PubMed Central

    Boswell, T. C.; Kudesia, G.

    1992-01-01

    Sera from 50 patients with culture-proven campylobacter gastroenteritis were examined for the presence of antibodies to Legionella pneumophila. Ten patients (20%) had a positive titre (> or = 16) as measured by indirect immunofluorescence. Antibodies were detected in only 1 of 36 acute sera but in 10 of 14 (71%) sera obtained more than 10 days after the onset of symptoms. All positive sera contained specific IgM antibodies but specific IgG or IgA could not be detected in any sample. No legionella antibodies could be detected in sera from 42 similar patients with salmonella gastroenteritis. These results were shown to be due to serological cross-reaction between L. pneumophila and campylobacter. PMID:1397117

  1. Indirect Comparisons and Network Meta-Analyses.

    PubMed

    Kiefer, Corinna; Sturtz, Sibylle; Bender, Ralf

    2015-11-20

    Systematic reviews provide a structured summary of the results of trials that have been carried out on any particular subject. If the data from multiple trials are sufficiently homogenous, a meta-analysis can be performed to calculate pooled effect estimates. Traditional meta-analysis involves groups of trials that compare the same two interventions directly (head to head). Lately, however, indirect comparisons and network metaanalyses have become increasingly common. Various methods of indirect comparison and network meta-analysis are presented and discussed on the basis of a selective review of the literature. The main assumptions and requirements of these methods are described, and a checklist is provided as an aid to the evaluation of published indirect comparisons and network meta-analyses. When no head-to-head trials of two interventions are available, indirect comparisons and network metaanalyses enable the estimation of effects as well as the simultaneous analysis of networks involving more than two interventions. Network meta-analyses and indirect comparisons can only be useful if the trial or patient characteristics are similar and the observed effects are sufficiently homogeneous. Moreover, there should be no major discrepancy between the direct and indirect evidence. If trials are available that compare each of two treatments against a third one, but not against each other, then the third intervention can be used as a common comparator to enable a comparison of the other two. Indirect comparisons and network metaanalyses are an important further development of traditional meta-analysis. Clear and detailed documentation is needed so that findings obtained by these new methods can be reliably judged.

  2. Superresolution microscopy for microbiology

    PubMed Central

    Coltharp, Carla; Xiao, Jie

    2014-01-01

    Summary This review provides a practical introduction to superresolution microscopy from the perspective of microbiological research. Because of the small sizes of bacterial cells, superresolution methods are particularly powerful and suitable for revealing details of cellular structures that are not resolvable under conventional fluorescence light microscopy. Here we describe the methodological concepts behind three major categories of super-resolution light microscopy: photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) and stimulated emission-depletion (STED) microscopy. We then present recent applications of each of these techniques to microbial systems, which have revealed novel conformations of cellular structures and described new properties of in vivo protein function and interactions. Finally, we discuss the unique issues related to implementing each of these superresolution techniques with bacterial specimens and suggest avenues for future development. The goal of this review is to provide the necessary technical background for interested microbiologists to choose the appropriate super-resolution method for their biological systems, and to introduce the practical considerations required for designing and analysing superresolution imaging experiments. PMID:22947061

  3. Indirect combustion noise of auxiliary power units

    NASA Astrophysics Data System (ADS)

    Tam, Christopher K. W.; Parrish, Sarah A.; Xu, Jun; Schuster, Bill

    2013-08-01

    Recent advances in noise suppression technology have significantly reduced jet and fan noise from commercial jet engines. This leads many investigators in the aeroacoustics community to suggest that core noise could well be the next aircraft noise barrier. Core noise consists of turbine noise and combustion noise. There is direct combustion noise generated by the combustion processes, and there is indirect combustion noise generated by the passage of combustion hot spots, or entropy waves, through constrictions in an engine. The present work focuses on indirect combustion noise. Indirect combustion noise has now been found in laboratory experiments. The primary objective of this work is to investigate whether indirect combustion noise is also generated in jet and other engines. In a jet engine, there are numerous noise sources. This makes the identification of indirect combustion noise a formidable task. Here, our effort concentrates exclusively on auxiliary power units (APUs). This choice is motivated by the fact that APUs are relatively simple engines with only a few noise sources. It is, therefore, expected that the chance of success is higher. Accordingly, a theoretical model study of the generation of indirect combustion noise in an Auxiliary Power Unit (APU) is carried out. The cross-sectional areas of an APU from the combustor to the turbine exit are scaled off to form an equivalent nozzle. A principal function of a turbine in an APU is to extract mechanical energy from the flow stream through the exertion of a resistive force. Therefore, the turbine is modeled by adding a negative body force to the momentum equation. This model is used to predict the ranges of frequencies over which there is a high probability for indirect combustion noise generation. Experimental spectra of internal pressure fluctuations and far-field noise of an RE220 APU are examined to identify anomalous peaks. These peaks are possible indirection combustion noise. In the case of the

