Science.gov

Sample records for indirect immunofluorescence microscopy

  1. 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. PMID:26982471

  2. Immunofluorescence Microscopy of Schizosaccharomyces pombe Using Chemical Fixation.

    PubMed

    Hagan, Iain M

    2016-01-01

    Establishing the subcellular distribution of molecules of interest and the dynamics of their spatial control underpins all areas of cell and developmental biology. Although the ability to monitor the distribution of fluorescent fusion proteins has revolutionized cell and developmental biology, indirect immunofluorescence microscopy of fixed samples remains an essential complement to this approach. Immunofluorescence is often a more appropriate approach for the study of subcellular architecture. It avoids potential artifacts caused by studying fusion proteins, which might show altered function under stressful imaging conditions. Furthermore, the quantitative analysis of multiple cells in an unperturbed population by immunofluorescence invariably provides a more accurate assessment of the spatial and temporal control of a particular process than does the analysis of individual cells that is the hallmark of live-cell imaging. Parallel studies of living and fixed cells often provide complementary data sets, both of which can be considered necessary for a comprehensive understanding of molecular function. This protocol provides a method for the visualization of the Schizosaccharomyces pombe microtubule cytoskeleton by indirect immunofluorescence microscopy following chemical fixation with formaldehyde and glutaraldehyde. It includes discussion of common modifications used to monitor the distribution of other fission yeast antigens and forms a basis from which to develop protocols to localize new molecules of interest. PMID:27371599

  3. Clinical aspects of indirect immunofluorescence for autoimmune diseases.

    PubMed

    Ghanadan, Alireza; Saghazadeh, Amene; Jahanzad, Issa; Rezaei, Nima

    2015-05-01

    Because the most common term used in conversations considering autoimmunity is autoantibodies, it is well-expected that the indirect immunofluorescence assay, which detects antibodies directed against various antigens, is one of our most impressive techniques for investigating autoimmune diseases (AIDs). Roughly speaking, the current literature corroborates that this immunopathologic investigation means that autoantibodies detection makes a considerable contribution to both diagnostic and prognostic aspects of AIDs in the clinical setting. However, it varies between different AIDs, autoantibodies, ethnicities or detection methodologies. Directly focusing on the indirect immunofluorescence assay, we present evidence to support this multidimensional variation regarding the subject via reviewing briefly the best-investigated autoantibodies in the well-documented AIDs, including vasculitis, inflammatory bowel disease, scleroderma, autoimmune hepatitis, primary biliary cirrhosis, systemic lupus erythematosus and Sjögren's syndrome. PMID:25786676

  4. Adaptive automatic segmentation of Leishmaniasis parasite in Indirect Immunofluorescence images.

    PubMed

    Ouertani, F; Amiri, H; Bettaib, J; Yazidi, R; Ben Salah, A

    2014-01-01

    This paper describes the first steps for the automation of the serum titration process. In fact, this process requires an Indirect Immunofluorescence (IIF) diagnosis automation. We deal with the initial phase that represents the fluorescence images segmentation. Our approach consists of three principle stages: (1) a color based segmentation which aims at extracting the fluorescent foreground based on k-means clustering, (2) the segmentation of the fluorescent clustered image, and (3) a region-based feature segmentation, intended to remove the fluorescent noisy regions and to locate fluorescent parasites. We evaluated the proposed method on 40 IIF images. Experimental results show that such a method provides reliable and robust automatic segmentation of fluorescent Promastigote parasite. PMID:25571049

  5. Dense fine speckled indirect immunofluorescence pattern in an Australian population.

    PubMed

    Broadfoot, Andrew; Sivertsen, Terri; Baumgart, Karl

    2016-04-01

    The dense fine speckled (DFS) pattern is an antinuclear antibody (ANA) pattern that has recently become of interest. This particular pattern has not been associated with systemic autoimmune rheumatic disorders (SARD) but has been associated with other inflammatory conditions such as interstitial nephritis, autoimmune thyroid disease and atopic eczema as well as being found in healthy individuals. We have been reporting this pattern in our laboratory for the past 3 years. The objective of this study was to determine the frequency of the DFS pattern as detected on indirect immunofluorescence (IIF) in an Australian population and to assess association of this pattern with other laboratory autoimmune markers. ANA tests performed by IIF from July 2012 until June 2014 were reviewed and the frequency of DFS pattern was determined. All DFS positive samples that had undergone concurrent testing for antibodies to extractable nuclear antigens (ENA), anti-dsDNA antibodies, rheumatoid factor (RF), anti-citrullinated protein antibodies (CCP) and anti- phospholipid antibodies were compared. Over the 2 year period, 181,819 patients had ANA tests performed and 51,905 were ANA positive. The DFS pattern was found in 5.7% of ANA positive patients. Within this group of patients, only 1.8% were positive for antibodies to ENA and only 0.7% had anti-dsDNA antibodies level greater than 9 IU/mL. RF and anti-CCP antibodies were positive in 6.3% and 4.1% of DFS positive samples, respectively. There were only two samples positive for anti-phospholipid antibodies when the DFS pattern was present. The presence of the DFS pattern as detected by IIF is infrequently associated with autoimmune markers of SARD which is consistent with international studies. PMID:27020500

  6. USE OF IMMUNOFLUORESCENCE AND PHASE-CONTRAST MICROSCOPY FOR DETECTION AND IDENTIFICATION OF 'GIARDIA' CYSTS IN WATER SAMPLES

    EPA Science Inventory

    A method was developed in which indirect immunofluorescence and phase-contrast microscopy are used for rapid detection and identification of Giardia cysts in raw and finished water supplies. When anti-Giardia cyst antiserum and fluorescein conjugate were applied to known Giardia ...

  7. 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. PMID:25801895

  8. Epidermal innervation morphometry by immunofluorescence and bright-field microscopy.

    PubMed

    Nolano, Maria; Biasiotta, Antonella; Lombardi, Raffaella; Provitera, Vincenzo; Stancanelli, Annamaria; Caporaso, Giuseppe; Santoro, Lucio; Merkies, Ingemar S J; Truini, Andrea; Porretta-Serapiglia, Carla; Cazzato, Daniele; Dacci, Patrizia; Vitale, Dino F; Lauria, Giuseppe

    2015-12-01

    We investigated the agreement between simple indirect immunofluorescence (IF) and bright-field immunohistochemistry (BFI) on free-floating sections for intraepidermal nerve fiber density (IENFD) quantification. Fifty-five healthy subjects and 63 patients with probable small fiber neuropathy (SFN) underwent two adjacent skin biopsies at the distal leg processed by IF and BFI technique. Agreement between IENFD pairs obtained by each method was assessed by Bland-Altman testing. The area under the curve of the receiving operating characteristics (ROC) curves was used to compare the discrimination ability. The diagnostic judgment was based on sex and age-adjusted normative values. IF and BFI showed good correlation (r = 0.81), with a ratio of about 2:1 and a mean difference of 5.5 ± 3.0 IENF per millimeter between paired measures, as demonstrated by linear regression and Bland-Altman test analyses. The square root transformation confirmed a Poisson distribution of the data and a fixed bias between IF and BFI measurements. The ROC curves analysis demonstrated a striking overlap between IF and BFI (0.83 and 0.82; p = 0.72). The diagnosis of SFN disagreed in only 6.7% of cases when the judgment was based on a difference of >1 IENF from 5% cut-off value. IF and BFI showed comparable diagnostic efficiency when referred to appropriate normative reference values. PMID:26309146

  9. Indirect immunofluorescence detection of E. coli O157:H7 with fluorescent silica nanoparticles.

    PubMed

    Chen, Ze-Zhong; Cai, Li; Chen, Min-Yan; Lin, Yi; Pang, Dai-Wen; Tang, Hong-Wu

    2015-04-15

    A method of fluorescent nanoparticle-based indirect immunofluorescence assay using either fluorescence microscopy or flow cytometry for the rapid detection of pathogenic Escherichia coli O157:H7 was developed. The dye-doped silica nanoparticles (NPs) were synthesized using W/O microemulsion methods with the combination of 3-aminopropyltriethoxysilane (APTES) and fluorescein isothiocyanate (FITC) and polymerization reaction with carboxyethylsilanetriol sodium salt (CEOS). Protein A was immobilized at the surface of the NPs by covalent binding to the carboxyl linkers and the surface coverage of Protein A on NPs was determined by the Bradford method. Rabbit anti-E. Coli O157:H7 antibody was used as primary antibody to recognize E. coli O157:H7 and then antibody binding protein (Protein A) labeled with FITC-doped silica NPs (FSiNPs) was used to generate fluorescent signal. With this method, E. Coli O157:H7 in buffer and bacterial mixture was detected. In addition, E. coli O157:H7 in several spiked background beef samples were measured with satisfactory results. Therefore, the FSiNPs are applicable in signal-amplified bioassay of pathogens due to their excellent capabilities such as brighter fluorescence and higher photostability than the direct use of conventional fluorescent dyes. PMID:25460888

  10. A micro-capture ELISA for detecting Mycoplasma pneumoniae IgM: comparison with indirect immunofluorescence and indirect ELISA.

    PubMed Central

    Wreghitt, T. G.; Sillis, M.

    1985-01-01

    A mu-capture ELISA was developed for detecting Mycoplasma pneumoniae-specific IgM, and compared with an indirect immunofluorescent antibody (IFA) technique and an indirect ELISA. mu-capture ELISA and IFA compared well and were found to be the most sensitive assays. The IFA test can be completed in 2 h whilst the results of the mu-capture ELISA can be available in 24 h. Both tests are amenable to routine diagnostic use and have similar sensitivity. Indirect ELISA was found to be less sensitive and less specific, giving high assay values with several sera having undetectable M. pneumoniae CF antibody or CF antibody in low titre. Serum samples obtained from 11 patients at various times after M. pneumoniae infection showed maximum antibody levels within the first month by all assays, with a gradual fall in amount of IgM with time when assayed by mu-capture ELISA, a more gradual decline by IFA and hardly any decline with indirect ELISA. It was concluded that the indirect ELISA is unsuitable for the investigation of possible M. pneumoniae infection because the sustained high assay values with serum samples taken many months after infection, make interpretation of the test results very difficult. PMID:3921607

  11. 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. PMID:26895535

  12. Localization of Ribulose Bisphosphate Carboxylase in the Guard Cells by an Indirect, Immunofluorescence Technique 1

    PubMed Central

    Madhavan, Soundararajan; Smith, Bruce N.

    1982-01-01

    Ribulose bisphosphate carboxylase, a key enzyme in the photosynthetic carboxylation process, has been localized through an indirect immunofluorescent technique in the guard cells of some of the 41 species of plants examined. This sample includes 17 families of both dicotyledons and monocotyledons, one gymnosperm, and one pteridophyte. Plants were selected to represent all of the three major photosynthetic categories, namely C3, C4, and Crassulacean acid metabolism. Antibodies raised against tobacco (Nicotiana tabacum L.) ribulose bisphosphate carboxylase were used for this immunofluorescent study. A good degree of fluorescence was observed in the guard cells of seven out of 21 species exhibiting Crassulacean acid metabolism. C3 plants exhibited a very low degree (almost negligible) of fluorescence, while the C4 species did not exhibit any fluorescence. Images PMID:16662174

  13. 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. PMID:25614095

  14. [Determination of anti-rabies antibodies by means of the indirect immunofluorescence method].

    PubMed

    Jiran, E; Závora, M

    1975-06-01

    The paper presents a description of the method of indirect immunofluorescence (IFRA test) for the demonstration of post-vaccination anti-rabies antibodies. The model of immune hamster serum was used to study the reproducibility of the test. The dynamics of antibody formation was quantitatively examined in rabbits immunized by the Flury-LEP virus. For brief information, the test was also used for the demonstration of post-vaccination antibodes against rabies in dogs. The method can be recommended as an additional and orientation test for the evaluation of the immunogenic properties of vaccines against rabies. PMID:810938

  15. 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.

  16. Original Approach for Automated Quantification of Antinuclear Autoantibodies by Indirect Immunofluorescence

    PubMed Central

    Bertin, Daniel; Jourde-Chiche, Noémie; Bongrand, Pierre

    2013-01-01

    Introduction. Indirect immunofluorescence (IIF) is the gold standard method for the detection of antinuclear antibodies (ANA) which are essential markers for the diagnosis of systemic autoimmune rheumatic diseases. For the discrimination of positive and negative samples, we propose here an original approach named Immunofluorescence for Computed Antinuclear antibody Rational Evaluation (ICARE) based on the calculation of a fluorescence index (FI). Methods. We made comparison between FI and visual evaluations on 237 consecutive samples and on a cohort of 25 patients with SLE. Results. We obtained very good technical performance of FI (95% sensitivity, 98% specificity, and a kappa of 0.92), even in a subgroup of weakly positive samples. A significant correlation between quantification of FI and IIF ANA titers was found (Spearman's ρ = 0.80, P < 0.0001). Clinical performance of ICARE was validated on a cohort of patients with SLE corroborating the fact that FI could represent an attractive alternative for the evaluation of antibody titer. Conclusion. Our results represent a major step for automated quantification of IIF ANA, opening attractive perspectives such as rapid sample screening and laboratory standardization. PMID:24454469

  17. Monitoring the Localization of MAP1LC3B by Indirect Immunofluorescence.

    PubMed

    Ktistakis, Nicholas T

    2015-08-01

    The autophagy protein MAP1LC3B (microtubule-associated proteins 1A/1B light chain 3B, hereafter referred to as LC3B), which is one of several mammalian homologs of yeast Atg8, is one of the most popular markers for autophagosome formation because its distribution changes from cytosolic/diffuse to punctate upon the induction of autophagy. In many settings, plasmids encoding fluorescently tagged LC3B are introduced into cells, and the subsequent autophagy response is monitored. However, for a variety of reasons, it would be desirable also to have a protocol to monitor the localization of endogenous LC3B under various conditions. This protocol provides such a methodology for the staining of endogenous LC3B by indirect immunofluorescence, such that autophagy responses can be monitored in mammalian cells. PMID:26240409

  18. An indirect immunofluorescent test for detection of rabies virus antibodies in foxes.

    PubMed

    Hostnik, P; Grom, J

    1997-01-01

    The blood-containing fluids in the thoracic cavity or blood from the heart from 177 red foxes (Vulpes vulpes) in Slovenia were evaluated for rabies antibodies by rapid fluorescent focus inhibition test (RFFIT) and an adapted indirect immunofluorescent test (IIF) in 1994. We evaluated the usefulness of anti-dog fluorescein-isothiocyanate (FITC) conjugate instead of anti-fox FITC conjugate in detection of antibodies against rabies virus in fox sera. In the RFFIT test, 92 (52%) of the fox samples were positive and 70 (40%) samples were negative for rabies antibodies; 15 (8.5%) samples were not suitable for examination in this test. In the IIF test, 98 (55%) fox samples were positive and 79 (45%) sera were negative. The IIF test was suitable for the rapid detection of antibodies against rabies virus in foxes, as often required for vaccine efficacy trials. PMID:9027703

  19. Indirect Immunofluorescence of Proteins in Oogenic Germ Cells of Caenorhabditis elegans.

    PubMed

    Brenner, John L; Schedl, Tim

    2016-01-01

    Formation of full-grown oocytes requires the control and coordination of a number of processes (e.g., oocyte growth) through multiple stages, where disruption at any one step can result in infertility. Numerous proteins are required for the regulation and execution of the various oogenic processes as well as functioning as maternal products needed for embryogenesis. Immunofluorescence microscopy combined with staining using antibodies against specific proteins, or their posttranslationally modified forms, is a standard approach to determine the temporal and spatial location of gene products that function in oocyte development. The simple linear organization of the germline in the model organism Caenorhabditis elegans allows easy correlation of protein localization and germ cell developmental stage, thus aiding in our understanding of protein function during gametogenesis. Here we outline co-immunofluorescence staining for two major regulators of C. elegans germline development, the translational repressor GLD-1 and activated form of MPK-1 (dpMPK-1) ERK MAP kinase in dissected gonads from adult C. elegans. Worms are first dissected and the extruded gonads are fixed and permeabilized before being bathed in primary antibodies against GLD-1 and dpMPK-1. Secondary antibodies conjugated to fluorophore dyes and that target the IgG domains of the primary antibody reagents are then used to provide a fluorescent signal that corresponds to the position of GLD-1 and dpMPK-1. The outlined procedure is amenable to many other proteins expressed in C. elegans germ cells. PMID:27557570

  20. 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. PMID:26000128

  1. [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. PMID:10758696

  2. 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. PMID:27026659

  3. Automated Indirect Immunofluorescence Evaluation of Antinuclear Autoantibodies on HEp-2 Cells

    PubMed Central

    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. PMID:23251220

  4. Measuring NLR Oligomerization II: Detection of ASC Speck Formation by Confocal Microscopy and Immunofluorescence.

    PubMed

    Beilharz, Michael; De Nardo's, Dominic; Latz, Eicke; Franklin, Bernardo S

    2016-01-01

    Inflammasome assembly results in the formation of a large intracellular protein scaffold driven by the oligomerization of the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). Following inflammasome activation, ASC polymerizes to form a large singular structure termed the ASC "speck," which is crucial for recruitment of caspase-1 and its inflammatory activity. Hence, due to the considerably large size of these structures, ASC specks can be easily visualized by microscopy as a simple upstream readout for inflammasome activation. Here, we provide two detailed protocols for imaging ASC specks: by (1) live-cell imaging of monocyte/macrophage cell lines expressing a fluorescently tagged version of ASC and (2) immunofluorescence of endogenous ASC in cell lines and human immune cells. In addition, we outline a protocol for increasing the specificity of ASC antibodies for use in immunofluorescence. PMID:27221487

  5. Indirect immunofluorescence localization of beta-adrenergic receptors and G-proteins in human A431 cells.

    PubMed Central

    Wang, H Y; Berrios, M; Malbon, C C

    1989-01-01

    Polyclonal antibodies directed against (i) rodent lung beta 2-adrenergic receptor, (ii) a synthetic fragment of an extracellular domain of the receptor, and (iii) human placenta G-protein beta-subunits, were used to localize these antigens in situ in intact and permeabilized human epidermoid carcinoma A431 cells. Antibodies directed against beta 2-adrenergic receptors showed a punctate immunofluorescence staining throughout the cell surface of fixed intact cells. Punctate staining was also observed in clones of Chinese hamster ovary cells transfected with an expression vector harbouring the gene for the hamster beta 2-adrenergic receptor. The immunofluorescence observed with anti-receptor antibodies paralleled the level of receptor expression. In contrast, the beta-subunits common to G-proteins were not stained in fixed intact cells, presumably reflecting their intracellular localization. In detergent-permeabilized fixed cells, strong punctate staining of G beta-subunits was observed throughout the cytoplasm. This is the first indirect immunofluorescence localization of beta-adrenergic receptors and G-proteins. Punctate immunofluorescence staining suggests that both antigens are distributed in clusters. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. p528-a Fig. 9. Fig. 10. PMID:2556996

  6. 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.