  4. Super resolution fluorescence microscopy

    PubMed Central

    Huang, Bo; Bates, Mark; Zhuang, Xiaowei

    2010-01-01

    Achieving a spatial resolution that is not limited by the diffraction of light, recent developments of super-resolution fluorescence microscopy techniques allow the observation of many biological structures not resolvable in conventional fluorescence microscopy. New advances in these techniques now give them the ability to image three-dimensional (3D) structures, measure interactions by multicolor colocalization, and record dynamic processes in living cells at the nanometer scale. It is anticipated that super-resolution fluorescence microscopy will become a widely used tool for cell and tissue imaging to provide previously unobserved details of biological structures and processes. PMID:19489737

  5. [Indirect costs in health technology assessment].

    PubMed

    Jakubczyk, Michał; Wrona, Witold; Macioch, Tomasz; Golicki, Dominik; Niewada, Maciej; Hermanowski, Tomasz

    2010-01-01

    In the health technology assessment it is crucial to define the perspective of the analysis. When the societal perspective is chosen it is necessary to include all the costs incurred by the society, also the costs of lost productivity resulting from absence of sick employees from work or their reduced efficiency at work. The aim of this article is to present the notion of indirect costs, their importance in health technology assessment and the methods of calculation. The economic literature has been reviewed for the state of knowledge on indirect costs. Three methods of calculation are described: human capital method, friction cost method or health state valuation. Indirect costs in Western European countries can amount to more than half of total costs attributed to the illness and its treatment. In the literature there is no consensus regarding the proper method of indirect costs calculation. It is necessary to conduct further theoretical and empirical research in the area of indirect costs and enhance discussion among Polish pharmacoeconomists.

  6. Immunofluorescence assay reactivity patterns of serum samples presenting indeterminate Western blot results for antibodies to HIV-1 and HTLV-I/II in Cordoba, Argentina.

    PubMed

    Gastaldello, R; Gallego, S; Isa, M B; Maturano, E; Sileoni, S; Nates, S; Medeot, S

    2001-01-01

    Serum samples (n: 110) from blood donors and high risk individuals from Cordoba, Argentina with indeterminate HIV-1 and HTLV-I/II Wb profiles were studied for specific antibodies to HTLV-I/II and HIV-1 by indirect immunofluorescence assay (IFA) and for the presence or absence of HIV-1 and HTLV-I/II specific bands by Wb. This study was carried out in order to characterize their putative reactions with HIV-1 and HTLV-I/II proteins and to resolve the retrovirus infection status of these individuals. Results indicated that blood donors sera displaying indeterminate HIV-1 or HTLV-I/II Wb patterns were not immunoreactive to HTLV-I/II and HIV-1 on IFA. However, a high rate of indeterminate HIV-1 and HTLV-I/II Wb samples from high risk individuals had positive HTLV-I/II and HIV-1 IFA results respectively. Our study supports the growing evidence that HTLV-HIV indeterminate seroreactivity in low risk population is due to a cross reaction against nonviral antigens, and in high risk populations the indeterminate samples show serological cross-recognition between HIV-1 proteins and HTLV-I/II proteins on Wb. These results point out the necessity to investigate the HTLV-I/II reactivity in indeterminate HIV-1 samples and vice versa in order to confirm the diagnosis. Finally, this study shows the potential usefulness of IFA in elucidating the status of HIV-1 and HTLV-I/II infection of individuals with indeterminate Wb profiles, thus enabling resolution of retrovirus infection status.

  7. Cross-reactive antibodies in convalescent SARS patients' sera against the emerging novel human coronavirus EMC (2012) by both immunofluorescent and neutralizing antibody tests.

    PubMed

    Chan, Kwok-Hung; Chan, Jasper Fuk-Woo; Tse, Herman; Chen, Honglin; Lau, Candy Choi-Yi; Cai, Jian-Piao; Tsang, Alan Ka-Lun; Xiao, Xincai; To, Kelvin Kai-Wang; Lau, Susanna Kar-Pui; Woo, Patrick Chiu-Yat; Zheng, Bo-Jiang; Wang, Ming; Yuen, Kwok-Yung