  7. Immunofluorescence Staining.

    PubMed

    Donaldson, Julie G

    2015-01-01

    This unit provides a protocol for indirect immunofluorescence, which is a method that provides information about the locations of specific molecules and the structure of the cell. Antibody molecules for a specific target molecule are exposed to the cell or tissue being investigated. The binding of these molecules is detected by incubating the sample with a secondary antibody specific for immunoglobulin molecules and conjugated to a fluorophore. This provides both a visible signal and amplification of the signal and the results are observed with a fluorescence microscope. This unit describes the widely used and powerful technique of localization of proteins in cells by immunofluorescence. The location can be determined by double labeling with an antibody directed against a protein of known location. The technique can be used as a supplement to immunolocalization by electron microscopy and subcellular fractionation. It allows not only identification of the antigen distribution in the cell but also a survey of the dynamic aspects of protein movements in the cell-on and off membranes, into and out of the nucleus, and through membrane traffic pathways. PMID:26621373

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

    PubMed

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

    2016-01-01

    Microtubules (MTs) 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 labeling, 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

  9. 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

  10. 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.

  11. 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

  12. Identification of Ancient Silk Using an Enzyme-linked Immunosorbent Assay and Immuno-fluorescence Microscopy.

    PubMed

    Liu, Miaomiao; Xie, Jun; Zheng, Hailing; Zhou, Yang; Wang, Bing; Hu, Zhiwen

    2015-01-01

    The identification of ancient silk is of great importance in both archaeology and academia. In the present work, a specific antibody having the characteristics of low cost, easy operation and extensive applicability was developed directly through immunizing rabbits with complete antigen (silk fibroin, SF). Then, antibody-based immunoassays, i.e. enzyme-linked immunosorbent assay (ELISA) and immuno-fluorescence microscopy (IFM), were established and conducted in tandem to identify the corresponding protein in ancient silks. The anti-SF antibody exhibits high sensitivity and specificity for the identification of modern and ancient silks. The detection limit of the ELISA method is about 0.1 ng/mL, and no cross-reactions with other possible interference antigens have been noted. IFM makes it possible to localize target proteins in archaeological samples, and also ensure the reliability of the ELISA results. Based on these advantages, immunological techniques have the potential to become powerful analytical tools at archaeological sites and conservation science laboratories. PMID:26656824

  13. Pathologic features of renal biopsies based on H & E, immunofluorescence and electron microscopy.

    PubMed

    Zahir, S Taghipour; Hosseini, E

    2014-01-01

    Kidneys are complex organs with multiple vital functions. They are an essential part of the urinary system and also are necessary for regulation of body homeostasis like electrolytes, acid base balance and blood pressure. Diagnosis of renal injuries is based on clinical and histopathologic features. In a descriptive cross-sectional study conducted in Shahid Sadoughi Hospital, Yazd, Iran, pathology reports of all renal biopsies, by light microscopic examination, immunofluorecence and electron microscopy (EM) were perused. Data were registered in a questionnaire with questions on patients' demographic information such as age, sex and also questions on clinical signs and symptoms and pathologic findings such as H & E, Immunofluorescence (IF) and electron microscopy. All data were analyzed by SPSS-15 software with descriptive analysis. A total of 80 patients were included in this study, 42 men (52.5%) and 38 women (47.5%), aged 19-73 years (mean: 40.59 ± 16.36). Based on H & E, IF and electron microscopic findings, it seems that in 26.4% of cases the IFM was necessary and in 67.6% was helpful and in 6% was unnecessary for diagnosis. Between 42 patients, EM was necessary in 12% of patients, while in 71.5% was helpful and in 16.5% was unnecessary. Based on IFM the most common renal disease was FSGS (focal segmental glomerulosclerosis) with mean age of 41.3 years. IFM was necessary in RPGN, chronic glomerulonephritis, mesangial hypercellularity, minimal change disease, IgA nephropathy, and was helpful in FSGS, MPGN, tubulointerstitial nephritis, diffuse sclerosing glomerulonephritis and membranous glomerulopathy but was unnecessary in lupus nephritis. EM was necessary in mesangial hypercellularity, chronic glomerulonephritis and diffuse sclerosing glomerulopathy and was helpful in FSGS, MPGN, lupus nephritis and membranous glomerulopathy while it was unnecessary in minimal change disease and IgA nephropathy. PMID:25726629

  14. Basics in standardization and practical applications of immunofluorescent microscopy: standardization of antinuclear antibody tests.

    PubMed

    Beutner, E H; Kumar, V; Greenlee, P

    1983-01-01

    ANA tests are, at present, the primary example of the diagnostic use of an indirect IF method. Appropriately standardized and interpreted ANA tests afford the primary sero-diagnostic screening test for certain connective tissue diseases. In the present state of the art of ANA testing, it appears possible to achieve appropriate standardization in up to 70% of laboratories, however, further work remains to be done to achieve a 90% or higher frequency of reliable testing among clinical laboratories for a given antigenic substrate. The present indications for the preparation and use of this and other IF methods assay systems for clinical laboratory studies are as follows: a. For each antigen substrate used for ANA tests, the manufacturers of kits or the laboratories which prepare their own ANA test reagents, should take responsibility for assuring that the sensitivity of their test systems as measured by ANA titers falls in the range expected for that particular antigenic substrate. b. If adequate assurance of the appropriate sensitivity level of a given ANA test system is provided both by their manufacturers and users, then physicians should be supplied with data on the frequencies of biologic false positives for different age groups of males and females as well as frequencies of biologic false negatives for at least the major diseases for which ANA are of diagnostic significance. Part II of this report (Chorzelski et al., in press) presents data on this point. c. Since ANA tests detect a heterogeneous population of antibody specificities, several of which are now recognized as having distinct clinical significance (Tan, 1981), appropriately standardized tests for each of these diagnostically relevant antibodies to identified nuclear antigens needs to be made available to physicians by clinical laboratories. They need to be provided with data on the frequencies of false negatives, biologic false positives and, importantly, with data on the kinetics or dynamics of the

  15. Flow cytometry compared with indirect immunofluorescence for rapid detection of dengue virus type 1 after amplification in tissue culture.

    PubMed

    Kao, C L; Wu, M C; Chiu, Y H; Lin, J L; Wu, Y C; Yueh, Y Y; Chen, L K; Shaio, M F; King, C C

    2001-10-01

    Dengue virus (DV) was detected early in infected mosquito C6/36 cells by using indirect immunofluorescence (IF) in conjunction with flow cytometry. Three fixation-permeabilization methods and three DV serotype 1 (DEN-1)-specific monoclonal antibodies, 8-8 (anti-E), 16-4 (anti-NS1), and 15F3-1 (anti-NS1), were evaluated for the detection of DEN-1 in infected C6/36 cells. We found that these three monoclonal antibodies were capable of detecting DV in C6/36 cells as early as 24 h postinoculation by using a conventional indirect IF stain. Both 8-8 and 16-4 detected DV earlier and showed a greater number of DV-positive cells than 15F3-1. In flow cytometry, 3% paraformaldehyde plus 0.1% Triton X-100 with 16-4, the best fixation-permeabilization method for testing DV, showed higher sensitivity (up to 1 PFU) than indirect IF stain. The higher sensitivity of 16-4 in detecting DEN-1 was found with both IF and flow cytometry. Flow cytometry, which had a sensitivity similar to that of nested reverse transcription-PCR, was more sensitive in detecting DV in the infected mosquito cells 10 h earlier than the conventional IF stain. When clinical specimens were amplified in mosquito C6/36 cells and then assayed for DV using flow cytometry and conventional virus isolation at day 7 postinfection, both methods had 97.22% (35 out of 36) agreement. Moreover, among 12 positive samples which were detected by conventional culture method, the flow cytometry assay could detect DV in 58.33% (7 out of 12) of samples even at day 3 postinfection. In conclusion, both monoclonal antibodies 8-8 and 16-4 can be used for the early detection of DEN-1-infected C6/36 cells, with 16-4 (anti-NS1) being the best choice for the rapid diagnosis of DV by both the IF staining and flow cytometry methods. PMID:11574589

  16. Detection of Cell Proliferation Markers by Immunofluorescence Staining and Microscopy Imaging in Paraffin-Embedded Tissue Sections.

    PubMed

    Eminaga, Seda; Teekakirikul, Polakit; Seidman, Christine E; Seidman, Jonathan G

    2016-01-01

    This unit describes a step-by-step protocol to detect and quantify proliferating cells in paraffin-embedded tissue sections. Two well-established markers of proliferation (incorporation of BrdU into newly synthesized DNA and expression of the nuclear protein Ki67) are detected after antigen-retrieval and subsequent immunofluorescence staining and confocal microscopy. © 2016 by John Wiley & Sons, Inc. PMID:27366888

  17. DETECTION AND IDENTIFICATION OF 'GIARDIA' CYSTS USING IMMUNOFLUORESCENCE AND PHASE CONTRAST MICROSCOPY

    EPA Science Inventory

    Detection and identification of Giardia cysts in water samples has been improved by the development of an immunofluorescent method that specifically stains Giardia cysts bright green and allows their easy detection against a black background. The report discusses aspects of the m...

  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. PMID:27311277

  19. Small Quantum Dots Conjugated to Nanobodies as Immunofluorescence Probes for Nanometric Microscopy

    PubMed Central

    2015-01-01

    Immunofluorescence, a powerful technique to detect specific targets using fluorescently labeled antibodies, has been widely used in both scientific research and clinical diagnostics. The probes should be made with small antibodies and high brightness. We conjugated GFP binding protein (GBP) nanobodies, small single-chain antibodies from llamas, with new ∼7 nm quantum dots. These provide simple and versatile immunofluorescence nanoprobes with nanometer accuracy and resolution. Using the new probes we tracked the walking of individual kinesin motors and measured their 8 nm step sizes; we tracked Piezo1 channels, which are eukaryotic mechanosensitive channels; we also tracked AMPA receptors on living neurons. Finally, we used a new super-resolution algorithm based on blinking of (small) quantum dots that allowed ∼2 nm precision. PMID:25397889

  20. Interpretation of ANA Indirect Immunofluorescence Test Outside the Darkroom Using NOVA View Compared to Manual Microscopy

    PubMed Central

    Copple, Susan S.; Jaskowski, Troy D.; Giles, Rashelle; Hill, Harry R.

    2014-01-01

    Objective. To evaluate NOVA View with focus on reading archived images versus microscope based manual interpretation of ANA HEp-2 slides by an experienced, certified medical technologist. Methods. 369 well defined sera from: 44 rheumatoid arthritis, 50 systemic lupus erythematosus, 35 scleroderma, 19 Sjögren's syndrome, and 10 polymyositis patients as well as 99 healthy controls were examined. In addition, 12 defined sera from the Centers for Disease Control and 100 random patient sera sent to ARUP Laboratories for ANA HEp-2 IIF testing were included. Samples were read using the archived images on NOVA View and compared to results obtained from manual reading. Results. At a 1 : 40/1 : 80 dilution the resulting comparison demonstrated 94.8%/92.9% positive, 97.4%/97.4% negative, and 96.5%/96.2% total agreements between manual IIF and NOVA View archived images. Agreement of identifiable patterns between methods was 97%, with PCNA and mixed patterns undetermined. Conclusion. Excellent agreements were obtained between reading archived images on NOVA View and manually on a fluorescent microscope. In addition, workflow benefits were observed which need to be analyzed in future studies. PMID:24741573

  1. Improved detection of lacZ reporter gene expression in transgenic epithelia by immunofluorescence microscopy.

    PubMed

    Mahony, Donna; Karunaratne, Seetha; Rothnagel, Joseph A

    2002-04-01

    The bacterial lacZ gene is commonly used as a reporter for the in vivo analysis of gene regulation in transgenic mice. However, several laboratories have reported poor detection of beta-galactosidase (the lacZ gene product) using histochemical techniques, particularly in skin. Here we report the difficulties we encountered in assessing lacZ expression in transgenic keratinocytes using classic X-gal histochemical protocols in tissues shown to express the transgene by mRNA in situ hybridization. We found that lacZ reporter gene expression could be reliably detected in frozen tissue sections by immunofluorescence analysis using a beta-galactosidase-specific antibody. Moreover, we were able to localize both transgene and endogenous gene products simultaneously using double-label immunofluorescence. Our results suggest that antibody detection of beta-galactosidase should be used to verify other assays of lacZ expression, particularly where low expression levels are suspected or patchy expression is observed. PMID:11994142

  2. 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.

  3. [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

  4. Rapid Identification of Candida dubliniensis by Indirect Immunofluorescence Based on Differential Localization of Antigens on C. dubliniensis Blastospores and Candida albicans Germ Tubes

    PubMed Central

    Bikandi, Joseba; Millán, Rosario San; Moragues, María D.; Cebas, Gontzal; Clarke, Mary; Coleman, David C.; Sullivan, Derek J.; Quindós, Guillermo; Pontón, José

    1998-01-01

    There is a clear need for the development of a rapid and reliable test for the identification of Candida dubliniensis and for the discrimination of this species from Candida albicans. In the present study we have investigated the potential use of C. dubliniensis-specific antigens as a basis for its identification. We produced an anti-C. dubliniensis serum which, after adsorption with C. albicans blastospores, was found to differentially label C. dubliniensis isolates in an indirect immunofluorescence test. In this test, the antiserum reacted with blastospores and germ tubes of C. dubliniensis and with blastospores of Candida krusei and Rhodotorula rubra but did not react with blastospores of several other Candida species including C. albicans. The antiserum also reacted with C. albicans germ tubes. The anti-C. dubliniensis adsorbed serum reacted with specific components of 25, 28, 37, 40, 52, and 62 kDa in the C. dubliniensis extract and with a variety of antigens from other yeast species. The antigens from non-C. dubliniensis yeasts showing reactivity with the anti-C. dubliniensis adsorbed serum are mostly expressed within the cell walls of these yeast species, and this reactivity does not interfere with the use of the anti-C. dubliniensis adsorbed serum in an indirect immunofluorescence test for the rapid identification of C. dubliniensis. PMID:9705368

  5. Immunofluorescence-guided atomic force microscopy to measure the micromechanical properties of the pericellular matrix of porcine articular cartilage.

    PubMed

    Wilusz, Rebecca E; DeFrate, Louis E; Guilak, Farshid

    2012-11-01

    The pericellular matrix (PCM) is a narrow region that is rich in type VI collagen that surrounds each chondrocyte within the extracellular matrix (ECM) of articular cartilage. Previous studies have demonstrated that the chondrocyte micromechanical environment depends on the relative properties of the chondrocyte, its PCM and the ECM. The objective of this study was to measure the influence of type VI collagen on site-specific micromechanical properties of cartilage in situ by combining atomic force microscopy stiffness mapping with immunofluorescence imaging of PCM and ECM regions in cryo-sectioned tissue samples. This method was used to test the hypotheses that PCM biomechanical properties correlate with the presence of type VI collagen and are uniform with depth from the articular surface. Control experiments verified that immunolabelling did not affect the properties of the ECM or PCM. PCM biomechanical properties correlated with the presence of type VI collagen, and matrix regions lacking type VI collagen immediately adjacent to the PCM exhibited higher elastic moduli than regions positive for type VI collagen. PCM elastic moduli were similar in all three zones. Our findings provide further support for type VI collagen in defining the chondrocyte PCM and contributing to its biological and biomechanical properties. PMID:22675162

  6. Localization of Cell Division Protein FtsQ by Immunofluorescence Microscopy in Dividing and Nondividing Cells of Escherichia coli

    PubMed Central

    Buddelmeijer, Nienke; Aarsman, Mirjam E. G.; Kolk, Arend H. J.; Vicente, Miguel; Nanninga, Nanne

    1998-01-01

    The localization of cell division protein FtsQ in Escherichia coli wild-type cells was studied by immunofluorescence microscopy with specific monoclonal antibodies. FtsQ could be localized to the division site in constricting cells. FtsQ could also localize to the division site in ftsQ1(Ts) cells grown at the permissive temperature. A hybrid protein in which the cytoplasmic domain and the transmembrane domain were derived from the γ form of penicillin-binding protein 1B and the periplasmic domain was derived from FtsQ was also able to localize to the division site. This result indicates that the periplasmic domain of FtsQ determines the localization of FtsQ, as has also been concluded by others for the periplasmic domain of FtsN. Noncentral FtsQ foci were found in the area of the cell where the nucleoid resides and were therefore assumed to represent sites where the FtsQ protein is synthesized and simultaneously inserted into the cytoplasmic membrane. PMID:9829918

  7. [Serological demonstration of experimental round worm infections-Ascaris suum, Toxocara canis--in swine by means of the indirect immunofluorescence antibody test].

    PubMed

    Buchwalder, R; Matthes, H F; Hiepe, T

    1981-11-01

    By means of indirect immunofluorescent antibody reaction (IFAR), using serum of experimentally infected pigs, various antigens were studied with regard to their usefulness for serological verification of prepatent Ascaris suum and Toxocara canis infections. Eggs, egg larvae, larvae received from livers, lungs and brains of experimentally infected white mice and sections of adult T. canis as well as eggs, egg larvae, liver larvae and sections of frozen adult A. suum proved to be not suitable for the reliable serum diagnosis of the infections. On the other hand, A. suum larvae, isolated from lungs of white mice or guinea pigs days after experimental infection, represent an antigen applicable to IFAR for the evidence of prepatent A. suum infections in pigs. The antigen, stored at -20 degree C, is durable without substantial impairment of its reactivity at least 7 months. PMID:7039425

  8. Immunofluorescence microscopy of SNAP23 in human skeletal muscle reveals colocalization with plasma membrane, lipid droplets, and mitochondria.