    2013-08-01

    A severe acute respiratory syndrome (SARS)-like disease due to a novel betacoronavirus, human coronavirus EMC (HCoV-EMC), has emerged recently. HCoV-EMC is phylogenetically closely related to Tylonycteris-bat-coronavirus-HKU4 and Pipistrellus-bat-coronavirus-HKU5 in Hong Kong. We conducted a seroprevalence study on archived sera from 94 game-food animal handlers at a wild life market, 28 SARS patients, and 152 healthy blood donors in Southern China to assess the zoonotic potential and evidence for intrusion of HCoV-EMC and related viruses into humans. Anti-HCoV-EMC and anti-SARS-CoV antibodies were detected using screening indirect immunofluorescence (IF) and confirmatory neutralizing antibody tests. Two (2.1%) animal handlers had IF antibody titer of ≥ 1:20 against both HCoV-EMC and SARS-CoV with neutralizing antibody titer of <1:10. No blood donor had antibody against either virus. Surprisingly, 17/28 (60.7%) of SARS patients had significant IF antibody titers with 7/28 (25%) having anti-HCoV-EMC neutralizing antibodies at low titers which significantly correlated with that of HCoV-OC43. Bioinformatics analysis demonstrated a significant B-cell epitope overlapping the heptad repeat-2 region of Spike protein. Virulence of SARS-CoV over other betacoronaviruses may boost cross-reactive neutralizing antibodies against other betacoronaviruses. Convalescent SARS sera may contain cross-reactive antibodies against other betacoronaviruses and confound seroprevalence study for HCoV-EMC. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  8. [Immunofluorescence assay with Crithidia luciliae for the detection of anti-DNA antibodies. Atypical images and their relationship with Chagas' disease and leishmaniasis].

    PubMed

    Griemberg, Gloria; Ferrarotti, Nidia F; Svibel, Graciela; Ravelli, Maria R; Taranto, Nestor J; Malchiodi, Emilio L; Pizzimenti, Maria C

    2006-01-01

    Anti-native DNA antibodies can be detected by indirect immunofluorescence assay with Crithidia luciliae, displaying an annular image due to a kinetoplast containing double stranded DNA. Other structures such as membrane, flagellum and basal corpuscle can be stained as well, showing what is called atypical fluorescent images. As C. luciliae belongs to the Trypanosomatidae family, which include the human pathogens Trypanosoma cruzi and Leishmania spp., it was considered that these atypical images could be caused by cross-reactions. Serological studies for Chagas' disease were performed in 105 serum samples displaying atypical images. Sixty four percent of the samples from non endemic and 78.3% from endemic areas for Chagas' disease showed fluorescence in both, membrane and flagellum (joint image). Fifty samples from normal blood donors and 57 samples from patients with conective tissue diseases were tested with C. luciliae. None of them presented the joint image except for two patients with lupus who were also chagasic. In addition, 54 samples from chagasic patients were studied and all of them presented the joint image. We also studied 46 samples from patients with leishmaniasis from whom 28 were coinfected with T. cruzi. The joint image was observed in 88.0% of the samples with leishmaniasis and in 89.3% of the co-infected samples. The results suggest that C. luciliae could be used as an economical, and of low risk, alternative substrate for the serological diagnosis of Chagas' disease, even though it does not discriminate for Leishmania spp. infection. This study also suggests that whenever atypical images are observed in C. luciliae during the search for anti-DNA antibodies, it would be convenient to submit the patient to clinical and serological tests for the diagnosis of leishmaniosis and Chagas' disease.

  9. A Clinicopathological Study of Pemphigus in Eastern India with Special Reference to Direct Immunofluorescence

    PubMed Central

    Chowdhury, Joyeeta; Datta, Pijush Kanti; Chowdhury, Satyendra Nath; Das, Nilay Kanti

    2016-01-01

    Background: Pemphigus is a group of chronic autoimmune vesico-bullous disorders in which the epidermis and the basement membrane zone are the focus of attack resulting in cutaneous and mucosal blister formation. Direct immunofluorescence (DIF) test is a very sensitive test for the diagnosis Aim: To study the clinico histopathological patterns of pemphigus in eastern India. The study also aims to correlate DIF with clinical and histologic findings as well as severity of skin involvement [scoring systems]. Materials and Methods: Total 41 patients were studied over a period of 1 year in the Post-graduate centre of Dermatology in Eastern India. DIF, histopathology and clinical data were correlated. Results: In our study Pemphigus vulgaris (PV) was the predominant type with 32 cases followed by 8 cases of pemphigus foliaceus (PF) and a single case of IgA pemphigus. Mean age at presentation was late middle age. Majority of the patients, 26 (63.41%) initially had cutaneous involvement followed by mucosal involvement. In this study group 36 (87.80%) patients showed acantholytic cells on histopathological examination. Most patients of PV showed suprabasal blister 20 (62.50%) followed by intraspinous 5 (15.62%) and subcorneal 5 (15.62%) blister. In majority 28 (87.50%) of the PV patients IgG and C3 antibodies were deposited throughout the epidermis. The strength of antibody positivity was strong in most of the patients (71.87%). In cases of PF mostly IgG 6 (75%) antibodies were deposited in the upper epidermis. DIF intensity had poor correlation with disease activity/severity except in PF. Conclusion: Almost 85.36% cases of pemphigus were diagnosed clinicopathologically. But 6 cases couldn’t be diagnosed accurately on clinicopathological basis and in them DIF was confirmatory. Two cases of pure mucosal PV and 1 case of IgA pemphigus was confirmed by DIF. Two cases of bullous pemphigoid clinico-histologically mimicking PV were also excluded by DIF. So it appears from our