    PubMed

    Strauss, Juliette A; Shaw, Christopher S; Bradley, Helen; Wilson, Oliver J; Dorval, Thierry; Pilling, James; Wagenmakers, Anton J M

    2016-01-01

    Synaptosomal-associated protein 23 (SNAP23) is a SNARE protein expressed abundantly in human skeletal muscle. Its established role is to mediate insulin-stimulated docking and fusion of glucose transporter 4 (GLUT4) with the plasma membrane. Recent in vitro research has proposed that SNAP23 may also play a role in the fusion of growing lipid droplets (LDs) and the channeling of LD-derived fatty acids (FAs) into neighboring mitochondria for β-oxidation. This study investigates the subcellular distribution of SNAP23 in human skeletal muscle using immunofluorescence microscopy to confirm that SNAP23 localization supports the three proposed metabolic roles. Percutaneous biopsies were obtained from the m. vastus lateralis of six lean, healthy males in the rested, overnight fasted state. Cryosections were stained with antibodies targeting SNAP23, the mitochondrial marker cytochrome c oxidase and the plasma membrane marker dystrophin, whereas intramuscular LDs were stained using the neutral lipid dye oil red O. SNAP23 displayed areas of intense punctate staining in the intracellular regions of all muscle fibers and continuous intense staining in peripheral regions of the cell. Quantitation of confocal microscopy images showed colocalization of SNAP23 with the plasma membrane marker dystrophin (Pearson's correlation coefficient r = 0.50 ± 0.01). The intense punctate intracellular staining colocalized primarily with the mitochondrial marker cytochrome C oxidase (r = 0.50 ± 0.012) and to a lesser extent with LDs (r = 0.21 ± 0.01) visualized with oil red O. We conclude that the observed subcellular distribution of SNAP23 in human skeletal muscle supports the three aforementioned metabolic roles. PMID:26733245

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

    PubMed Central

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

    2014-01-01

    Abstract 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. PMID:25052490

  10. 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. PMID:24211046

  11. 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. PMID:25455225

  12. 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

  13. The use of chosen serological diagnostic methods in Lyme disease in horses. Part I. Indirect immunofluorescence and enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Dzierzecka, M; Kita, J

    2002-01-01

    The investigations aimed to establish the reliability of the chosen serological tests designed for the diagnosis of Lyme borreliosis in horses. The investigations were carried out in five Horse Breeding Centres (OHK). Statistical analysis methods were used to determine sample size for particular centres: Krasne (Kr)--49, Łack (Ł)--21, Walewice (W)--111, BogusŁawice (B)--17, Kozienice (K)--61. The experimental material comprised the chosen horses from which blood samples were collected in order to obtain sera. The test used for indirect immunofluorescence assay (IFA No 75941, Bio-Mérieux) is commercially designed for the investigation of human sera and thus needed a prior species adaptation and standardization; ELISA (MRL DIAGNOSTICS, No EL0400G) which was also species adapted and stardandized and ELISA commercially assigned for the examination of dog or horse sera (Die System Diagnostica GmbH Borrelia burgdorferi Veterinary ELISA No. 122.00 Genzyme Virotech GmbH). In the IFA test the highest share of positive results was obtained in respect of the sera from OHK in (K)--60.7% and then in (B)--52.9%, (Ł)--42.9%, (W)--40.5%, (Kr)--38.7%. In the standardized ELISA the highest percent of positive results, amounting to 33.3%, was obtained in respect of the sera from (Ł), and then from (W)--20.7%, (K)--11.5%, (Kr)--10.2% and (B)--5.9%. The percent of positive results obtained in the commercial ELISA also agreement on a high level: the sera originating from (W) were positive in 18.9%, from (K)--9.8%, (Ł)--9.5%. (B)--5.9% and (Kr)--4.1%. Both ELISAs showed high agreement although the standardized test was characterized by a greater tendency for suggesting the presence of B. burgdorferi infection and the agreement of these two ELISAs with the IFA was not so strong. The IFA showed the highest tendency for suggesting the presence of the B. burgdorferi infection, being characterized by the highest percent of false positive results. PMID:12189952

  14. Estimation of changes in vimentin filaments induced by etoposide and doxorubicin in human leukemia cell line K-562 by using immunofluorescence microscopy.

    PubMed

    Grzanka, A

    2001-01-01

    This study was undertaken to examine the influence of etoposide and doxorubicin on the distribution of vimentin in cells of human leukemia cell line K-562 by using immunofluorescence microscopy. The cells were cultured with 5 different doses of etoposide: 0.02, 0.2, 2, 20, 200 microM/l and three doses of doxorubicin: 0.5, 5, 10 microM/l. Changes in vimentin filaments were dependent on concentration of drugs compared to untreated control cells. Cells treated with 20 microM/l, especially with 200 microM/l etoposide were much bigger from other cells exposed to lower doses of etoposide and control cells, and their number decreased. In most control cells vimentin was seen as a ring with the increased concentration on one pole of the cells. In 20 microM/l and 200 microM/l etoposide the cells showed rather a diffuse cytoplasmic staining pattern. Vimentin filaments were organized as a dense network in cytoplasm of these cells. Immunofluorescence studies on K-562 cells treated with doxorubicin showed that cells incubated with 5 microM/l doxorubicin have much diffuse staining pattern of vimentin with delicate reticular structure and with intense staining near one pool of the cells. Addition of 10 microM/l doxorubicin to cells resulted in increasing of fluorescence staining, which appeared in the cells as enough dense network with intense staining rather in the centre of the cell. PMID:11712680

  15. Comparison of sensitivity and specificity of 4 methods for detection of Giardia duodenalis in feces: immunofluorescence and PCR are superior to microscopy of concentrated iodine-stained samples.

    PubMed

    Gotfred-Rasmussen, Helle; Lund, Marianne; Enemark, Heidi L; Erlandsen, Mogens; Petersen, Eskild

    2016-03-01

    For decades, microscopy of feces after formol-ethylacetate (FEA) concentration and iodine staining has been the routine test for intestinal protozoa. Lately, polymerase chain reaction or fluorescence-labeled parasite-specific antibodies have been introduced, but their place in everyday routine diagnostics has not yet been established. We compared FEA and salt-sugar flotation (SSF) concentration followed by microscopy of iodine-stained concentrate and immunofluorescence assay (IFA) and real-time polymerase chain reaction (qPCR) for detection of Giardia duodenalis in human feces. The median number of Giardia cysts found by FEA in 19 Giardia-positive samples was 50 cysts per gram (CPG), by SSF 350 CPG, by IFA 76,700 CPG, and by qPCR 316,000 CPG. We next tested 455 consecutive samples for presence of Giardia cysts. Using IFA as reference, qPCR had a sensitivity of 91%, specificity of 95.1%, a false-positive rate of 50%, a false-negative rate of 0.48%, a positive predictive value of 50%, and a negative predictive value of 99.5%. In conclusion, qPCR and IFA were significantly more sensitive than microscopy of iodine-stained concentrates using either FEA or SSF. We suggest, when using qPCR, that positive samples are verified by IFA to prevent false-positive results. PMID:26707069

  16. 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. PMID:27302205

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

    DOE PAGESBeta

    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 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

  18. Immunofluorescence in dermatology.

    PubMed

    Chhabra, Seema; Minz, Ranjana Walker; Saikia, Biman

    2012-01-01

    Direct immunofluorescence (DIF) and indirect immunofluorescence (IIF) tests on skin biopsy are being done mostly in academic teaching hospitals. These tests provide a useful diagnostic aid to dermatologists. Immunohistology and serology can, in conjunction with histology, provide considerable help in delineation and diagnosis of various skin disorders as well as systemic diseases with skin involvement, e.g. systemic lupus erythematosus. Immunofluorescence (IF) studies have now become an invaluable supplement to clinical and histological examination in a variety of dermatological diseases. These skin diseases now include not only bullous and connective tissue disorders, vasculitides, and conditions such as lichen planus, but also the scaling dermatoses, notably psoriasis. In this review article, we share our experience of providing such a diagnostic facility for more than 30 years in a large tertiary care health center in North India and also help to outline the conditions, which can be diagnosed confidently, and others where IF can help in confirming a diagnosis or the immune component of the disease. The article also deals with handling of skin biopsy specimens and interpretation of biopsy findings on DIF and IIF examination. PMID:23075636

  19. 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. PMID:25064337

  20. 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

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

    PubMed

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

    2015-09-01

    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

  2. Immunofluorescent Staining of Mouse Intestinal Stem Cells

    PubMed Central

    O’Rourke, Kevin P.; Dow, Lukas E; Lowe, Scott W

    2016-01-01

    Immunofluorescent staining of organoids can be performed to visualize molecular markers of cell behavior. For example, cell proliferation marked by incorporation of nucleotide (EdU), or to observe markers of intestinal differentiation including paneth cells, goblet cells, or enterocytes (see Figure 1). In this protocol we detail a method to fix, permeabilize, stain and mount intestinal organoids for analysis by immunofluorescent confocal microscopy.

  3. 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

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

    2016-06-01

    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

  4. Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus.

    PubMed

    Allaire, Andréa; Picard-Jean, Frédéric; Bisaillon, Martin

    2015-01-01

    Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed. PMID:26485289

  5. Quantitative studies of immunofluorescent staining

    PubMed Central

    Wick, G.; Beutner, E. H.

    1970-01-01

    The antiperinuclear factor (APF) is found in a high percentage of sera from patients with rheumatoid arthritis. It can be demonstrated by direct immunofluorescence using the keratohyaline granules of human buccal mucosa as antigenic substrate. Mixing of some normal goat sera with an APF positive serum from a patient with rheumatoid arthritis resulted in an inhibition of the APF titre of the patient's serum. However, there was no clear cut correlation between the APF-positivity of normal goat sera and their inhibitory effect on the APF-reactivity of a human rheumatoid arthritis patient's serum. In reciprocal screening tests the human rheumatoid arthritis serum blocked only one of the APF-reactive goat sera. The reciprocal blocking activity of this goat serum and the patient's serum could be more exactly evaluated by the use of chessboard titrations in an indirect immunofluorescence blocking test. This test consisted of mixing equal volumes of serial dilutions of a goat serum and the patient's serum and subsequent examination of the mixtures for APF using an anti-human IgG conjugate and an anti-goat immunoglobulin conjugate, respectively. The results point to an antibody nature for the APF in preimmune, normal goat sera and to the value of chessboard titrations of this type in demonstrating the identity, non-identity, partial identity (or very close proximity of antigenic determinants) of the antibodies in different antisera which cannot be distinguished by their immunofluorescent staining patterns. ImagesFIG. 1FIG. 2 PMID:4913803

  6. The immunofluorescence techniques in the diagnosis of endocrine autoimmune diseases.

    PubMed

    Betterle, Corrado; Zanchetta, Renato

    2012-08-01

    In the study of autoimmune diseases, the laboratory plays a very important role. We describe the immunofluorescence techniques (direct, indirect, complement-fixing, double) for determining the presence of autoantibodies and their role in the autoimmune endocrine diseases. PMID:26000129

  7. 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.

  8. Development of an immunofluorescence focus assay for Ebola virus.

    PubMed Central

    Truant, A L; Regnery, R L; Kiley, M P

    1983-01-01

    A 48-h indirect immunofluorescence focus assay for the quantitation of Ebola virus was developed, utilizing HeLa-229 cell monolayers. The dose dependency and the sensitivity of this assay as compared with conventional assays are reported. This indirect immunofluorescence focus assay can be used as a rapid, quantitative test for the detection of Ebola virus, an agent from Africa known to cause hemorrhagic fever. Images PMID:6352735

  9. Validation of the MycAssay Pneumocystis kit for detection of Pneumocystis jirovecii in bronchoalveolar lavage specimens by comparison to a laboratory standard of direct immunofluorescence microscopy, real-time PCR, or conventional PCR.

    PubMed

    McTaggart, Lisa R; Wengenack, Nancy L; Richardson, Susan E

    2012-06-01

    Pneumocystis jirovecii pneumonia is a significant cause of morbidity and mortality in AIDS patients as well as those with non-HIV immunosuppressive diseases. To aid diagnosis, the commercial MycAssay Pneumocystis real-time PCR assay (Myconostica, Ltd., Manchester, United Kingdom) targeting the mitochondrial ribosomal large subunit (mtLSU) has been developed to detect P. jirovecii in bronchoalveolar lavage (BAL) specimens. Here, we validated this assay against a laboratory standard of direct immunofluorescence microscopy, a cdc2 real-time PCR assay, or conventional PCR and sequencing of mtLSU. While more sensitive than any of these three assays analyzed individually, the MycAssay Pneumocystis assay demonstrated 100% sensitivity, 100% specificity, a 100% negative predictive value, and a 100% positive predictive value for detecting the presence of P. jirovecii in BAL specimens compared to the laboratory standard. Of note, two samples with positive cycle threshold (C(T)) values according to the MycAssay Pneumocystis assay lacked exponential amplification curves and thus were deemed negative. Also negative according to the laboratory standard, these samples highlight the importance of examining the amplification curves, in addition to noting the C(T) values, when interpreting positive results. Comparison of the MycAssay Pneumocystis assay to a laboratory standard establishes this assay to be a highly sensitive and specific method for the detection of P. jirovecii in bronchoalveolar lavage specimens. The approach may also be useful for the clinical laboratory validation of other sensitive real-time PCR assays. PMID:22422855

  10. 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.

  11. 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.

  12. An immunofluorescence assay to detect soybean rust urediniospores

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An indirect immunofluorescence assay (IF) was developed to detect urediniospores of Phakopsora pachyrhizi and P. meibomiae, utilizing rabbit polyclonal antisera produced in response to intact non-germinated (SBR1A) or germinated (SBR2) urediniospores of P. pachyrhizi. Both antisera were specific to ...

  13. Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins.

    PubMed

    Niikura, Yohei; Kitagawa, Katsumi

    2016-01-01

    "Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.(1-4) Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells. PMID:26967065

  14. Unsupervised HEp-2 mitosis recognition in indirect immunofluorescence imaging.

    PubMed

    Tonti, Simone; Di Cataldo, Santa; Macii, Enrico; Ficarra, Elisa

    2015-08-01

    Automated HEp-2 mitotic cell recognition in IIF images is an important and yet scarcely explored step in the computer-aided diagnosis of autoimmune disorders. Such step is necessary to assess the goodness of the HEp-2 samples and helps the early diagnosis of the most difficult or ambiguous cases. In this work, we propose a completely unsupervised approach for HEp-2 mitotic cell recognition that overcomes the problem of mitotic/non-mitotic class imbalance due to the limited number of mitotic cells. Our technique automatically selects a limited set of candidate cells from the HEp-2 slide and then applies a clustering algorithm to identify the mitotic ones based on their texture. Finally, a second stage of clustering discriminates between positive and negative mitoses. Experiments on public IIF images demonstrate the performance of our technique compared to previous approaches. PMID:26738182

  15. Mouse Cochlear Whole Mount Immunofluorescence

    PubMed Central

    Akil, Omar; Lustig, Lawrence R.

    2016-01-01

    This protocol comprises the entire process of immunofluorescence staining on mouse cochlea whole mount, starting from tissue preparation to the mounting of the tissue. This technique provides “three-dimensional” views of the stained components in order to determine the localization of a protein of interest in the tissue in its natural state and environment. PMID:27547786

  16. Comparison of rapid immunofluorescence procedure with TestPack RSV and Directigen FLU-A for diagnosis of respiratory syncytial virus and influenza A virus.

    PubMed Central

    Todd, S J; Minnich, L; Waner, J L

    1995-01-01

    A rapid immunofluorescence format requiring 20 min for completion was as effective as conventional indirect and direct immunofluorescence procedures for detecting respiratory syncytial virus and influenza A virus antigens in clinical specimens. Rapid immunofluorescence was more sensitive than TestPack RSV and comparable to Directigen FLU-A immunosorbent assays, which require 20 min for completion. PMID:7650206

  17. Wheat germ agglutinin as a counterstain for immunofluorescence studies of equine hoof lamellae.

    PubMed

    Clark, Robert K; Galantino-Homer, Hannah L

    2014-09-01

    Equine laminitis is a common, painful, debilitating condition of the hoof that is a leading cause of disability in horses, often necessitating euthanasia. The equine hoof represents an extreme evolutionary adaptation of an epidermal structure homologous to the human or murine nail units. Immunohistochemistry is frequently utilized in the study of the pathophysiology of laminitis. The complex, multilayered, extensively interdigitated epidermal-dermal lamellar interface renders precise interpretation of immunofluorescence localization difficult, especially when effective technique and reagents render non-reactive tissues completely dark. Fluorescent-conjugated wheat germ agglutinin (WGA) selectively labels dermal extracellular matrix fibres and epidermal cell membranes in tissue sections of horse hoof lamellae, is compatible with indirect immunofluorescence and augments interpretation of indirect immunofluorescence antigen localization. The current report details the use of WGA as a rapid, simple, economical counterstain for immunofluorescence studies of the equine hoof and may have application to other complex epidermal tissue structures. PMID:25040657

  18. 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. PMID:26795470

  19. An Immunofluorescence Assay to Detect Urediniospores of the Soybean Rust Pathogen, Phakopsora pachyrhizi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An indirect immunofluorescence spore assay (IFSA) was developed to detect urediniospores of Phakopsora pachyrhizi, utilizing rabbit polyclonal antisera produced in response to intact non-germinated (SBR1A) or germinated (SBR2) urediniospores of P. pachyrhizi. Both antisera were specific to Phakopso...

  20. Cryosectioning of undecalcified tissues for immunofluorescence.

    PubMed

    Rijntjes, N V; Van de Putte, L B; Van der Pol, M; Guelen, P J

    1979-01-01

    The present report describes a procedure for preparing 4--6 micrometers cryostat sections of undecalcified fresh frozen tissue which contain hard tissue, for immunofluorescence. The apparatus used is a cryomicrotome originally designed for cutting sections for whole body autoradiography. To obtain cryostat sections suitable for tissue immunofluorescence the standard procedure was modified with respect to the hardness and edges of the microtome knife, the temperature of the cryostat and the carboxymethyl cellulose concentration of the embedding material. PMID:387879

  1. DETECTION OF GIARDIA MURIS AND GIARDIA LAMBLIA CYSTS BY IMMUNOFLUORESCENCE IN ANIMAL TISSUES AND FECAL SAMPLES SUBJECTED TO CYCLES OF FREEZING AND THAWING

    EPA Science Inventory

    The effects of freezing and thawing on the detection of selected Giardia spp. cysts were investigated using immunofluorescence, bright field microscopy, and low voltage scanning electron microscopy (SEM). iardia muris cysts were obtained from either animal carcasses, fecal pellet...

  2. Localization of the neurofilament protein in neuroblastoma cells by immunofluorescent staining.

    PubMed

    Jorgensen, A O; Subrahmanyan, L; Turnbull, C; Kalnins, V I

    1976-09-01

    Neurofilament protein (54,000-56,000 daltons) has been localized in murine neuroblastoma cells by indirect immunofluorescent staining with antisera to purified calf brain neurofilament protein. In some cells with only short processes, specific staining of fibrous material was present in the perinuclear region while in other cells similar fibers, coiled to varying degrees, were present in other regions of the cytoplasm. In cells with longer processes a stained fiber extended throughout each process. The staining pattern observed followed the distribution of bundles of 100 A filaments as determined by electron microscopy. The fibers did not stain with antisera to tubulin or tropomyosin. The observations reported strongly indicate (i) that neurofilament protein isolated from calf brain is antigenically related to a component of the bundles of 100 A filaments in neuroblastoma cells, and (ii) that the neurofilament protein is an integral part of bundles of 100 A filaments in neuroblastoma cells, while neither tubulin nor tropomyosin is present in these bundles. PMID:787987

  3. 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. PMID:25530004

  4. 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.