  10. Sensitivity of direct immunofluorescence in oral diseases. Study of 125 cases.

    PubMed

    Sano, Susana Mariela; Quarracino, María Cecilia; Aguas, Silvia Cristina; González, Ernestina Jesús; Harada, Laura; Krupitzki, Hugo; Mordoh, Ana

    2008-05-01

    Direct immunofluorescence (DIF) is widely used for the diagnosis of bullous diseases and other autoimmune pathologies such as oral lichen planus. There is no evidence in the literature on how the following variants influence the detection rate of DIF: intraoral site chosen for the biopsy, perilesional locus or distant site from the clinical lesion, number of biopsies and instrument used. to determine if the following variants influenced the sensitivity (detection rate): intraoral site chosen for the biopsy, perilesional or distant site from the clinical lesion, number of biopsies and instrument used (punch or scalpel). A retrospective study was done at the Cátedra de Patología y Clínica Bucodental II at the Facultad de Odontología, Universidad de Buenos Aires; 136 clinical medical histories were revised for the period March 2000 - March 2005 corresponding to patients with clinical diagnosis of OLP and bullous diseases (vulgar pemphigus, bullous pemphigoid and cicatricial pemphigoid). DIF detection rate was 65.8% in patients with OLP, 66.7% in cicatricial pemphigoid patients, in bullous pemphigoid 55.6%, in pemphigus vulgaris 100%, and in those cases in which certain diagnosis could not be obtained, the DIF positivity rate was 45.5% (Pearson chi(2) (4)= 21.5398 Pr= 0.000). There was no statistically significant difference between the different sites of biopsy (Fisher exact test: 0.825). DIF detection rate in perilesional biopsies was 66.1% and in those distant from the site of clinical lesion was 64.7% (Pearson chi(2) v1)= 0.0073 Pr= 0.932. When the number of biopsies were incremented, DIF detection rate also incremented (Pearson chi(2) = 8.7247 Pr= 0.003). The biopsies taken with punch had a higher detection rate than those taken with scalpel (39.1% versus 71.7%) (Pearson chi(2) = 49.0522 Pr= 0.000). While not statistically significant, the tendency outlined in this study indicates there are intraoral regions in which the detection rate of the DIF technique is

  11. Dark matter dynamics and indirect detection

    SciTech Connect

    Bertone, Gianfranco; Merritt, David; /Rochester Inst. Tech.

    2005-04-01

    Non-baryonic, or ''dark'', matter is believed to be a major component of the total mass budget of the universe. We review the candidates for particle dark matter and discuss the prospects for direct detection (via interaction of dark matter particles with laboratory detectors) and indirect detection (via observations of the products of dark matter self-annihilations), focusing in particular on the Galactic center, which is among the most promising targets for indirect detection studies. The gravitational potential at the Galactic center is dominated by stars and by the supermassive black hole, and the dark matter distribution is expected to evolve on sub-parsec scales due to interaction with these components. We discuss the dominant interaction mechanisms and show how they can be used to rule out certain extreme models for the dark matter distribution, thus increasing the information that can be gleaned from indirect detection searches.

  12. Resonant indirect optical absorption in germanium

    NASA Astrophysics Data System (ADS)

    Menéndez, José; Noël, Mario; Zwinkels, Joanne C.; Lockwood, David J.

    2017-09-01

    The optical absorption coefficient of pure Ge has been determined from high-accuracy, high-precision optical measurements at photon energies covering the spectral range between the indirect and direct gaps. The results are compared with a theoretical model that fully accounts for the resonant nature of the energy denominators that appear in perturbation-theory expansions of the absorption coefficient. The model generalizes the classic Elliott approach to indirect excitons, and leads to a predicted optical absorption that is in excellent agreement with the experimental values using just a single adjustable parameter: the average deformation potential DΓ L coupling electrons at the bottom of the direct and indirect valleys in the conduction band. Remarkably, the fitted value, DΓ L=4.3 ×108eV /cm , is in nearly perfect agreement with independent measurements and ab initio predictions of this parameter, confirming the validity of the proposed theory, which has general applicability.

  13. Household Health Costs: Direct, Indirect and Intangible

    PubMed Central

    YOUSEFI, Mehdi; ASSARI ARANI, Abbas; SAHABI, Bahram; KAZEMNEJAD, Anoshirvan; FAZAELI, Somayeh

    2014-01-01

    Abstract Background This study aimed at identifying components of the household health costs. Methods This study was a qualitative research conducted in two main phases. The first phase consisted of interviews with sample households selected in eight provinces of Iran. They were to identify components of the household health costs. In the second phase, components were determined as direct, indirect and intangible based on a content analysis. Results In the first phase of the study, 93 components of households’ health costs were identified. According to the content analysis, 44 components were categorized as direct costs, 10 components were indirect and 39 components were categorized as intangible. Conclusion All components of households’ health costs including: direct, indirect and intangible costs, should be considered in the planning and policy-making in the health system. PMID:26060744

  14. [Indirect costs in idiopathic Parkinson's disease].

    PubMed

    Barth, F; Baum, B; Bremen, D; Meuser, T; Jost, W H

    2005-04-01

    The economic impact of parkinsonism has been getting more significant due to the increasing prevalence of Parkinson's disease and the modern therapies available nowadays. The present study is supposed to update the existing databases and to provide a sound foundation for rational decision-making in the health care sector. It does not only focus on the direct costs of this disease incurred by 75 patients over a longer period, but also and for the first time, takes a look on the indirect cost as well. The study shows that the expenses for PD-related house rebuilding and early retirement make up for a substantial share among the indirect costs. In the overall analysis, the ratio between both, direct and indirect costs appear to be relatively balanced with slight domination of the direct costs.

  15. Immunofluorescent Staining for the Detection of the Hepatitis B Core Antigen in Frozen Liver Sections of Human Liver Chimeric Mice.

    PubMed

    Allweiss, Lena; Lütgehetmann, Marc; Dandri, Maura

    2017-01-01

    The hepatitis B virus (HBV) is the causative agent for chronic hepatitis B infection, which affects an estimate of 240 million people worldwide and puts them at risk of developing terminal liver disease. The life cycle of the virus and its interactions with the host immune system are still incompletely understood, and currently available treatment options rarely achieve a cure. Therefore, basic research and new drug development are needed. One parameter for measuring the intrahepatic activity of the virus is monitoring the production of the HBV core antigen (HBcAg), which not only serves as the main structural protein of its nucleocapsid but is also recruited to the covalently closed circular DNA (cccDNA), the nuclear HBV genome responsible for infection persistence. Here, we report a sensitive immunofluorescence staining method to detect HBcAg in cryopreserved liver sections. The method combines conventional immunofluorescence staining procedures with the Tyramide Signal Amplification (TSA) system.

  16. Clinical specular microscopy

    SciTech Connect

    Hirst, L.W.; Laing, R.A.

    1987-01-01

    This book provides the general ophthalmologist with a guide to the clinical applications of specular microscopy. Important material is included on laser injury, cataract surgery, corneal transplants, glaucoma, uveitis, and trauma.

  17. Photothermal imaging scanning microscopy

    DOEpatents

    Chinn, Diane; Stolz, Christopher J.; Wu, Zhouling; Huber, Robert; Weinzapfel, Carolyn

    2006-07-11

    Photothermal Imaging Scanning Microscopy produces a rapid, thermal-based, non-destructive characterization apparatus. Also, a photothermal characterization method of surface and subsurface features includes micron and nanoscale spatial resolution of meter-sized optical materials.

  18. Stimulated Emission Depletion Microscopy.

    PubMed

    Blom, Hans; Widengren, Jerker

    2017-06-14

    Despite its short history, diffraction-unlimited fluorescence microscopy techniques have already made a substantial imprint in the biological sciences. In this review, we describe how stimulated emission depletion (STED) imaging originally evolved, how it compares to other optical super-resolution imaging techniques, and what advantages it provides compared to previous golden-standards for biological microscopy, such as diffraction-limited optical microscopy and electron microscopy. We outline the prerequisites for successful STED imaging experiments, emphasizing the equally critical roles of instrumentation, sample preparation, and photophysics, and describe major evolving strategies for how to push the borders of STED imaging even further in life science. Finally, we provide examples of how STED nanoscopy can be applied, within three different fields with particular potential for STED imaging experiments: neuroscience, plasma membrane biophysics, and subcellular clinical diagnostics. In these areas, and in many more, STED imaging can be expected to play an increasingly important role in the future.