  5. Analyzing maize meiotic chromosomes with super-resolution structured illumination microscopy.

    PubMed

    Wang, Chung-Ju Rachel

    2013-01-01

    The success of meiosis depends on intricate coordination of a series of unique cellular processes to ensure proper chromosome segregation. Many proteins involved in these cellular events are directly or indirectly associated with chromosomes, especially those required for homologous recombination. These meiotic processes have been explored extensively by conventional light microscopy. However, many features of interest, such as chromatin organization, recombination nodules, or the synaptonemal complex are beyond the resolution of conventional wide-field microscopy. Moreover, in most sample preparation techniques for light microscopy, meiotic cells are squashed, which destroys the spatial organization of the nucleus. Here, I describe a protocol to analyze maize meiotic chromosomes by three-dimensional structured illumination microscopy (3D-SIM), a recently developed high-resolution microscopy technique. This protocol can be used to examine protein localizations at a high resolution level by immunofluorescence. PMID:23559203

  6. Immunofluorescence profile of discoid lupus erythematosus.

    PubMed

    Bharti, Shreekant; Dogra, Sunil; Saikia, Biman; Walker, Ranjana Minz; Chhabra, Seema; Saikia, Uma Nahar

    2015-01-01

    The direct immunofluorescence (DIF) of skin in conjunction with histopathology gives the best diagnostic yield. It is invaluable in confirming the diagnosis of small vessel vasculitides and bullous lesions of the skin and can be used as an additional tool to pinpoint the diagnosis of systemic and localized autoimmune diseases involving the skin. This study was undertaken to analyze the strength of DIF vis-à -vis histopathology in the diagnosis of discoid lupus erythematosus (DLE) and at the same time to elaborate the specific immunofluorescence findings in the lesions of DLE. The clinical profile and cutaneous lesions of 75 patients with DLE are described. DIF was positive in 68% and histopathology in 60% of cases. The most common immunoreactant was IgG at the dermoepidermal junction, followed by IgM and IgA. A conclusive diagnosis of DLE could be achieved satisfactorily in 64 cases (85%) by a combination of the two techniques. PMID:26549071

  7. Morphological and biochemical analysis by atomic force microscopy and scanning near-field optical microscopy techniques of human keratinocytes (HaCaT) exposed to extremely low frequency 50 Hz magnetic field

    NASA Astrophysics Data System (ADS)

    Rieti, Sabrina; Manni, Vanessa; Lisi, Antonella; Grimaldi, Settimio; Generosi, Renato; Luce, Marco; Perfetti, Paolo; Cricenti, Antonio; Pozzi, Deleana; Giuliani, Livio

    2002-10-01

    We studied the effect of the interaction of electromagnetic radiation with human keratinocytes (HaCaT), at low (50 Hz, 1 mT) frequency using both atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM) techniques. AFM analysis showed modifications in shape and morphology in exposed cells, while SNOM indirect immunofluorescence analysis revealed an increase of segregation of β4 integrin (an adhesion marker) in the cell membrane of the same cells, suggesting that a higher percentage of the exposed cells shows a modified pattern of this adhesion marker.

  8. Immunofluorescent Detection of Herpesvirus Antigens in Exfoliated Cells from Human Cervical Carcinoma

    PubMed Central

    Royston, Ivor; Aurelian, Laure

    1970-01-01

    Exfoliated cells from patients with squamous carcinoma of the cervix contain antigens related to herpesvirus subtype 2, as revealed by direct or indirect immunofluorescent techniques. Normal squamous cells from the same subjects and from controls without the disease, and cells from a small number of tumors at sites other than the cervix, did not react with anti-herpesvirus subtype 2 serum. Antisera to adenovirus 18 or mycoplasma orale did not react with the exfoliated cells. Images PMID:4318779

  9. 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.

  10. Specific immunofluorescence staining of Treponema pallidum in smears and tissues.

    PubMed

    Ito, F; Hunter, E F; George, R W; Swisher, B L; Larsen, S A

    1991-03-01

    To date, tissue sections prepared from Formalin-fixed tissues have not been successfully stained with Treponema pallidum subspecies-specific antibody in a direct fluorescent-antibody assay. While current methods stain T. pallidum, they do not distinguish T. pallidum from other spirochetes such as Borrelia burgdorferi (E. F. Hunter, P. W. Greer, B. L. Swisher, A. R. Simons, C. E. Farshy, J. A. Crawford, and K. R. Sulzer, Arch. Pathol. Lab. Med. 108:878-880, 1984). Because trypsin pretreatment of tissue sections has enhanced other immunofluorescent-antibody (IFA) applications, we compared the use of the trypsin digestion method with the current 1% ammonium hydroxide (NH4OH) method as a means to obtain specific staining of T. pallidum in tissues by both direct and indirect IFA techniques. Pretreated T. pallidum-infected tissues sections from rabbits, hamsters, and humans were quantitatively examined with the direct fluorescent-antibody-T. pallidum test conjugate absorbed with Treponema phagedenis, the Reiter treponeme. For indirect staining, a serum specimen from a patients with syphilis absorbed by affinity chromatography with T. phagedenis was used as the primary reagent, and a fluorescein isothiocyanate-labeled rabbit anti-human globulin was used as the secondary reagent. Serum specificity was established first by examining antigen smears of T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, B. burgdorferi, T. phagedenis, and Treponema denticola MRB and then by examining tissues infected with these pathogens plus those infected with four Leptospira serovars. When we stained tissue using the direct IFA method that is currently a standard method for the examination of chancre smears, we found it to be unsuitable for use with tissue. Trypsin digestion did not offer an improvement over the NH4OH pretreatment method in the specific identification of T. pallidum by direct IFA. However, specific identification of T. pallidum in tissue sections was obtained by the

  11. Indirect Imaging

    NASA Astrophysics Data System (ADS)

    Kundu, Mukul R.

    This book is the Proceedings of an International Symposium held in Sydney, Australia, August 30-September 2, 1983. The meeting was sponsored by the International Union of Radio Science and the International Astronomical Union.Indirect imaging is based upon the principle of determining the actual form of brightness distribution in a complex case by Fourier synthesis, using information derived from a large number of Fourier components. The main topic of the symposium was how to get the best images from data obtained from telescopes and other similar imaging instruments. Although the meeting was dominated by radio astronomers, with the consequent dominance of discussion of indirect imaging in the radio domain, there were quite a few participants from other disciplines. Thus there were some excellent discussions on optical imaging and medical imaging.

  12. Immunofluorescence testing in the diagnosis of autoimmune blistering diseases: overview of 10-year experience*

    PubMed Central

    Arbache, Samia Trigo; Nogueira, Tarsila Gasparotto; Delgado, Lívia; Miyamoto, Denise; Aoki, Valéria

    2014-01-01

    BACKGROUND Immunofluorescence testing is an important tool for diagnosing blistering diseases. OBJECTIVE To characterize the immunofluorescence findings in patients diagnosed with autoimmune blistering skin diseases. METHODS We retrospectively analyzed immunofluorescence results encompassing a 10-year period. RESULTS 421 patients were included and divided into 2 groups: group 1- intraepidermal blistering diseases (n=277) and 2- subepidermal blistering diseases (n=144). For group 1, positive DIF findings demonstrated: predominance of IgG intercellular staining (ICS) and C3 for pemphigus foliaceus-PF (94% and 73% respectively), pemphigus vulgaris-PV (91.5%-79.5%) and paraneoplastic pemphigus-PNP (66%-33%); ICS IgA in 100% of IgA pemphigus cases, and IgG deposits in the basement membrane zone (BMZ) along with ICS in one Hailey-Hailey patient. The IIF findings revealed mean titers of 1:2.560 for PV and 1:1.280 for PF. For paraneoplastic pemphigus, IIF was positive in 2 out of 3 cases with rat bladder substrate. In group 2, positive DIF findings included multiple deposits at basement membrane zone for epidermolysis bullosa acquisita-EBA (C3-89%,IgG-79%,IgA-47%,IgM-21%) mucous membrane pemphigoid-MMP (C3,IgG,IgA,IgM-80%) and bullous pemphigoid-BP (C3-91%,IgG-39%,IgA-11%,IgM-6%), and IgA at basement membrane zone for IgA linear disease (99%) and dermatitis herpetiformis-DH (dermal papillae in 84.6%). For lichen planus pemphigoides, there was C3 (100%) and IgG (50%) deposition at basement membrane zone. indirect immunofluorescence positive findings revealed basement membrane zone IgG deposits in 46% of BP patients, 50% for EBA, 15% for IgA linear dermatosis and 50% for LPP. Indirect immunofluorescence positive results were higher for BP and EBA with Salt-Split skin substrate. CONCLUSION Our results confirmed the importance of immunofluorescence assays in diagnosing autoimmune blistering diseases, and higher sensitivity for indirect immunofluorescence when Salt-split skin

  13. 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

    2016-01-01

    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. PMID:27616137

  14. Role of direct immunofluorescence in dermatological disorders

    PubMed Central

    Mysorekar, Vijaya V.; Sumathy, T. K.; Shyam Prasad, A. L.

    2015-01-01

    Background: Direct immunofluorescence (DIF) test for tissue-bound autoantibodies, has been found to be of value in the diagnosis of several dermatological disorders. The location and pattern of deposition of immunoreactants helps in classifying various immune-mediated diseases. Aims and Objectives: The aim of this study was to analyze the concordance between the clinical, histopathological and DIF diagnosis in bullous and nonbullous lesions of the skin, and thus determine the impact of immunofluorescence on diagnosis. Materials and Methods: A total of 215 skin biopsies performed in suspected immune-mediated vesiculobullous disease, vasculitis or dermatosis, were studied. Histopathological examination was done along with DIF study for deposits of immunoglobulin G(IgG), IgA, IgM, and C3. Results: Direct immunofluorescence was positive in 103/215 cases. There was very good concordance between the clinical, histological and DIF results (observed agreement = 93.4%, κ =0.90, with 95% confidence interval = 0.86–0.94). The overall sensitivity of DIF in immune-mediated skin disorders was 98.0%. DIF was positive in 52/53 cases (98.1%) in the pemphigus group and 24/25 (96.0%) bullous pemphigoid cases. None of the clinically suspected cases of dermatitis herpetiformis showed DIF positivity. A positive lupus band test was seen in 9/9 (100%) cases of lupus erythematosus. DIF was positive in 10/10 (100%) clinically suspected cases of Henoch–Schönlein purpura. In 110 cases, negative DIF results helped to rule out immune-mediated vesiculobullous disorders, lupus erythematosus and vasculitis, and the final diagnosis was made on the basis of the clinical features and/or histopathology. Conclusion: Direct immunofluorescence is a useful supplement for the accurate diagnosis of immune-mediated dermatological disorders, and helps to classify various autoimmune bullous disorders. When the clinical features/histopathology are inconclusive, the diagnosis often can be made on the basis

  15. Probing the role of the actin cytoskeleton during regulated exocytosis by intravital microscopy

    PubMed Central

    Milberg, Oleg; Tora, Muhibullah; Shitara, Akiko; Masedunskas, Andrius

    2015-01-01

    Summary The actin cytoskeleton plays a fundamental role in controlling several steps during regulated exocytosis. Here we describe a combination of procedures that are aimed at studying the dynamics and the mechanism of the actin cytoskeleton in the salivary glands of live rodents, a model for exocrine secretion. Our approach relies on intravital microscopy, an imaging technique that enables imaging biological events in live animals at a subcellular resolution, and it is complemented by the use of pharmacological agents and indirect immunofluorescence in the salivary tissue. PMID:24947398

  16. Localization by immunofluorescence and by light- and electron-microscopic immunoperoxidase techniques of Tamm–Horsfall glycoprotein in adult hamster kidney

    PubMed Central

    Sikri, Krishan L.; Foster, Charles L.; Bloomfield, Frederick J.; Marshall, R. Derek

    1979-01-01

    1. Tamm–Horsfall glycoprotein was isolated from hamster urine and antiserum against it was produced in rabbits. Immunoglobulin G was isolated from the antiserum. 2. Indirect methods of immunofluorescence staining were applied to kidney sections previously fixed by both perfusion and immersion methods. Tamm–Horsfall glycoprotein was identified associated with only the cells of the ascending limb of the loop of Henle and the distal convoluted tubule. Maculae densae were free of the glycoprotein. 3. Indirect immunoperoxidase procedures with light microscopy were applied to kidney sections. The results extended those found by immunofluorescence by showing that the glycoprotein is largely associated with the plasma membrane of the cells. Macula densa cells were shown to be free of the glycoprotein, although the luminal surface of the remaining cells in the transverse section of the nephron at that region was shown to contain it. 4. A variety of immuno-electron-microscopic techniques were applied to sections previously fixed in a number of ways. Providing periodate/lysine/paraformaldehyde was used as the fixative, the glycoprotein was often seen to be present not only on the luminal surface of the cells of the thick ascending limb of the loop of Henle and of the distal convoluted tubule, but also on the basal plasma membrane, including the infoldings. 5. It is generally accepted that the hyperosmolarity in the medulla of the kidney results from passage of Cl− ions with their accompanying Na+ ions across the single cell layer of the lumen of the thick ascending limb of the loop of Henle, a region of the nephron with relatively high impermeability to water. We suggest that Tamm–Horsfall glycoprotein operates as a barrier to decrease the passage of water molecules by trapping the latter at the membrane of the cells. Our hypothesis requires the glycoprotein on the basal plasma membrane also. ImagesFig. 1.PLATE 4PLATE 1PLATE 3PLATE 2PLATE 6PLATE 5 PMID:391220

  17. 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.

  18. Immunofluorescent localization of adhesive glycoproteins in resting and thrombin-stimulated platelets.

    PubMed Central

    Wencel-Drake, J. D.; Plow, E. F.; Zimmerman, T. S.; Painter, R. G.; Ginsberg, M. H.

    1984-01-01

    The distribution and transport of thrombospondin (TSP), fibrinogen (Fbg), fibronectin (Fn), and Factor VIII-related antigen (VIII:RAg) in resting and thrombin-stimulated platelets was investigated by immunofluorescence microscopy. In resting intact cells, little surface staining was seen for these proteins. In permeable resting cells, punctate staining similar to that reported for platelet factor 4 was observed. Double-label immunofluorescence staining for Fbg and either beta-thromboglobulin (beta TG), TSP, or Fn demonstrated co-localization, indicating their presence in the same intracellular structures. VIII:RAg showed general co-localization; however, the staining was finer, suggesting a possible differential intragranular localization. Thrombin stimulation induced the appearance of larger (approximately 0.5 mu) immunofluorescent masses of these proteins. In thrombin-stimulated cells, co-localization of all proteins in these masses was observed by double label immunofluorescence. Thus, TSP, Fbg, Fn, and beta TG are localized in the same structure in resting cells. Thrombin stimulates formation of common larger masses of these proteins prior to their release, suggesting that they reach the cell surface through a common intermediate. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:6232852

  19. Purification, characterization, and immunofluorescence localization of Saccharomyces cerevisiae capping protein.

    PubMed

    Amatruda, J F; Cooper, J A

    1992-06-01

    Capping protein binds the barbed ends of actin filaments and nucleates actin filament assembly in vitro. We purified capping protein from Saccharomyces cervisiae. One of the two subunits is the product of the CAP2 gene, which we previously identified as the gene encoding the beta subunit of capping protein based on its sequence similarity to capping protein beta subunits in chicken and Dictyostelium (Amatruda, J. F., J. F. Cannon, K. Tatchell, C. Hug, and J. A. Cooper. 1990. Nature (Lond.) 344:352-354). Yeast capping protein has activity in critical concentration and low-shear viscometry assays consistent with barbed-end capping activity. Like chicken capping protein, yeast capping protein is inhibited by PIP2. By immunofluorescence microscopy yeast capping protein colocalizes with cortical actin spots at the site of bud emergence and at the tips of growing buds and shmoos. In contrast, capping protein does not colocalize with actin cables or with actin rings at the site of cytokinesis. PMID:1315784

  20. Detection of CXCR2 cytokine receptor surface expression using immunofluorescence.

    PubMed

    Lam, Clarissa; Pavel, Mahmud Arif; Kashyap, Parul; Salehi-Najafabadi, Zahra; Valentino, Victoria; Yu, Yong

    2014-01-01

    The interleukin-8 (IL-8, CXCL8) chemokine, also known as the neutrophil chemotactic factor, is a cytokine that plays a key role in inflammatory response, cell proliferation, migration, and survival. IL-8 expression is increased not only in inflammatory disorders, but also in many types of cancer, including prostate cancer. IL-8 acts as a ligand for the C-X-C chemokine receptor 2 (CXCR2) protein present on the cell plasma membrane. Binding of the IL-8 ligand to the CXCR2 receptor results in an intracellular signaling pathway mediated by GTP binding proteins coupled to the receptor itself. Knowledge of the CXCR2 expression levels facilitates the understanding of the role and function of IL-8. In this chapter, we describe a protocol that uses the immunofluorescence method and confocal microscopy to analyze the CXCR2 surface expression in human prostate cancer cells. However, this protocol is easily adaptable to analyze the surface expression of other cytokine receptors in different cell types. PMID:24908306

  1. Direct immunofluorescence testing in the diagnosis of immunobullous disease, collagen vascular disease, and vascular injury syndromes.

    PubMed

    Magro, Cynthia M; Roberts-Barnes, Jennifer; Crowson, A Neil

    2012-10-01

    Direct and indirect immunofluorescence (IF) plays a role in the evaluation of immunobullous diseases and their mimics, and in the investigation of vascular injury syndromes and autoimmune connective tissue disease (CTD). IF mapping may be an important adjunct in the assessment of congenital epidermolysis bullosa syndromes and in Alport disease, in which antibodies are directed at certain components of the basement membrane zone to assay for their deficiency. In many cases of immunobullous and autoimmune CTDs, correlation with direct IF results is useful and often decisive in lesional evaluation and thus in patient management. PMID:23021058

  2. [Immunofluorescent diagnosis of Entamoeba histolytica trophocoites in preserved stool specimens of patients (author's transl)].

    PubMed

    Hess, U; Fröhlich, A

    1979-09-01

    A fixative and examination technique is described for identification of Entamoeba histolytica trophocoites in faeces with the aid of the indirect immunofluorescence technique. Magnaforms in bloody-dysenteric specimens and Minutaforms in spontaneous or saline purged specimens give equally good fluorescence. The specifity of the rabbit antiserum against axenically grown E. hist. is good: There is strong positive reaction only with trophocoites of E. hist. Weak crossreactions are encountered with trophocoites of E. coli, E. hartmanni and E. polecki. Negative reactions are encountered with trophocoites of Endolimax nana, Jodamoeba bāutschlii, and Dientamoeba fragilis. Amebic cysts, flagellates, leucocytes, epithelial cells and Blastocytis give negative reactions, too. PMID:94475

  3. Immunofluorescent staining of septins in primary cilia.

    PubMed

    Kim, M S; Froese, C D; Xie, H; Trimble, W S

    2016-01-01

    Primary cilia are cellular antennae that receive and transduce extracellular cues. These microtubule-rich structures are comprised of at least three distinct ciliary compartments: basal bodies, transition zone, and axoneme. Septins have been implicated in cilia function at the transition zone, but accumulating evidence suggests that they localize predominantly within the axoneme. Here, we describe three fixation conditions that preserve the substructure of primary cilia and demonstrate known ciliary proteins that localize to these distinct ciliary substructures. Finally, we show immunostaining and live microscopy methods to detect septins within the axoneme. PMID:27473914

  4. Rapid diagnosis of mumps virus infections by immunofluorescence methods.

    PubMed

    Lennette, D A; Emmons, R W; Lennette, E H

    1976-08-01

    Mumps and its complications, particularly meningoencephalitis, is an important disease problem, and more rapid diagnostic methods are desirable. A study was made of immunofluorescence methods for the early detection of mumps virus isolated in cell cultures, or adsorbed directly from clinical specimens onto guinea pig erythrocytes. A specific diagnosis could be made in hours to 2 or 3 days utilizing immunofluorescence methods, in contrast to about 6 days by standard methods. Details of the direct immunofluorescence methods are presented, to encourage wider application in clinical virology laboratories. PMID:787002

  5. 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. PMID:27441410

  6. 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

  7. 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

    2014-01-01

    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. PMID:24589329

  8. Methods of Immunohistochemistry and Immunofluorescence: Converting Invisible to Visible.

    PubMed

    Mori, Hidetoshi; Cardiff, Robert D

    2016-01-01

    Observing changes in pathophysiological tissue samples often relies on immunohistochemical or immunofluorescence analysis. These techniques show target microanatomy by visualizing marker molecules on cells and their microenvironment. Here, we describe the "pros and cons" in each method, along with alternative procedures and the suggested imaging equipment. PMID:27581010

  9. 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...

  10. 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 (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460...

  11. 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...

  12. 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...

  13. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 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...

  14. 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 (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460...

  15. 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 (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3460...

  16. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 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...

  17. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 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...

  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...

  19. 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.

  20. Use of immunofluorescence technique in cultured fibroblasts from Mediterranean cetaceans as new "in vitro" tool to investigate effects of environmental contaminants.

    PubMed

    Marsili, Letizia; Casini, Silvia; Bucalossi, Daniela; Porcelloni, Serena; Maltese, Silvia; Fossi, Maria Cristina

    2008-07-01

    The aim of the present study was to propose the immunofluorescence technique in cultured fibroblasts from Mediterranean cetaceans as a new "in vitro" tool to explore the susceptibility of these marine mammals to different xenobiotic compounds. The cell lines were cultured from integument biopsies of free-ranging and stranded cetaceans (dead within 12h). Using the indirect immunofluorescence assay, we detected endogenous proteins induced by different contaminants. Here we present the method used for qualitative and quantitative evaluation of cytochromes P450 (CYP1A1 and CYP2B) induced by some POPs (DDTs and PCBs) and emerging contaminants (PBDEs) in fibroblast cell cultures of striped dolphin (Stenella coeruleoalba) and bottlenose dolphin (Tursiops truncatus). Immunofluorescence was quantified with a specially designed Olympus macro, DetectIntZ. A major result was the possibility of using this "in vitro" assay to quantify induction of endogenous proteins. PMID:18396327

  1. An assessment of the immunofluorescence technique as a method for demonstrating the histological localization of tetrahydrocannabinol in mammalian tissues

    SciTech Connect

    Morley, M.; Gee, D.J.

    1982-10-01

    The use of the indirect immunofluorescence technique as a method for demonstrating the histological localization of tetrahydrocannabinol (delta-THC) has been examined. The experimental protocol was designed in order that optimal staining conditions with respect to temperature, the length of time of incubations and washes, and the dilution of the antisera should be defined. No marked differences were detected between frozen sections of liver from normal and delta-THC-injected mice. Results from radiotracer experiments using human liver suggest that the success of the method is dependent upon the solubility characteristics of the antigen-antibody complex.

  2. Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method.

    PubMed

    Lin, Jia-Ren; Fallahi-Sichani, Mohammad; Sorger, Peter K

    2015-01-01

    Single-cell analysis reveals aspects of cellular physiology not evident from population-based studies, particularly in the case of highly multiplexed methods such as mass cytometry (CyTOF) able to correlate the levels of multiple signalling, differentiation and cell fate markers. Immunofluorescence (IF) microscopy adds information on cell morphology and the microenvironment that are not obtained using flow-based techniques, but the multiplicity of conventional IF is limited. This has motivated development of imaging methods that require specialized instrumentation, exotic reagents or proprietary protocols that are difficult to reproduce in most laboratories. Here we report a public-domain method for achieving high multiplicity single-cell IF using cyclic immunofluorescence (CycIF), a simple and versatile procedure in which four-colour staining alternates with chemical inactivation of fluorophores to progressively build a multichannel image. Because CycIF uses standard reagents and instrumentation and is no more expensive than conventional IF, it is suitable for high-throughput assays and screening applications. PMID:26399630

  3. Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method

    PubMed Central

    Lin, Jia-Ren; Fallahi-Sichani, Mohammad; Sorger, Peter K.

    2015-01-01

    Single-cell analysis reveals aspects of cellular physiology not evident from population-based studies, particularly in the case of highly multiplexed methods such as mass cytometry (CyTOF) able to correlate the levels of multiple signalling, differentiation and cell fate markers. Immunofluorescence (IF) microscopy adds information on cell morphology and the microenvironment that are not obtained using flow-based techniques, but the multiplicity of conventional IF is limited. This has motivated development of imaging methods that require specialized instrumentation, exotic reagents or proprietary protocols that are difficult to reproduce in most laboratories. Here we report a public-domain method for achieving high multiplicity single-cell IF using cyclic immunofluorescence (CycIF), a simple and versatile procedure in which four-colour staining alternates with chemical inactivation of fluorophores to progressively build a multichannel image. Because CycIF uses standard reagents and instrumentation and is no more expensive than conventional IF, it is suitable for high-throughput assays and screening applications. PMID:26399630

  4. Identification of Mesenchymal Stem Cell Marker STRO-1 in Oral Reactive Lesions by Immunofluorescence Method

    PubMed Central

    Dehghani Nazhvani, Ali; Hosseini, Seyed-Mojtaba; Tahoori, Bita; Tavangar, Maryam-Sadat; Attar, Armin

    2015-01-01

    Statement of the Problem Stem cells are considered as new implement for tissue regeneration. Several niches in adult human body are colonized by multipotent stem cells but access to these potential reservoirs is often limited. Although human dental pulp stem cells isolated from healthy teeth have been extensively characterized, it is still unknown whether stem cells also exist in reactive lesions of oral cavity such as pyogenic granuloma and peripheral ossifying fibroma which are deliberated as inflammatory proliferation of different cell families. Purpose The aim of this study was to explore for clues to see whether pyogenic granuloma or peripheral ossifying fibroma contain dental mesenchymal stem cell (DMSC). Materials and Method Four pyogenic granuloma and four peripheral ossifying fibroma specimens were collected by excisional biopsy and preserved in PBS-EDTA at -86 °C. Then we cut them in 5µm diameter using Cryostat. Having been rinsed with PBS, the samples were stained with a primary mouse anti-human STRO-1 monoclonal IgM antibody. Afterward, a secondary goat anti-mouse IgM-FITC antibody was applied to detect STRO-1+ cells as probable stem cells by immunofluorescence technique. Results Immunofluorescence microscopy revealed presence of STRO-1+ cells in these lesions, particularly localized on perivascular zone. The negative control group was not glowing. Conclusion Based on these results, it was found that reactive lesions of pyogenic granuloma and peripheral ossifying fibroma have STRO-1 positive cells, which raises the possibility that these cells may be DMSCs. PMID:26535404

  5. Pulmonary fibrosis following pneumonia due to acute Legionnaires' disease. Clinical, ultrastructural, and immunofluorescent study.

    PubMed

    Chastre, J; Raghu, G; Soler, P; Brun, P; Basset, F; Gibert, C

    1987-01-01

    During a recent nosocomial outbreak, 20 critically ill patients with acute Legionnaires' disease were admitted to the intensive care unit of Hopital Bichat, Paris. Pulmonary specimens were obtained at surgery or immediately after death in 12 patients and were examined by light, immunofluorescent, and electron microscopy. Five of these 12 patients showed evidence of pulmonary fibrosis. In all of these five patients, infection with Legionella pneumophila was evidenced by bacteriologic methods, and other diseases known to cause fibrosis were excluded. The condition of four patients deteriorated rapidly with respiratory failure, and they died with pulmonary fibrosis. Only one patient finally recovered but was left with pulmonary sequelae. Two distinctive morphologic patterns were observed, one in which interstitial fibrosis was predominant and one in which intra-alveolar organization and fibrosis were also present. The alveolar epithelial lining and the basement membranes were disrupted in all patients, as evidenced by ultrastructural observations and by immunofluorescent studies showing gaps in the distribution of type 4 collagen and laminin. Types 1 and 3 collagen accumulated in areas corresponding to thickened interstitium and intra-alveolar fibrosis. Thus, some patients who survive the acute pneumonia of Legionnaires' disease may develop pulmonary fibrosis, and this process may lead to functional impairment or death despite prompt and appropriate treatment. PMID:3539546

  6. Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

    PubMed Central

    Yamada, Norishige; Ogawa, Akiyo; Ogawa, Yuya

    2014-01-01

    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. PMID:25489864

  7. 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.

  8. An immunofluorescence diagnostic test for feline viral rhinotracheitis.

    PubMed

    Carlson, J H; Scott, F W

    1978-03-01

    Hyperimmune serum against feline viral rhinotracheitis was produced in a goat and conjugated with a fluorescent dye. Cell cultures infected with rhinotracheitis virus had positive immunofluorescence. Cell cultures infected with other feline viruses and herpesviruses of other species did not fluoresce. In cats experimentally infected with rhinotracheitis virus, the virus was isolated from nasal and conjunctival swabs 1 to 9 days after inoculation. Nasal smears stained with the conjugated antiserum fluoresced 1 to 9 days after inoculation when clinical disease was most apparent. Conjunctival smears had positive immunofluorescence 1 to 6 days, but not 9 days, after inoculation. On postinoculation day 23, rhinotracheitis virus was not isolated from nasal or conjunctival swabs and nasal and conjunctival smears did not fluoresce. Rhinotracheitis virus or feline calicivirus was isolated from naturally infected cats with upper respiratory tract disease. Nasal and conjunctival smears from rhinotracheitis virus-infected cats had positive immunofluorescence in all cast showing clinical illness. Smears from 1 clinically normal cat from which rhinotracheitis virus was isolated did not fluoresce. Nasal and conjunctival smears from calicivirus-infected cats did not fluoresce. PMID:205147

  9. Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

    PubMed Central

    Wilsbacher, Lisa D.; Coughlin, Shaun R.

    2015-01-01

    During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects. PMID:25866997

  10. 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. PMID:24070488

  11. 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. PMID:27342035

  12. The classification of Crithidia luciliae immunofluorescence test (CLIFT) using a novel automated system

    PubMed Central

    2014-01-01

    Introduction In recent years, there has been an increased demand for computer-aided diagnosis (CAD) tools to support clinicians in the field of indirect immunofluorescence. To this aim, academic and industrial research is focusing on detecting antinuclear, anti-neutrophil, and anti-double-stranded (anti-dsDNA) antibodies. Within this framework, we present a CAD system for automatic analysis of dsDNA antibody images using a multi-step classification approach. The final classification of a well is based on the classification of all its images, and each image is classified on the basis of the labeling of its cells. Methods We populated a database of 342 images—74 positive (21.6%) and 268 negative (78.4%)— belonging to 63 consecutive sera: 15 positive (23.8%) and 48 negative (76.2%). We assessed system performance by using k-fold cross-validation. Furthermore, we successfully validated the recognition system on 83 consecutive sera, collected by using different equipment in a referral center, counting 279 images: 92 positive (33.0%) and 187 negative (67.0%). Results With respect to well classification, the system correctly classified 98.4% of wells (62 out of 63). Integrating information from multiple images of the same wells recovers the possible misclassifications that occurred at the previous steps (cell and image classification). This system, validated in a clinical routine fashion, provides recognition accuracy equal to 100%. Conclusion The data obtained show that automation is a viable alternative for Crithidia luciliae immunofluorescence test analysis. PMID:24625089

  13. Association of Immunofluorescence pattern of Antinuclear Antibody with Specific Autoantibodies in the Bangladeshi Population.

    PubMed

    Sharmin, S; Ahmed, S; Abu Saleh, A; Rahman, F; Choudhury, M R; Hassan, M M

    2014-08-01

    Antinuclear antibody (ANA) is useful in the diagnosis of connective tissue disorder (CTD). Association of specific autoantibodies with the immunofluorescence pattern of ANA in CTD, noted in western literature has been considered as reference in all over the world. However, in Bangladesh no such research work or data correlating the autoantibodies and their ANA patterns is found. Objective of the study was to identify an association between immunofluorescence patterns of antinuclear antibody on HEp-2 cell and more specific antinuclear reactivities (e.g. anti-dsDNA and anti-extractable nuclear antigen) in the serum samples of CTD patients. Serum samples of 152 CTD patients (Systemic lupus erythematosus, Rhumatoid arthritis, Sjogren's syndrome, Systemic sclerosis, Polymyositis, Mixed connective tissue disease) were diagnosed clinically, attending at Bangabandhu Sheikh Mujib Medical University (BSMMU) during the study period of January, 2010 to December, 2010. Samples were subjected for ANA testing by Indirect Immunofluorescence (IIF) on HEp-2 cell (ALPHADIA) in dilution of 1:40, anti-dsDNA by ELISA and anti- extractable nuclear antigen (anti-ENA) by Dot Immunoblot. Dot blot strips were tested for anti-Sm, anti-RNP, anti-SSA/Ro, anti-SSB/La, anti-Scl-70 and anti-Jo-1. Out of 152 patients 110 (72.3%) cases were ANA positive by IIF on HEp-2 cell. ANA positive sera exhibited four fluorescence patterns such as speckled (50.8%), peripheral (21.6%) , homogenous (18.1%) and nucleolar pattern (9%). Peripheral pattern and homogenous pattern was predominantly associated with anti-dsDNA (p < 0.05). Speckled pattern was significantly associated with anti-ENA (p < 0.05).The most commonly identified antinuclear autoreactivity was directed towards anti-RNP (25.7%) then anti-Scl-70 (20%), anti-SSA (14.2%) and anti-SSB (5.7%). Multiple anti-ENA reactivities were identified in 34.28% cases. Peripheral and homogenous pattern is strongly associated with anti-dsDNA and speckled pattern may

  14. Role of Protein A in Nonspecific Immunofluorescence of Staphylococcus aureus

    PubMed Central

    Forsgren, Arne; Forsum, Urban

    1970-01-01

    γG-globulin from nonimmunized rabbits and from rabbits immunized with various bacteria reacted in the immunofluorescence technique with protein A-containing Staphylococcus aureus. Pepsin digestion of most immunoglobulin preparations eliminated the reaction, thus showing that the Fc fragment is involved and that the reaction is not a true antigen-antibody reaction. As the specific immunological activity of the immunoglobulin molecules was intact after digestion, it is suggested that the method be used to eliminate reactions with S. aureus in the fluorescent-antibody technique. PMID:16557850

  15. Cell cycle imaging with quantitative differential interference contrast microscopy

    NASA Astrophysics Data System (ADS)

    Kostyk, Piotr; Phelan, Shelley; Xu, Min

    2013-02-01

    We report a microscopic approach for determining cell cycle stages by measuring the nuclear optical path length (OPL) with quantitative differential interference contrast (DIC) microscopy. The approach is validated by the excellent agreement between the proportion of proliferating-to-quiescent cancerous breast epithelial cells obtained from DIC microscopy, and that from a standard immunofluorescence assay.

  16. 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.

  17. Serologic immunoreactivity to Neospora caninum antigens in dogs determined by indirect immunofluorescence, western blotting and dot-ELISA.

    PubMed

    Pinheiro, A M; Costa, M F; Paule, B; Vale, V; Ribeiro, M; Nascimento, I; Schaer, R E; Almeida, M A O; Meyer, R; Freire, S M

    2005-06-10

    Neospora caninum, is a coccidian protozoan known as a major cause of bovine abortion and canine neuropathies. The aim of the present study was to develop a reliable and quick test to detect antibodies to N. caninum in dog sera. Sixty-five serum samples from dogs, including 35 positive and 30 negative for N. caninum antibodies were used for standardization of the test. In parallel, immunoreactivity of the sera to Toxoplasma gondii antigens was investigated using a passive agglutination test. A dot-ELISA test, using soluble extract of N. caninum tachyzoites on nitrocellulose ester membranes, was developed and standardized. SDS-PAGE and complementary analysis of reactivity by Western blotting were used for the characterization of the immunoreactive fractions of all tested sera. The sensitivity and specificity of the dot-ELISA were 94 and 73%, respectively, compared to IFAT at a cut-off of 1:50, and 87 and 100% compared to IFAT at a cut-off of 1:25. Among the sera that tested positively for both IFAT and dot-ELISA, only 8.6% were reactive to T. gondii. The most immunoreactive fractions in Western blots were the 14-, 33-, 42- and 55 kDa bands, with percentages of 42, 60, 42 and 37%, respectively. The 60 kDa band showed a non-specific reaction in 43% of neosporosis-negative animals by both dot-ELISA and IFAT. These results indicate that the dot-ELISA using N. caninum antigen present good sensitivity and specificity, and might be used as a screening test to detect antibodies to N. caninum in dogs. PMID:15893072

  18. 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

  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. 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.

  1. Fluorescence Microscopy

    PubMed Central

    Sanderson, Michael J.; Smith, Ian; Parker, Ian; Bootman, Martin D.

    2016-01-01

    Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. PMID:25275114

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

    PubMed Central

    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%). PMID:27247573

  3. 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. PMID:1101770

  4. Chemical Pretreatment of Growth Plate Cartilage Increases Immunofluorescence Sensitivity

    PubMed Central

    Ahrens, Molly J.; Dudley, Andrew T.