  19. Multicolor Immunofluorescent Imaging of Complex Cellular Mixtures on Micropallet Arrays Enables the Identification of Single Cells of Defined Phenotype.

    PubMed

    Westerhof, Trisha M; Li, Guann-Pyng; Bachman, Mark; Nelson, Edward L

    2016-04-06

    A Micropallet-Array-based strategy allowing the identification of cells of defined phenotype in complex mixtures, such as would be obtained from a tissue biopsy, is presented. Following the distribution of single adherent cells from the mixture on individual pedestals, termed "micropallets", immunofluorescent confocal imaging is applied to interrogate the expression of five cell surface molecules. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Biomass Indirect Liquefaction Strategy Workshop Summary Report

    SciTech Connect

    none,

    2014-07-01

    This report is based on the proceedings of the U.S. Department of Energy Bioenergy Technologies Office Biomass Indirect Liquefaction Strategy Workshop. The workshop, held March 20–21, 2014, in Golden, Colorado, discussed and detailed the research and development needs for biomass indirect liquefaction. Discussions focused on pathways that convert biomass-based syngas (or any carbon monoxide, hydrogen gaseous stream) to liquid intermediates (alcohols or acids) and further synthesize those intermediates to liquid hydrocarbons that are compatible as either a refinery feed or neat fuel.

  1. Quantification of progress in indirect coal liquefaction

    SciTech Connect

    Gray, D.; ElSawy, A.; Tomlinson, G.

    1991-01-01

    The objective of this study is to quantify the economic and technical impact of incorporating various advanced technologies into the indirect coal liquefaction system. These advanced technologies include entrained flow Shell gasification and slurry-phase Fischer-Tropsch (F-T) synthesis. This objective was accomplished by substituting the Shell entrained goal gasifier system for the Lurgi and the advanced slurry F-T reactor for the Synthol and ARGE F-T systems in a SASOL-type indirect liquefaction facility. 4 refs., 3 figs., 2 tabs.

  2. Immunofluorescent detection of 8-oxo-dG and PAH bulky adducts in fish liver and mussel digestive gland.

    PubMed

    Machella, Nicola; Regoli, Francesco; Santella, Regina M

    2005-03-04

    Development of methodologies to detect DNA damage induced by pollutants is of increasing concern in marine ecotoxicology. We previously described an immunoperoxidase method for revealing 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-dG) in marine organisms. In this work, the approach was extended to immunofluorescence detection and to the use of another antibody for polycyclic aromatic hydrocarbon (PAH)-DNA adducts. Specimens of European eel (Anguilla anguilla) and Mediterranean mussel (Mytilus galloprovincialis) were exposed to benzo(a)pyrene (B[a]P), as model chemical carcinogen to induce both oxidized bases and B[a]P diol-epoxide-deoxyguanosine adducts. Cryostat sections of liver and digestive gland from both species were immunostained, and DNA damage was semiquantitatively evaluated by an image analysis system. Compared to untreated organisms, B[a]P-exposed organisms exhibited increased levels of oxidative DNA damage; in eels, which rapidly metabolize PAHs, the occurrence of B[a]P-DNA adducts was also detected. The immunofluorescent assay maintained all the advantages previously reported for the immunoperoxidase protocol. Both methods were tested on the same eel specimens and the immunofluorescence method showed a greater extent of relative DNA damage and a higher sensitivity. Although field validation is being carried out, our results indicate the utility of antibodies to rapidly detect DNA alterations in aquatic organisms, and to investigate the risk associated with genotoxins in marine environment.

  3. Naturally Occurring Immune-Complex Glomerulonephritis in Cynomolgus Monkeys (Macaca irus). I. Light, Immunofluorescence, and Electron Microscopic Studies.

    DTIC Science & Technology

    IMMUNOGLOBULINS, *VETERINARY MEDICINE, *MONKEYS, PATHOLOGY, FLUORESCENT ANTIBODY TECHNIQUES, DISEASES , GAMMA GLOBULIN, ANTIGENS, ANTIBODIES, HISTOLOGY, ETIOLOGY, ELECTRON MICROSCOPY, MICROSCOPES, IMMUNOLOGY.

  4. Direct immunofluorescence and enzyme-linked immunosorbent assays for evaluating chlorinated hydrocarbon degrading bacteria

    SciTech Connect

    Brigmon, R.L.; Franck, M.M.; Brey, J.; Fliermans, C.B.; Scott, D.; Lanclos, K.