    2011-01-01

    Immunofluorescence detection of proteins in growth plate cartilage is often unsuccessful because of innate autofluorescence, fixative-induced fluorescence, and dense cartilage matrix, which can inhibit antibody penetration. To overcome these limitations, the authors have tested various chemical pretreatments, including the autofluorescence quencher sodium borohydride, the antigen retrieval method of boiling sodium citrate, sugar-degrading enzymes (hyaluronidase, heparinase, and chondroitinase), and the proteolytic enzyme protease XXIV. Here the authors show that, in most cases, background fluorescence in cartilage is the primary obstacle to high-quality imaging. Blocking intrinsic fluorescence of the specimen in combination with specific pretreatments allows visualization using antibodies that previously did not generate a robust signal in the growth plate. Each antibody requires a specific combination of chemical pretreatments that must be empirically determined to achieve optimal staining levels. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. PMID:21411811

  5. Immunofluorescence studies of disseminated Hantaan virus infection of suckling mice.

    PubMed Central

    Kurata, T; Tsai, T F; Bauer, S P; McCormick, J B

    1983-01-01

    Hantaan virus, the etiological agent of Korean hemorrhagic fever, was inoculated intracerebrally or intraperitoneally into suckling mice, and the course of the infection was followed by infectivity titration and immunofluorescence studies. Mice became ill and were moribund by 13 to 14 days postinfection. In mice inoculated either intracerebrally or intraperitoneally, virus antigen was present in brain, heart, lungs, liver, and kidney. Less consistently, specific fluorescence was observed in spleen, pituitary gland, thymus, lymph nodes, adrenal, pancreas, salivary glands, trigeminal ganglia, adipose tissue, intestine, and muscle. In all of these tissues, the primary target of infection was the capillary endothelium. In mice inoculated intracerebrally, virus antigen was present mainly in choroid plexus, hippocampal nuclei, and meninges, but in mice inoculated intraperitoneally, central nervous system infection was marked by antigen accumulation in cortical nuclei and thalamus. Images PMID:6134678

  6. 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. PMID:23689693

  7. 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. PMID:12050861

  8. Dermoscopy and direct immunofluorescence findings of elastosis perforans serpiginosa.

    PubMed

    Ramírez-Bellver, J L; Bernárdez, C; Macías, E; Moya, L; Molina-Ruiz, A M; Cannata Ortiz, P; Requena, L

    2016-08-01

    Elastosis perforans serpiginosa (EPS) is a rare skin disorder characterized by transepidermal elimination of abnormal elastic fibres. We present a new case of D-penicillamine (DPA)-induced EPS, and describe the clinical, dermoscopic, histopathological and direct immunofluorescence (DIF) findings. A 33-year-old woman receiving treatment with DPA presented with annular skin lesions. Digital dermoscopy of the lesions showed a central area of pink and yellowish discolouration with keratotic papules in the periphery, surrounded by a white halo, disposed in a way that resembled the islands of an archipelago. Other lesions showed a white to yellow central colouration and 'chrysalides' surrounding the keratotic plugs. Linear and granular deposits of IgG attached to the abnormal elastic fibres were seen with DIF. Dermoscopy can be helpful in the diagnosis of EPS. Moreover, DIF findings in skin biopsies of this case support the immune-mediated pathogenesis of EPS. PMID:27378586

  9. Clinical Application of Immunofluorescence I. Grouping β-Hemolytic Streptococci

    PubMed Central

    Smith, Thomas B.

    1965-01-01

    Smith, Thomas B. (Armed Forces Institute of Pathology, Washington, D.C.). Clinical application of immunofluorescence. I. Grouping β-hemolytic streptococci. J. Bacteriol. 89:198–204. 1965.—Procedures are described for the production of antistreptococcal serum in rabbits and for the preparation of group-specific conjugates for Lancefield groups A, C, and G. A modification of the conventional technique of absorption and inhibition to prevent cross-reactions with common antigens was used with excellent results. In addition, a promising new approach to eliminating cross-reactions of group A conjugate with antigens of groups C and G by dilution with group A-variant antiserum was tested. A complete method is introduced that enables the clinical laboratory to report whether group A streptococci are present in a given throat culture well within 24 hr after the physician collects the sample. Images PMID:14255663

  10. Quantitative Immunofluorescence Analysis of Nucleolus-Associated Chromatin.

    PubMed

    Dillinger, Stefan; Németh, Attila

    2016-01-01

    The nuclear distribution of eu- and heterochromatin is nonrandom, heterogeneous, and dynamic, which is mirrored by specific spatiotemporal arrangements of histone posttranslational modifications (PTMs). Here we describe a semiautomated method for the analysis of histone PTM localization patterns within the mammalian nucleus using confocal laser scanning microscope images of fixed, immunofluorescence stained cells as data source. The ImageJ-based process includes the segmentation of the nucleus, furthermore measurements of total fluorescence intensities, the heterogeneity of the staining, and the frequency of the brightest pixels in the region of interest (ROI). In the presented image analysis pipeline, the perinucleolar chromatin is selected as primary ROI, and the nuclear periphery as secondary ROI. PMID:27576710

  11. IMMUNOFLUORESCENCE DETECTION OF 'CRYPTOSPORIDIUM' OOCYSTS IN FECAL SMEARS

    EPA Science Inventory

    An indirect fluorescent antibody (IFA) procedure was developed for the detection of Cryptosporidium sp. oocysts in human, nonhuman primate, and bovine fecal smears. The procedure, which takes about 90 min to perform, involves the use of a rabbit antiserum against Cryptosporidium ...

  12. 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)

  13. A rapid and highly specific immunofluorescence method to detect Escherichia coli O157:H7 in infected meat samples.

    PubMed

    Balakrishnan, Baskar; Barizuddin, Syed; Wuliji, Tumen; El-Dweik, Majed

    2016-08-16

    Developing rapid and sensitive methods for the detection of pathogenic Escherichia coli O157:H7 remains a major challenge in food safety. The present study attempts to develop an immunofluorescence technique that uses Protein-A-coated, magnetic beads as the platform. The immunofluorescence technique described here is a direct detection method in which E. coli O157:H7 cells are labeled with tetramethylrhodamine (TRITC) fluorescent dye. TRITC-labeled bacteria are captured by the desired antibody (Ab), which is immobilized on the Protein-A magnetic beads. Fluorescence of the captured cells is recorded in a fluorescence spectrophotometer, where the fluorescence values are shown to be directly proportional to the number of bacteria captured on the immunobead. The formation of an immunocomplex is evidenced by the fluorescence of the beads under microscopy. The Ab immobilization procedure is also evidenced by microscopy using fluorescein isothiocyanate (FITC)-labeled Ab. The total experimental time, including preparation of the sample, is just 1h. The minimum bacterial concentration detected by this method is 1.2±0.06×10(3)CFUml(-1). The high specificity of this method was proved by using the specific monoclonal Ab (MAb) in the test. The proposed protocol was successfully validated with E. coli O157:H7-infected meat samples. This approach also opens the door for the detection of other bacterial pathogens using Protein-A magnetic beads as a detection platform. PMID:27209618

  14. 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

  15. Indirect visual cryptography scheme

    NASA Astrophysics Data System (ADS)

    Yang, Xiubo; Li, Tuo; Shi, Yishi

    2015-10-01

    Visual cryptography (VC), a new cryptographic scheme for image. Here in encryption, image with message is encoded to be N sub-images and any K sub-images can decode the message in a special rules (N>=2, 2<=K<=N). Then any K of the N sub-images are printed on transparency and stacked exactly, the message of original image will be decrypted by human visual system, but any K-1 of them get no information about it. This cryptographic scheme can decode concealed images without any cryptographic computations, and it has high security. But this scheme lacks of hidden because of obvious feature of sub-images. In this paper, we introduce indirect visual cryptography scheme (IVCS), which encodes sub-images to be pure phase images without visible strength based on encoding of visual cryptography. The pure phase image is final ciphertexts. Indirect visual cryptography scheme not only inherits the merits of visual cryptography, but also raises indirection, hidden and security. Meanwhile, the accuracy alignment is not required any more, which leads to the strong anti-interference capacity and robust in this scheme. System of decryption can be integrated highly and operated conveniently, and its process of decryption is dynamic and fast, which all lead to the good potentials in practices.

  16. Indirect mechanisms of genotoxicity.

    PubMed

    Kirsch-Volders, Micheline; Vanhauwaert, Annelies; Eichenlaub-Ritter, Ursula; Decordier, Ilse

    2003-04-11

    Indirect mechanisms of genotoxicity correspond to interactions of mutagens with non-DNA targets, and are expected to show threshold concentration-effect response curves. If these thresholds can be proven experimentally they may provide a third alternative for risk assessment, besides the No Effect Level/Safety Factor approach and the low dose linear extrapolation method. We contributed significantly to the in vitro assessment of thresholds in human lymphocytes exposed to the spindle inhibitors nocodazole and carbendazim showing dose dependency and existence of lower thresholds for induction of non-disjunction as compared to chromosome loss. Micronuclei correlated with p53-independent or p53-dependent apoptosis and elimination of aneuploid cells. Extrapolation from in vitro threshold values to the in vivo situation remains unsolved. Comparing the in vitro threshold values for griseofulvin in human and rat lymphocytes with in vivo NOAEL/LOAEL in bone marrow/gut/erythrocytes suggests that the in vitro human system is the most sensitive. The threshold for induction of non-disjunction in in vitro maturing, nocodazole-exposed mouse oocytes was in the same low range. Regulators (UK Committee on Mutagenicity, http://www.doh.gov.uk/com/com.htm) considered the importance of thresholds for indirect mechanisms of genotoxicity. Acceptance of a non-linear extrapolation for mutagens requires mechanistic studies identifying the mutagen/target interactions. Moreover appropriate risk evaluation will require additional studies on individual susceptibility for indirect mutagenic effects and on interactions of aneugens in complex mixtures. PMID:12676452

  17. Direct and indirect inversions

    NASA Astrophysics Data System (ADS)

    Virieux, Jean; Brossier, Romain; Métivier, Ludovic; Operto, Stéphane; Ribodetti, Alessandra

    2016-06-01

    A bridge is highlighted between the direct inversion and the indirect inversion. They are based on fundamental different approaches: one is looking after a projection from the data space to the model space while the other one is reducing a misfit between observed data and synthetic data obtained from a given model. However, it is possible to obtain similar structures for model perturbation, and we shall focus on P-wave velocity reconstruction. This bridge is built up through the Born approximation linearizing the forward problem with respect to model perturbation and through asymptotic approximations of the Green functions of the wave propagation equation. We first describe the direct inversion and its ingredients and then we focus on a specific misfit function design leading to a indirect inversion. Finally, we shall compare this indirect inversion with more standard least-squares inversion as the FWI, enabling the focus on small weak velocity perturbations on one side and the speed-up of the velocity perturbation reconstruction on the other side. This bridge has been proposed by the group led by Raul Madariaga in the early nineties, emphasizing his leading role in efficient imaging workflows for seismic velocity reconstruction, a drastic requirement at that time.

  18. Construction of cell surface-engineered yeasts displaying antigen to detect antibodies by immunofluorescence and yeast-ELISA.

    PubMed

    Tang, Yu Qian; Han, Shuang Yan; Zheng, Hong; Wu, Lin; Ueda, Mitsuyoshi; Wang, Xiao Ning; Lin, Ying

    2008-07-01

    In order to detect monoclonal antibodies (MAbs) from insufficient and unavailable human proteins, yeast cells were engineered to display human antigens on their surface and consequently endowed with the ability to specifically bind antibodies. Thus, a fusion gene for the expression of the human proteasome subunit alpha 6 (hPSA6) and human profilin I (hProI) were assembled, respectively, with a His.tag marker at the C-terminal and displayed on yeast surface. With anti-His.tag MAb as the primary antibody and the fluorescein isothiocyanate-conjugated goat anti-mouse Immunoglobulin G as the second antibody, the surface display of hPSA6 and hProI were verified by immunofluorescence labeling. The antigen-displayed yeast particles were used for MAbs detection from ascites through both immunofluorescence and yeast-enzyme-linked immunosorbent assay (ELISA) methods. The results were verified by Western blotting and indirect ELISA. By improving the sensitivity, the novel MAbs detection can be applied in the generation and screening of positive hybridoma. It is suggested that by combining the DNA immunization, the present study can evolve into a quick and protein-free way of MAbs production for insufficient and unavailable antigen. PMID:18542951

  19. 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

  20. Machine-based method for multiplex in situ molecular characterization of tissues by immunofluorescence detection

    PubMed Central

    Yarilin, Dmitry; Xu, Ke; Turkekul, Mesruh; Fan, Ning; Romin, Yevgeniy; Fijisawa, Sho; Barlas, Afsar; Manova-Todorova, Katia

    2015-01-01

    Immunofluorescent staining is an informative tool that is widely used in basic research. Automation of immunostaining improves reproducibility and quality of the results. Up to now, use of automation in immunofluorescent staining was mostly limited to one marker. Here we present tyramide signal amplification based method of multiple marker immunofluorescent detection, including detection of antibodies, raised in the same species, in tissue sections and cultured cells. This method can be beneficial for both basic and clinical research. PMID:25826597

  1. 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.

  2. Multiplexed immunofluorescence delineates proteomic cancer cell states associated with metabolism

    PubMed Central

    Sood, Anup; Miller, Alexandra M.; Brogi, Edi; Sui, Yunxia; Armenia, Joshua; McDonough, Elizabeth; Santamaria-Pang, Alberto; Carlin, Sean; Stamper, Aleksandra; Campos, Carl; Pang, Zhengyu; Li, Qing; Port, Elisa; Graeber, Thomas G.; Schultz, Nikolaus; Ginty, Fiona; Larson, Steven M.; Mellinghoff, Ingo K.

    2016-01-01

    The phenotypic diversity of cancer results from genetic and nongenetic factors. Most studies of cancer heterogeneity have focused on DNA alterations, as technologies for proteomic measurements in clinical specimen are currently less advanced. Here, we used a multiplexed immunofluorescence staining platform to measure the expression of 27 proteins at the single-cell level in formalin-fixed and paraffin-embedded samples from treatment-naive stage II/III human breast cancer. Unsupervised clustering of protein expression data from 638,577 tumor cells in 26 breast cancers identified 8 clusters of protein coexpression. In about one-third of breast cancers, over 95% of all neoplastic cells expressed a single protein coexpression cluster. The remaining tumors harbored tumor cells representing multiple protein coexpression clusters, either in a regional distribution or intermingled throughout the tumor. Tumor uptake of the radiotracer 18F-fluorodeoxyglucose was associated with protein expression clusters characterized by hormone receptor loss, PTEN alteration, and HER2 gene amplification. Our study demonstrates an approach to generate cellular heterogeneity metrics in routinely collected solid tumor specimens and integrate them with in vivo cancer phenotypes. PMID:27182557

  3. 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. PMID:26303944

  4. Deformability-based cell selection with downstream immunofluorescence analysis.

    PubMed

    Shaw Bagnall, Josephine; Byun, Sangwon; Miyamoto, David T; Kang, Joon Ho; Maheswaran, Shyamala; Stott, Shannon L; Toner, Mehmet; Manalis, Scott R

    2016-05-16

    Mechanical properties of single cells have been shown to relate to cell phenotype and malignancy. However, until recently, it has been difficult to directly correlate each cell's biophysical characteristics to its molecular traits. Here, we present a cell sorting technique for use with a suspended microchannel resonator (SMR), which can measure biophysical characteristics of a single cell based on the sensor's record of its buoyant mass as well as its precise position while it traverses through a constricted microfluidic channel. The measurement provides information regarding the amount of time a cell takes to pass through a constriction (passage time), as related to the cell's deformability and surface friction, as well as the particular manner in which it passes through. In the method presented here, cells of interest are determined based on passage time, and are collected off-chip for downstream immunofluorescence imaging. The biophysical single-cell SMR measurement can then be correlated to the molecular expression of the collected cell. This proof-of-principle is demonstrated by sorting and collecting tumor cells from cell line-spiked blood samples as well as a metastatic prostate cancer patient blood sample, identifying them by their surface protein expression and relating them to distinct SMR signal trajectories. PMID:26999591

  5. Borrelia burgdorferi visualized in Ixodes scapularis tick excrement by immunofluorescence.

    PubMed

    Patton, Toni G; Brandt, Kevin S; Gilmore, Robert D

    2012-11-01

    The enzootic cycle of Borrelia burgdorferi, the etiologic agent of Lyme disease, involves Ixodes spp. ticks and vertebrates. Resident tick Borrelia, harbored inside the midgut, are eventually expelled with the tick's saliva into the vertebrate host when a tick consumes a blood meal. During this 4- to 5-day feeding period I. scapularis will defecate onto the host's skin. Previously we detected borrelial DNA in tick feces throughout engorgement. In this study we report the microscopic examination for B. burgdorferi in nymphal excrement. Using immunofluorescence assays, we observed Borrelia in all mouse skin and capsule fecal swabs tested, although we could not culture the spirochetes. These results update our previous analysis by revealing that spirochetes can also be visualized in tick excrement. Furthermore, the results emphasize that borrelial contamination by defecation is a possibility, and that caution should be exercised by researchers investigating pathogen/host/vector interactions. The biological significance of the presence of non-culturable Borrelia in tick feces during engorgement is unclear. PMID:22651382

  6. 3D multiplexed immunoplasmonics microscopy.

    PubMed

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

    2016-07-21

    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

  7. 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

  8. 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.

  9. Immunofluorescent localization of actin in relation to transcription sites in mouse pronuclei.

    PubMed

    Nguyen, E; Besombes, D; Debey, P

    1998-07-01

    Previous biochemical and morphological studies have shown the presence of actin in the nucleus of different cell types where its role remains unclear. In this work, through fluorescence microscopy we studied the localization of actin in the nuclei of early mouse embryos with particular attention to its possible involvement in the onset of transcription occurring at the late one-cell stage. Fluorescent labelling of embryo sections showed that nuclear actin in abundant, in a non-filamentous state, in the whole nucleoplasm excluding the nucleolar precursor bodies. Immunofluorescence on permeabilized embryos revealed that insoluble nuclear actin accumulates in a few large aggregates in transcriptionally inert early one-cell embryos and progressively redistributes into many small aggregates in transcriptionally active late one-cell embryos. Interestingly, these actin aggregates clearly colocalize with transcription sites. Treatment of late one-cell embryos with cytochalasin D induces the formation of actin bundles network in the nucleoplasm but has no apparent effect on the transcriptional activity. In addition, the inhibition of transcription by alpha-amanitin does not modify the nuclear actin distribution. Hence, there does not appear to be a direct causal relationship between transcriptional activity and nuclear actin organization at the one-cell stage although nuclear actin aggregates appear associated with transcription sites. PMID:9621302

  10. 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.

  11. [An immunofluorescent analysis of bovine rotavirus during its isolation and adaptation to cell cultures].

    PubMed

    Skybyts'kyĭ, V H; Nosach, L M; Martynenko, D L; Onufriiev, V P

    1993-01-01

    The results from comparative studies in the reactions of immunofluorescence, complement binding, diffusion precipitation, hemagglutination, solid-phase immunoenzyme analysis, histochemical variant of immunoenzyme analysis as tests for detection of cattle rotavirus in the process of its isolation from pathological material and adaptation to cell cultures are presented. The immunofluorescence reaction is shown to have an advantage over the other reactions. PMID:8388533

  12. Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

    PubMed Central

    Chaumeil, Julie; Micsinai, Mariann; Skok, Jane A.

    2013-01-01

    Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei. PMID:23407477

  13. 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. PMID:26392518

  14. Correlative Microscopy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  15. 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

  16. 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. PMID:26779585

  17. 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

  18. Photoacoustic Microscopy

    PubMed Central

    Yao, Junjie; Wang, Lihong V.

    2012-01-01

    Photoacoustic microscopy (PAM) is a hybrid in vivo imaging technique that acoustically detects optical contrast via the photoacoustic effect. Unlike pure optical microscopic techniques, PAM takes advantage of the weak acoustic scattering in tissue and thus breaks through the optical diffusion limit (~1 mm in soft tissue). With its excellent scalability, PAM can provide high-resolution images at desired maximum imaging depths up to a few millimeters. Compared with backscattering-based confocal microscopy and optical coherence tomography, PAM provides absorption contrast instead of scattering contrast. Furthermore, PAM can image more molecules, endogenous or exogenous, at their absorbing wavelengths than fluorescence-based methods, such as wide-field, confocal, and multi-photon microscopy. Most importantly, PAM can simultaneously image anatomical, functional, molecular, flow dynamic and metabolic contrasts in vivo. Focusing on state-of-the-art developments in PAM, this Review discusses the key features of PAM implementations and their applications in biomedical studies. PMID:24416085

  19. Intravital microscopy

    PubMed Central

    Masedunskas, Andrius; Milberg, Oleg; Porat-Shliom, Natalie; Sramkova, Monika; Wigand, Tim; Amornphimoltham, Panomwat; Weigert, Roberto

    2012-01-01

    Intravital microscopy is an extremely powerful tool that enables imaging several biological processes in live animals. Recently, the ability to image subcellular structures in several organs combined with the development of sophisticated genetic tools has made possible extending this approach to investigate several aspects of cell biology. Here we provide a general overview of intravital microscopy with the goal of highlighting its potential and challenges. Specifically, this review is geared toward researchers that are new to intravital microscopy and focuses on practical aspects of carrying out imaging in live animals. Here we share the know-how that comes from first-hand experience, including topics such as choosing the right imaging platform and modality, surgery and stabilization techniques, anesthesia and temperature control. Moreover, we highlight some of the approaches that facilitate subcellular imaging in live animals by providing numerous examples of imaging selected organelles and the actin cytoskeleton in multiple organs. PMID:22992750

  20. SIMULTANEOUS DETECTION OF EDWARDSIELLA ICTALURI AND FLAVOBACTERIUM COLUMNARE BY DUAL IMMUNOFLUORESCENCE TEST

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A rapid immunofluorescence test with fluorescent conjugated-antibodies having different spectral properties (Alexa Fluor 488-emitting green fluorescence and Alexa Fluor 594-emitting red fluorescence) was compared with standard bacteriological culture for simultaneous detection of bacterial fish path...

  1. [Quantitative Analysis of Immuno-fluorescence of Nuclear Factor-κB Activation].

    PubMed

    Xiu, Min; He, Feng; Lou, Yuanlei; Xu, Lu; Xiong Jieqi; Wang, Ping; Liu, Sisun; Guo, Fei

    2015-06-01

    Immuno-fluorescence technique can qualitatively determine certain nuclear translocation, of which NF-κB/ p65 implicates the activation of NF-κB signal pathways. Immuno-fluorescence analysis software with independent property rights is able to quantitatively analyze dynamic location of NF-κB/p65 by computing relative fluorescence units in nuclei and cytoplasm. We verified the quantitative analysis by Western Blot. When we applied the software to analysis of nuclear translocation in lipopolysaccharide (LPS) induced (0. 5 h, 1 h, 2 h, 4 h) primary human umbilical vein endothelial cells (HUVECs) , we found that nuclear translocation peak showed up at 2h as with calculated Western blot verification results, indicating that the inventive immuno-fluorescence analysis software can be applied to the quantitative analysis of immuno-fluorescence. PMID:26485997

  2. 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.

  3. 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.

  4. Endoscopic Microscopy

    PubMed Central

    Sokolov, Konstantin; Sung, Kung-Bin; Collier, Tom; Clark, Anne; Arifler, Dizem; Lacy, Alicia; Descour, Michael; Richards-Kortum, Rebecca

    2002-01-01

    In vivo endoscopic optical microscopy provides a tool to assess tissue architecture and morphology with contrast and resolution similar to that provided by standard histopathology – without need for physical tissue removal. In this article, we focus on optical imaging technologies that have the potential to dramatically improve the detection, prevention, and therapy of epithelial cancers. Epithelial pre-cancers and cancers are associated with a variety of morphologic, architectural, and molecular changes, which currently can be assessed only through invasive, painful biopsy. Optical imaging is ideally suited to detecting cancer-related alterations because it can detect biochemical and morphologic alterations with sub-cellular resolution throughout the entire epithelial thickness. Optical techniques can be implemented non-invasively, in real time, and at low cost to survey the tissue surface at risk. Our manuscript focuses primarily on modalities that currently are the most developed: reflectance confocal microscopy (RCM) and optical coherence tomography (OCT). However, recent advances in fluorescence-based endoscopic microscopy also are reviewed briefly. We discuss the basic principles of these emerging technologies and their current and potential applications in early cancer detection. We also present research activities focused on development of exogenous contrast agents that can enhance the morphological features important for cancer detection and that have the potential to allow vital molecular imaging of cancer-related biomarkers. In conclusion, we discuss future improvements to the technology needed to develop robust clinical devices. PMID:14646041

  5. 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. PMID:26412738

  6. Moral assessment in indirect reciprocity

    PubMed Central

    Sigmund, Karl

    2012-01-01

    Indirect reciprocity is one of the mechanisms for cooperation, and seems to be of particular interest for the evolution of human societies. A large part is based on assessing reputations and acting accordingly. This paper gives a brief overview of different assessment rules for indirect reciprocity, and studies them by using evolutionary game dynamics. Even the simplest binary assessment rules lead to complex outcomes and require considerable cognitive abilities. PMID:21473870

  7. Indirect electroanalytical detection of phenols.

    PubMed

    Kolliopoulos, Athanasios V; Kampouris, Dimitrios K; Banks, Craig E

    2015-05-01

    A novel indirect electrochemical protocol for the electroanalytical detection of phenols is presented for the first time. This methodology is demonstrated with the indirect determination of the target analytes phenol, 2-chlorophenol, 4-chlorophenol and 2,4-dichlorophenol through an electrochemically adapted optical protocol. This electrochemical adaptation allows the determination of the above mentioned phenols without the use of any oxidising agents, as is the case in the optical method, where pyrazoline compounds (mediators) chemically react with the target phenols forming a quinoneimine product which is electrochemically active providing an indirect analytical signal to measure the target phenol(s). A range of commercially available pyrazoline substitution products, namely 4-dimethylaminoantipyrine, antipyrine, 3-methyl-1-(2-phenylethyl)-2-pyrazolin-5-one, 3-amino-1-(1-naphthylmethyl)-2-Pyrazolin-5-one, 4-amino-1,2-dimethyl-3-pentadecyl-3-pyrazolin-5-one hydrochloride, 3-amino-1-(2-amino-4-methylsulfonylphenyl)-2-pyrazolin-5-one hydrochloride and 4-aminoantipyrine are evaluated as mediators for the indirect detection of phenols. The indirect electrochemical detection of phenol, 2-chlorophenol, 4-chlorophenol and 2,4-dichlorophenol through the use of 4-aminoantipyrine as a mediator are successfully determined in drinking water samples at analytically useful levels. Finally, the comparison of the direct (no mediator) and the proposed indirect determination (with 4-aminoantipyrine) towards the analytical detection of the target phenols in drinking water is presented. The limitation of the proposed electroanalytical protocol is quantified for all the four target phenols. PMID:25771897

  8. 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

  9. 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.

  10. 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.

  11. Indirect comparisons of therapeutic interventions

    PubMed Central

    Schöttker, Ben; Lühmann, Dagmar; Boulkhemair, Dalila; Raspe, Heiner

    2009-01-01

    Health political background The comparison of the effectiveness of health technologies is not only laid down in German law (Social Code Book V, § 139 and § 35b) but also constitutes a central element of clinical guidelines and decision making in health care. Tools supporting decision making (e. g. Health Technology Assessments (HTA)) are therefore in need of a valid methodological repertoire for these comparisons. Scientific background Randomised controlled head-to-head trials which directly compare the effects of different therapies are considered the gold standard methodological approach for the comparison of the efficacy of interventions. Because this type of trial is rarely found, comparisons of efficacy often need to rely on indirect comparisons whose validity is being controversially debated. Research questions Research questions for the current assessment are: Which (statistical) methods for indirect comparisons of therapeutic interventions do exist, how often are they applied and how valid are their results in comparison to the results of head-to-head trials? Methods In a systematic literature research all medical databases of the German Institute of Medical Documentation and Information (DIMDI) are searched for methodological papers as well as applications of indirect comparisons in systematic reviews. Results of the literature analysis are summarized qualitatively for the characterisation of methods and quantitatively for the frequency of their application. The validity of the results from indirect comparisons is checked by comparing them to the results from the gold standard – a direct comparison. Data sets from systematic reviews which use both direct and indirect comparisons are tested for consistency by of the z-statistic. Results 29 methodological papers and 106 applications of indirect methods in systematic reviews are being analysed. Four methods for indirect comparisons can be identified: Unadjusted indirect comparisons include, independent of

  12. 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

  13. Identifying the Spatial Relationships of Thymic Stromal and Thymocyte Subsets by Immunofluorescence Analysis.

    PubMed

    Bain, Virginia; Richie, Ellen R

    2016-01-01

    Immunofluorescence analysis of thymic tissue sections is an indispensable technique for visualizing spatial relationships among thymocyte and stromal cell subsets. The thymus is organized into distinct microenvironmental zones in which particular thymic epithelial cell (TEC) subsets support specific stages of thymocyte maturation. Conversely, thymocytes and lymphoid tissue inducer cells support functional maturation of TECs. The composition and organization of TECs change during ontogeny to generate a maximally functional organ in the young adult. Deterioration of thymic architecture and stromal organization occurs with age as the thymus undergoes involution. Such changes can be monitored by immunofluorescent staining of thymic sections obtained at different ages throughout the life-span. Here we describe methods to generate frozen or paraffin-embedded thymic tissue sections for multicolor immunofluorescence staining using antibodies to surface and/or cytoplasmic antigens. PMID:26294399

  14. 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.

  15. 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. PMID:26842269

  16. 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. PMID:26079823

  17. Direct Detectors for Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Clough, R. N.; Moldovan, G.; Kirkland, A. I.

    2014-06-01

    There is interest in improving the detectors used to capture images in transmission electron microscopy. Detectors with an improved modulation transfer function at high spatial frequencies allow for higher resolution in images at lower magnification, which leads to an increased effective field of view. Detectors with improved detective quantum efficiency are important for low dose applications. One way in which these performance enhancements can be achieved is through direct detection, where primary electrons are converted directly into suitable electrical signals by the detector rather than relying on an indirect electron to photon conversion before detection. In this paper we present the characterisation of detector performance for a number of different direct detection technologies, and compare these technologies to traditional indirect detectors. Overall our results show that direct detection enables a significant improvement in all aspects of detector performance.

  18. 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...

  19. 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...

  20. 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...

  1. 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...

  2. 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...

  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 10.776 - 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...

  5. 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...

  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. 7 CFR 3430.54 - Indirect costs.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...-GENERAL AWARD ADMINISTRATIVE PROVISIONS Post-Award and Closeout § 3430.54 Indirect costs. Indirect cost... assistance regulations and cost principles, unless superseded by another authority. Use of indirect costs as... 7 Agriculture 15 2010-01-01 2010-01-01 false Indirect costs. 3430.54 Section 3430.54...

  8. 24 CFR 576.109 - Indirect costs.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 3 2014-04-01 2013-04-01 true Indirect costs. 576.109 Section 576... § 576.109 Indirect costs. (a) In general. ESG grant funds may be used to pay indirect costs in.... Indirect costs may be allocated to each eligible activity under § 576.101 through § 576.108, so long...

  9. 24 CFR 576.109 - Indirect costs.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 24 Housing and Urban Development 3 2013-04-01 2013-04-01 false Indirect costs. 576.109 Section 576... § 576.109 Indirect costs. (a) In general. ESG grant funds may be used to pay indirect costs in.... Indirect costs may be allocated to each eligible activity under § 576.101 through § 576.108, so long...

  10. 24 CFR 578.63 - Indirect costs.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 24 Housing and Urban Development 3 2013-04-01 2013-04-01 false Indirect costs. 578.63 Section 578... Indirect costs. (a) In general. Continuum of Care funds may be used to pay indirect costs in accordance with OMB Circulars A-87 or A-122, as applicable. (b) Allocation. Indirect costs may be allocated...

  11. 7 CFR 3430.54 - Indirect costs.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Post-Award and Closeout § 3430.54 Indirect costs. Indirect cost rates for grants and cooperative... 7 Agriculture 15 2011-01-01 2011-01-01 false Indirect costs. 3430.54 Section 3430.54 Agriculture..., unless superseded by another authority. Use of indirect costs as in-kind matching contributions...

  12. 7 CFR 3430.54 - Indirect costs.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Post-Award and Closeout § 3430.54 Indirect costs. Indirect cost rates for grants and cooperative... 7 Agriculture 15 2014-01-01 2014-01-01 false Indirect costs. 3430.54 Section 3430.54 Agriculture..., unless superseded by another authority. Use of indirect costs as in-kind matching contributions...

  13. 24 CFR 578.63 - Indirect costs.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 3 2014-04-01 2013-04-01 true Indirect costs. 578.63 Section 578... Indirect costs. (a) In general. Continuum of Care funds may be used to pay indirect costs in accordance with OMB Circulars A-87 or A-122, as applicable. (b) Allocation. Indirect costs may be allocated...

  14. 7 CFR 3430.54 - Indirect costs.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Post-Award and Closeout § 3430.54 Indirect costs. Indirect cost rates for grants and cooperative... 7 Agriculture 15 2013-01-01 2013-01-01 false Indirect costs. 3430.54 Section 3430.54 Agriculture..., unless superseded by another authority. Use of indirect costs as in-kind matching contributions...

  15. 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...

  16. 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...

  17. Water Rockets and Indirect Measurement.

    ERIC Educational Resources Information Center

    Inman, Duane

    1997-01-01

    Describes an activity that teaches a number of scientific concepts including indirect measurement, Newton's third law of motion, manipulating and controlling variables, and the scientific method of inquiry. Uses process skills such as observation, inference, prediction, mensuration, and communication as well as problem solving and higher-order…

  18. 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.

  19. 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.

  20. Ecology: Dynamics of Indirect Extinction.

    PubMed

    Montoya, Jose M

    2015-12-01

    The experimental identification of the mechanism by which extinctions of predators trigger further predator extinctions emphasizes the role of indirect effects between species in disturbed ecosystems. It also has deep consequences for the hidden magnitude of the current biodiversity crisis. PMID:26654371

  1. Feedback control indirect response models.

    PubMed

    Zhang, Yaping; D'Argenio, David Z

    2016-08-01

    A general framework is introduced for modeling pharmacodynamic processes that are subject to autoregulation, which combines the indirect response (IDR) model approach with methods from classical feedback control of engineered systems. The canonical IDR models are modified to incorporate linear combinations of feedback control terms related to the time course of the difference (the error signal) between the pharmacodynamic response and its basal value. Following the well-established approach of traditional engineering control theory, the proposed feedback control indirect response models incorporate terms proportional to the error signal itself, the integral of the error signal, the derivative of the error signal or combinations thereof. Simulations are presented to illustrate the types of responses produced by the proposed feedback control indirect response model framework, and to illustrate comparisons with other PK/PD modeling approaches incorporating feedback. In addition, four examples from literature are used to illustrate the implementation and applicability of the proposed feedback control framework. The examples reflect each of the four mechanisms of drug action as modeled by each of the four canonical IDR models and include: selective serotonin reuptake inhibitors and extracellular serotonin; histamine H2-receptor antagonists and gastric acid; growth hormone secretagogues and circulating growth hormone; β2-selective adrenergic agonists and potassium. The proposed feedback control indirect response approach may serve as an exploratory modeling tool and may provide a bridge for development of more mechanistic systems pharmacology models. PMID:27394724

  2. Periodic Acid-Schiff Staining Parallels the Immunoreactivity Seen By Direct Immunofluorescence in Autoimmune Skin Diseases

    PubMed Central

    Abreu Velez, Ana Maria; Upegui Zapata, Yulieth Alexandra; Howard, Michael S

    2016-01-01

    Background: In many countries and laboratories, techniques such as direct immunofluorescence (DIF) are not available for the diagnosis of skin diseases. Thus, these laboratories are limited in the full diagnoses of autoimmune skin diseases, vasculitis, and rheumatologic diseases. In our experience with these diseases and the patient's skin biopsies, we have noted a positive correlation between periodic acid-Schiff (PAS) staining and immunofluorescence patterns; however, these were just empiric observations. In the current study, we aim to confirm these observations, given the concept that the majority of autoantibodies are glycoproteins and should thus be recognized by PAS staining. Aims: To compare direct immunofluorescent and PAS staining, in multiple autoimmune diseases that are known to exhibit specific direct immunofluorescent patterns. Materials and Methods: We studied multiple autoimmune skin diseases: Five cases of bullous pemphigoid, five cases of pemphigus vulgaris, ten cases of cutaneous lupus, ten cases of autoimmune vasculitis, ten cases of lichen planus (LP), and five cases of cutaneous drug reactions (including one case of erythema multiforme). In addition, we utilized 45 normal skin control specimens from plastic surgery reductions. Results: We found a 98% positive correlation between DIF and PAS staining patterns over all the disease samples. Conclusion: We recommend that laboratories without access to DIF always perform PAS staining in addition to hematoxylin and eosin (H&E) staining, for a review of the reactivity pattern. PMID:27114972

  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. 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

  6. 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...

  7. 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...

  8. 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...

  9. 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...

  10. 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...

  11. The serological diagnosis of Mycoplasma pneumoniae infection: a comparison of complement fixation, haemagglutination and immunofluorescence.

    PubMed Central

    Rousseau, S. A.; Tettmar, R. E.