    1997-06-01

    Immunological procedures were developed to enumerate chlorinated hydrocarbon degrading bacteria. Polyclonal antibodies (Pabs) were produced by immunizing New Zealand white rabbits against 18 contaminant-degrading bacteria. These included methanotrophic and chlorobenzene (CB) degrading species. An enzyme-linked immunosorbent assay (ELISA) was used to test for specificity and sensitivity of the Pabs. Direct fluorescent antibodies (DFAs) were developed with these Pabs against select methanotrophic bacteria isolated from a trichloroethylene (TCE) contaminated landfill at the Savannah River Site (SRS) and cultures from the American Type Culture Collection (ATCC). Analysis of cross reactivity testing data showed some of the Pabs to be group specific while others were species specific. The threshold of sensitivity for the ELISA is 105 bacteria cells/ml. The DFA can detect as few as one bacterium per ml after concentration. Results from the DFA and ELISA techniques for enumeration of methanotrophic bacteria in groundwater were higher but not significantly different (P < 0.05) compared to indirect microbiological techniques such as MPN. These methods provide useful information on in situ community structure and function for bioremediation applications within 1--4 hours of sampling.

  5. Specificity of antigens from pathogenic Aspergillus species. I. Studies with ELISA and immunofluorescence.

    PubMed

    De Magaldi, S W; Mackenzie, D W

    1984-01-01

    Studies were made by enzyme linked immunosorbent assay (ELISA) and indirect fluorescent antibody (IFA) tests on the reactivities and specificities of 13 antigens prepared from four species of Aspergillus against antisera from immunized rabbits and 64 sera from patients with aspergillosis, other systemic mycoses and nocardiosis. Although reactions in both serological tests were invariably strongest with homologous antigen: antibody systems, antisera from rabbits immunized with A. fumigatus, Blastomyces dermatitidis, Candida albicans and Paracoccidioides brasiliensis reacted in the ELISA test with all of the Aspergillus antigens. In contrast, cross-reactivity was virtually non-existent with antiserum to Histoplasma capsulatum. Of five antigens prepared from A fumigatus tested by ELISA against human sera from patients with aspergillosis and other nocardial and systemic fungal infections, sensitivities varied from 81 to 100% for sera from 32 patients with aspergillosis, and specificities from 20 to 97% for sera from 30 patients with nocardiosis and other systemic mycoses. Purified A. fumigatus C antigen reacted weakly with sera from eight of these 30 patients, but the reactions were readily distinguishable from those obtained with sera from patients with aspergillosis. At optimal serum dilutions, cross-reactivities of A. fumigatus in the IFA studies were non-existent in the sera from 28 patients with candidosis, coccidioidomycosis, cryptococcosis, histoplasmosis, paracoccidioidomycosis and nocardiosis. Sensitivities of IFA were 94% for patients with aspergilloma and 83% for patients with allergic bronchopulmonary aspergillosis.

  6. Indirect processes in electron-ion scattering

    SciTech Connect

    Bottcher, C.; Griffin, D.C.; Pindzola, M.S.; Phaneuf, R.A.

    1983-10-01

    A summary is given of an informal workshop held at Oak Ridge National Laboratory on June 22-23, 1983, in which the current status of theoretical calculations of indirect processes in electron-ion scattering was reviewed. Processes of particular interest in astrophysical and fusion plasmas were emphasized. Topics discussed include atomic structure effects, electron-impact ionization, and dielectronic recombination.

  7. 19 CFR 18.43 - Indirect exportation.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    .... port to another for actual exportation at the second port, any export declarations required to be... 19 Customs Duties 1 2010-04-01 2010-04-01 false Indirect exportation. 18.43 Section 18.43 Customs... TRANSPORTATION IN BOND AND MERCHANDISE IN TRANSIT Merchandise Not Otherwise Subject to Customs Control...

  8. 19 CFR 18.26 - Indirect exportation.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... merchandise to be exported and provide such evidence of exportation as required by the port director under... 19 Customs Duties 1 2010-04-01 2010-04-01 false Indirect exportation. 18.26 Section 18.26 Customs... TRANSPORTATION IN BOND AND MERCHANDISE IN TRANSIT Exportation from Customs Custody of Merchandise Unentered...

  9. 19 CFR 10.1024 - Indirect materials.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 19 Customs Duties 1 2013-04-01 2013-04-01 false Indirect materials. 10.1024 Section 10.1024 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY ARTICLES CONDITIONALLY FREE, SUBJECT TO A REDUCED RATE, ETC. United States-Korea Free Trade...

  10. 19 CFR 10.1024 - Indirect materials.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 19 Customs Duties 1 2012-04-01 2012-04-01 false Indirect materials. 10.1024 Section 10.1024 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY ARTICLES CONDITIONALLY FREE, SUBJECT TO A REDUCED RATE, ETC. United States-Korea Free Trade...

  11. Indirect effects of recreation on wildlife

    Treesearch

    David N. Cole; Peter B. Landres

    1995-01-01

    Most of this book focuses on direct impacts to wildlife that result from contact with people. The purpose of our chapter is to provide a broad overview of the indirect influences that recreation has on wildlife. Recreational activities can change the habitat of an animal. This, in turn, affects the behavior, survival, reproduction, and distribution of individuals....

  12. 19 CFR 10.460 - Indirect materials.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 1 2010-04-01 2010-04-01 false Indirect materials. 10.460 Section 10.460 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY ARTICLES CONDITIONALLY FREE, SUBJECT TO A REDUCED RATE, ETC. United States-Chile Free Trade Agreement...

  13. 77 FR 41899 - Indirect Food Additives: Polymers

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-17

    ... HUMAN SERVICES Food and Drug Administration 21 CFR Part 177 Indirect Food Additives: Polymers AGENCY: Food and Drug Administration, HHS. ACTION: Final rule. SUMMARY: The Food and Drug Administration (FDA or the Agency) is amending the food additive regulations to no longer provide for the use of...

  14. Epidemic spreading through direct and indirect interactions

    NASA Astrophysics Data System (ADS)

    Ganguly, Niloy; Krueger, Tyll; Mukherjee, Animesh; Saha, Sudipta

    2014-09-01

    In this paper we study the susceptible-infected-susceptible epidemic dynamics, considering a specialized setting where popular places (termed passive entities) are visited by agents (termed active entities). We consider two types of spreading dynamics: direct spreading, where the active entities infect each other while visiting the passive entities, and indirect spreading, where the passive entities act as carriers and the infection is spread via them. We investigate in particular the effect of selection strategy, i.e., the way passive entities are chosen, in the spread of epidemics. We introduce a mathematical framework to study the effect of an arbitrary selection strategy and derive formulas for prevalence, extinction probabilities, and epidemic thresholds for both indirect and direct spreading. We also obtain a very simple relationship between the extinction probability and the prevalence. We pay special attention to preferential selection and derive exact formulas. The analysis reveals that an increase in the diversity in the selection process lowers the epidemic thresholds. Comparing the direct and indirect spreading, we identify regions in the parameter space where the prevalence of the indirect spreading is higher than the direct one.

  15. Time Spent in Indirect Nursing Care

    DTIC Science & Technology

    1983-09-01

    Part of the FY 83 Army Study Program and intended to augment the FY 81 completed study titled ’ Nursing Care Hours Standards’ by providing valid and...reliable percentages for hospital patient nursing care unit requirements (i.e., direct care, indirect care, and non-productive time). These data

  16. 19 CFR 10.776 - Indirect materials.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 19 Customs Duties 1 2012-04-01 2012-04-01 false Indirect materials. 10.776 Section 10.776 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY ARTICLES CONDITIONALLY FREE, SUBJECT TO A REDUCED RATE, ETC. United States-Morocco Free Trade...

  17. 48 CFR 1631.203 - Indirect costs.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 6 2013-10-01 2013-10-01 false Indirect costs. 1631.203 Section 1631.203 Federal Acquisition Regulations System OFFICE OF PERSONNEL MANAGEMENT FEDERAL EMPLOYEES HEALTH BENEFITS ACQUISITION REGULATION GENERAL CONTRACTING REQUIREMENTS CONTRACT COST PRINCIPLES AND...

  18. 48 CFR 1631.203 - Indirect costs.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 6 2014-10-01 2014-10-01 false Indirect costs. 1631.203 Section 1631.203 Federal Acquisition Regulations System OFFICE OF PERSONNEL MANAGEMENT FEDERAL EMPLOYEES HEALTH BENEFITS ACQUISITION REGULATION GENERAL CONTRACTING REQUIREMENTS CONTRACT COST PRINCIPLES AND...

  19. 48 CFR 1631.203 - Indirect costs.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Indirect costs. 1631.203 Section 1631.203 Federal Acquisition Regulations System OFFICE OF PERSONNEL MANAGEMENT FEDERAL EMPLOYEES HEALTH BENEFITS ACQUISITION REGULATION GENERAL CONTRACTING REQUIREMENTS CONTRACT COST PRINCIPLES AND...

  20. 48 CFR 1631.203 - Indirect costs.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 6 2012-10-01 2012-10-01 false Indirect costs. 1631.203 Section 1631.203 Federal Acquisition Regulations System OFFICE OF PERSONNEL MANAGEMENT FEDERAL EMPLOYEES HEALTH BENEFITS ACQUISITION REGULATION GENERAL CONTRACTING REQUIREMENTS CONTRACT COST PRINCIPLES AND...