    1985-01-01

    A total of 193 sera were examined for antibody to Mycoplasma pneumoniae by three techniques - complement fixation (CF), haemagglutination (HA) and immunofluorescence (IF), the last method being used to assess IgM, IgG and IgA antibodies. The most reliable single test for diagnosis was HA, and the most useful combination of tests was HA with IF (IgM and IgG). The IgA IF was not found to be diagnostically helpful. PMID:3934260

  12. 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. PMID:25307685

  13. Characterization of Antibodies to Products of Proinsulin Processing Using Immunofluorescence Staining of Pancreas in Multiple Species

    PubMed Central

    Asadi, Ali; Bruin, Jennifer E.

    2015-01-01

    The efficient processing of proinsulin into mature insulin and C-peptide is often compromised under conditions of beta cell stress, including diabetes. Impaired proinsulin processing has been challenging to examine by immunofluorescence staining in pancreas tissue because the characterization of antibodies specific for proinsulin, proinsulin intermediates, processed insulin and C-peptide has been limited. This study aimed to identify and characterize antibodies that can be used to detect products of proinsulin processing by immunofluorescence staining in pancreata from different species (mice, rats, dog, pig and human). We took advantage of several knockout mouse lines that lack either an enzyme involved in proinsulin processing or an insulin gene. Briefly, we report antibodies that are specific for several proinsulin processing products, including: a) insulin or proinsulin that has been appropriately processed at the B-C junction; b) proinsulin with a non-processed B-C junction; c) proinsulin with a non-processed A-C junction; d) rodent-specific C-peptide 1; e) rodent-specific C-peptide 2; and f) human-specific C-peptide or proinsulin. In addition, we also describe two ‘pan-insulin’ antibodies that react with all forms of insulin and proinsulin intermediates, regardless of the species. These antibodies are valuable tools for studying proinsulin processing by immunofluorescence staining and distinguishing between proinsulin products in different species. PMID:26216140

  14. 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. PMID:26206941

  15. 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...

  16. 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...

  17. 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...

  18. 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....

  19. 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....

  20. 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....

  1. 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...

  2. 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...

  3. 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...

  4. 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...

  5. 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...

  6. 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...

  7. 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... 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...

  8. 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....

  9. 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...

  10. 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...

  11. 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...

  12. 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....

  13. 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...

  14. 7 CFR 2500.044 - Indirect costs.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Closeout § 2500.044 Indirect costs. Indirect cost rates for grants and cooperative agreements shall be determined in accordance with the applicable assistance regulations and cost principles, unless superseded by... 7 Agriculture 15 2013-01-01 2013-01-01 false Indirect costs. 2500.044 Section 2500.044...

  15. 7 CFR 2500.044 - Indirect costs.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Closeout § 2500.044 Indirect costs. Indirect cost rates for grants and cooperative agreements shall be determined in accordance with the applicable assistance regulations and cost principles, unless superseded by... 7 Agriculture 15 2014-01-01 2014-01-01 false Indirect costs. 2500.044 Section 2500.044...

  16. 48 CFR 31.203 - Indirect costs.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... cost of that or any other final cost objective. (c) The contractor shall accumulate indirect costs by... for allocating indirect costs is the cost accounting period during which such costs are incurred and... 48 Federal Acquisition Regulations System 1 2014-10-01 2014-10-01 false Indirect costs....

  17. 38 CFR 17.261 - Indirect costs.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2013-07-01 2013-07-01 false Indirect costs. 17.261... Exchange of Information § 17.261 Indirect costs. The grantee shall allocate expenditures as between direct and indirect costs according to generally accepted accounting procedures. The amount allocated...

  18. 38 CFR 17.261 - Indirect costs.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2014-07-01 2014-07-01 false Indirect costs. 17.261... Exchange of Information § 17.261 Indirect costs. The grantee shall allocate expenditures as between direct and indirect costs according to generally accepted accounting procedures. The amount allocated...

  19. 48 CFR 31.203 - Indirect costs.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... cost of that or any other final cost objective. (c) The contractor shall accumulate indirect costs by... for allocating indirect costs is the cost accounting period during which such costs are incurred and... 48 Federal Acquisition Regulations System 1 2013-10-01 2013-10-01 false Indirect costs....

  20. 38 CFR 17.261 - Indirect costs.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2012-07-01 2012-07-01 false Indirect costs. 17.261... Exchange of Information § 17.261 Indirect costs. The grantee shall allocate expenditures as between direct and indirect costs according to generally accepted accounting procedures. The amount allocated...

  1. 38 CFR 17.261 - Indirect costs.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2010-07-01 2010-07-01 false Indirect costs. 17.261... Exchange of Information § 17.261 Indirect costs. The grantee shall allocate expenditures as between direct and indirect costs according to generally accepted accounting procedures. The amount allocated...

  2. 38 CFR 17.261 - Indirect costs.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2011-07-01 2011-07-01 false Indirect costs. 17.261... Exchange of Information § 17.261 Indirect costs. The grantee shall allocate expenditures as between direct and indirect costs according to generally accepted accounting procedures. The amount allocated...

  3. Indirect Cost Reimbursement: An Industrial View.

    ERIC Educational Resources Information Center

    Bolton, Robert

    1987-01-01

    The meaning of indirect costs in an industrial environment is discussed. Other factors considered are corporate policies; nature of work being supported; the uniqueness of the work; who is doing the negotiating for industry; and indirect rates. Suggestions are offered for approaches to indirect cost reimbursement. (Author/MLW)

  4. 19 CFR 10.924 - Indirect materials.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 19 Customs Duties 1 2012-04-01 2012-04-01 false Indirect materials. 10.924 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....

  5. 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...

  6. 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... 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...

  7. 19 CFR 10.541 - Indirect materials.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 19 Customs Duties 1 2012-04-01 2012-04-01 false Indirect materials. 10.541 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...

  8. Do infants detect indirect reciprocity?

    PubMed

    Meristo, Marek; Surian, Luca

    2013-10-01

    In social interactions involving indirect reciprocity, agent A acts prosocially towards B and this prompts C to act prosocially towards A. This happens because A's actions enhanced its reputation in the eyes of third parties. Indirect reciprocity may have been of central importance in the evolution of morality as one of the major mechanisms leading to the selection of helping and fair attitudes. Here we show that 10-month-old infants expect third parties to act positively towards fair donors who have distributed attractive resources equally between two recipients, rather than toward unfair donors who made unequal distributions. Infants' responses were dependent on the reciprocator's perceptual exposure to previous relevant events: they expected the reciprocator to reward the fair donor only when it had seen the distributive actions performed by the donors. We propose that infants were able to generate evaluations of agents that were based on the fairness of their distributive actions and to generate expectations about the social preferences of informed third parties. PMID:23887149

  9. 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

  10. 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. PMID:23123557

  11. 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. PMID:23713982

  12. 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

  13. 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

  14. Clinical features and in vivo confocal microscopy assessment in 12 patients with ocular cicatricial pemphigoid

    PubMed Central

    Long, Qin; Zuo, Ya-Gang; Yang, Xue; Gao, Ting-Ting; Liu, Jie; Li, Ying

    2016-01-01

    AIM To describe the clinical features and microstructural characteristics assessed by in vivo confocal microscopy (IVCM) in patients with ocular cicatricial pemphigoid (OCP). METHODS A descriptive, uncontrolled case series study. Patients diagnosed with OCP were examined by clinical history, slit-lamp biomicroscopy features and IVCM images. The results of direct immunofluorescence (DIF) biopsies and indirect immunofluorescence (IIF) were also recorded. Local and systemic immunosuppressive therapy were administered and adjusted according to response. RESULTS A total of 12 consecutive OCP patients (7 male, 5 female; mean age 60.42±10.39y) were recruited. All patients exhibited bilateral progressive conjunctival scarring and recurrent chronic conjunctivitis was the most frequent clinical pattern. The mean duration of symptoms prior to diagnosis of OCP was 2.95±2.85y (range: 5mo to 10y). The Foster classification varied from stage I to IV and 20 eyes (83%) were within or greater than Foster stage III on presentation. Two of the 12 patients (17%) demonstrated positive DIF; 3 of the 12 (25%) patients reported positive IIF. The mean duration of the follow-up period was 20.17±11.88mo (range: 6 to 48mo). IVCM showed variable degrees of abnormality in the conjuctiva-cornea and conjuctival scarring was detected in all the involved eyes. Corneal stromal cell activation and dendritic cell infiltration presented as ocular surface inflammation, ocular surface keratinization along with the destroyed Vogt palisades was noted in eyes with potential limbal stem cell deficiency. After treatment, remission of ocular surface inflammation was achieved in all the patients, 18 eyes (75%) remained stable, 6 eyes (25%) had recurrent conjunctivitis and cicatrization in 2 eyes (8%) was progressing. CONCLUSION As an autoimmune disease, OCP manifests as variable degrees of clinical and laboratory abnormalities with both local and systemic immunosuppressive treatment playing important roles

  15. 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.

  16. 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.

  17. 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

  18. Indirect conductimetric assay of antibacterial activities.

    PubMed

    Sawai, J; Doi, R; Maekawa, Y; Yoshikawa, T; Kojima, H

    2002-11-01

    The applicability of indirect conductimetric assays for evaluation of antibacterial activity was examined. The minimal inhibitory concentration (MIC) obtained by the indirect method was consistent with that by the direct conductimetric assay and the turbidity method. The indirect assay allows use of growth media, which cannot be used in the direct conductimetric assay, making it possible to evaluate the antibacterial activity of insoluble or slightly soluble materials with high turbidity, such as antibacterial ceramic powders. PMID:12407467

  19. Two stage indirect evaporative cooling system

    DOEpatents

    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.

  20. 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. PMID:26164838

  1. A Cross-sectional Study of Clinical, Histopathological and Direct Immunofluorescence Spectrum of Vesiculobullous Disorders

    PubMed Central

    S., Arundhathi; S., Ragunatha; K.C., Mahadeva

    2013-01-01

    Background: Accurate diagnosis of vesiculobullous lesions of skin requires evaluation of clinical, histopathologic and immunofluorescence findings. Methods: A cross-sectional study of 68 patients to evaluate the clinical, histopathological and direct immunofluorescence (DIF) features in the diagnosis of cutaneous vesiculobullous disorders. The patients with vesiculobullous lesions were subjected to clinical examination regarding socio-demographic and clinical data. Two biopsy specimens were taken, one from intact vesicle for histopathological study and another from perilesional normal looking skin or oral mucosa for DIF. Results: Vesiculobullous lesions constituted 22.08% of total number of skin biopsies. The most common clinical diagnosis was pemphigus vulgaris (PV) in 36 cases, followed by bullous pemphigoid (BP) in 8 cases, pemphigus foliaceous (PF) in 6 cases, and dermatitis herpetiformis (DH) in 4 cases. Characteristic histopathological features were present in 26 cases of PV, 9 cases of BP and 4 cases of PF, and 17.7% showed non- specific changes. DIF was positive in 24 cases of PV, 9 cases of BP and 3 cases of PF, and negative in 34.92% of cases. Conclusion: Clinical, histopathological and DIF features together or in combination help in the final diagnosis of vesiculobullous disorders. Individually, none of these methods are diagnostic in each and every case. PMID:24551638

  2. Intensive Immunofluorescence Staining Methods for Low Expression Protein: Detection of Intestinal Stem Cell Marker LGR5

    PubMed Central

    Yamazaki, Masaki; Kato, Atsuhiko; Zaitsu, Yoko; Watanabe, Takeshi; Iimori, Makoto; Funahashi, Shinichi; Kitao, Hiroyuki; Saeki, Hiroshi; Oki, Eiji; Suzuki, Masami

    2015-01-01

    Leucine-rich repeat-containing G-protein coupled receptor 5, or LGR5, is a molecule that recognizes stem cells in multiple organs and also in colon cancer. Previously, we have developed monoclonal antibodies specific to LGR5 protein that can be used for immunofluorescence staining, but because a very low level of LGR5 protein is expressed, the visualization technique needed to be enhanced. To develop procedures to detect LGR5 protein in various specimens by immunofluorescence staining, we evaluated the Alexa-labeled streptavidin biotin (LSAB), the Qdot, and the tyramide methods. The detection sensitivity was highest in the tyramide method followed by the Qdot method, whereas subcellular localization of the protein was most clear in the Qdot method, because the Qdot method gave a high S/N ratio that could show a low background. Thus, the tyramide method is superior to the Q-dot method for intensifying the signal of a low expression protein, and the Qdot method is superior to the tyramide method for identifying the subcellular localization of the target protein. The results of the present study will be helpful in providing more insight into the pathophysiological roles of LGR5-positive cancer stem cells and in developing therapeutic approaches for targeting cancer stem cells. PMID:26633908

  3. Evaluation of reflectance confocal microscopy in dermatophytosis.

    PubMed

    Hui, Dai; Xue-cheng, Sun; Ai-e, Xu

    2013-03-01

    Traditional diagnostic testing for dermatophyte infection currently requires skin scraping for light microscopy and/or fungal culture or skin biopsy. Immunofluorescent microscopy can also be used with calcofluor stain. All of these tests can be time-consuming to perform, require a waiting period for results and are invasive. This study aimed to define the in vivo reflectance confocal microscopy (RCM) features of superficial cutaneous fungal infections and to analyse concordance with microscopic examination. Totally, 45 patients, who were diagnosed with superficial cutaneous fungal infections according to the positive result of microscopic examination, were enrolled in this study. We selected three typical lesions examined by RCM, and then recorded the results. In the patients with the tinea manus and pedis, mycelium in stratum corneum was found by the RCM in 14 of 22 patients (14/22; 63.64%). In the patients with the tinea cruris, mycelium in stratum corneum was found by the RCM in 19 of 23 patients (19/23; 82.61%). RCM seems to be useful for microscopic evaluation of mycelium features and may have a scientific value in study of superficial cutaneous fungal infections. PMID:22963376

  4. 48 CFR 31.203 - Indirect costs.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... coverage, the contractor shall follow the criteria and guidance in 48 CFR 9904.406 for selecting the cost... REQUIREMENTS CONTRACT COST PRINCIPLES AND PROCEDURES Contracts With Commercial Organizations 31.203 Indirect... final cost objectives. No final cost objective shall have allocated to it as an indirect cost any...

  5. 29 CFR 452.119 - Indirect elections.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 29 Labor 2 2013-07-01 2013-07-01 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...

  6. 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...

  7. 29 CFR 452.119 - Indirect elections.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 2 2011-07-01 2011-07-01 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...

  8. 29 CFR 452.119 - Indirect elections.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 29 Labor 2 2012-07-01 2012-07-01 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...

  9. 27 CFR 6.26 - Indirect interest.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 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...

  10. 27 CFR 6.32 - Indirect interest.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Indirect interest. 6.32 Section 6.32 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL âTIED-HOUSEâ Unlawful Inducements Interest in Retail Property § 6.32 Indirect interest. Industry member interest in...

  11. 27 CFR 6.26 - Indirect interest.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Indirect interest. 6.26 Section 6.26 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL âTIED-HOUSEâ Unlawful Inducements Interest in Retail License § 6.26 Indirect interest. Industry member interest in...

  12. 27 CFR 6.26 - Indirect interest.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 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...

  13. 27 CFR 6.26 - Indirect interest.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Indirect interest. 6.26 Section 6.26 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL âTIED-HOUSEâ Unlawful Inducements Interest in Retail License § 6.26 Indirect interest. Industry member interest in...

  14. 27 CFR 6.32 - Indirect interest.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 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...

  15. 27 CFR 6.32 - Indirect interest.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 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...

  16. 27 CFR 6.32 - Indirect interest.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Indirect interest. 6.32 Section 6.32 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL âTIED-HOUSEâ Unlawful Inducements Interest in Retail Property § 6.32 Indirect interest. Industry member interest in...

  17. 19 CFR 10.1024 - Indirect materials.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... considered originating. Although non-originating material B must undergo the applicable tariff shift in order... 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 10.924 - Indirect materials.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... considered originating. Although non-originating material B must undergo the applicable tariff shift in order... 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....

  19. Indirect techniques in nuclear astrophysics: a review

    NASA Astrophysics Data System (ADS)

    Tribble, R. E.; Bertulani, C. A.; La Cognata, M.; 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.

  20. Indirect Costs of Federally Supported Research.

    ERIC Educational Resources Information Center

    Brown, Kenneth T.

    1981-01-01

    Addressed is the problem of increasing indirect costs in federally supported research at universities and colleges. Effects of this increase are examined, using data on National Institutes of Health grants to educational institutions for examples. Discussed is the establishment of uniform indirect cost rates to modify the present policy. (CS)

  1. 48 CFR 31.203 - Indirect costs.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 1 2012-10-01 2012-10-01 false Indirect costs. 31.203 Section 31.203 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION GENERAL CONTRACTING REQUIREMENTS CONTRACT COST PRINCIPLES AND PROCEDURES Contracts With Commercial Organizations 31.203 Indirect costs. (a) For contracts subject...

  2. 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…

  3. Indirect Costs of University Research: Background Information.

    ERIC Educational Resources Information Center

    Voet, Tony Vander

    This paper is intended to provide a solid base of information about the treatment of indirect university research costs in various jurisdictions and to highlight some of the factors that have contributed to increased interest in the issues surrounding the funding of indirect costs of research. University research in Ontario has continued to evolve…

  4. 7 CFR 2500.044 - Indirect costs.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 15 2012-01-01 2012-01-01 false Indirect costs. 2500.044 Section 2500.044 Agriculture Regulations of the Department of Agriculture (Continued) OFFICE OF ADVOCACY AND OUTREACH, DEPARTMENT OF AGRICULTURE OAO FEDERAL FINANCIAL ASSISTANCE PROGRAMS-GENERAL AWARD ADMINISTRATIVE PROCEDURES Post-Award and Closeout § 2500.044 Indirect...

  5. Indirect Cost Rate Composition and Myths.

    ERIC Educational Resources Information Center

    Selby, Stephen E.

    1984-01-01

    In response to criticism of the rise in indirect cost rates and their effect on federally funded research, the methods for calculating and applying indirect costs rates according to the new cost principles applicable to sponsored agreements are examined, and specific criticisms are addressed. (MSE)

  6. 19 CFR 10.603 - Indirect materials.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY... 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...

  7. 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 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CERTAIN BULK DANGEROUS CARGOES SAFETY STANDARDS FOR SELF-PROPELLED VESSELS CARRYING BULK LIQUEFIED GASES Special Design and Operating Requirements § 154.1720 Indirect refrigeration....

  8. 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 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CERTAIN BULK DANGEROUS CARGOES SAFETY STANDARDS FOR SELF-PROPELLED VESSELS CARRYING BULK LIQUEFIED GASES Special Design and Operating Requirements § 154.1720 Indirect refrigeration....

  9. 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... OF THE TREASURY LIQUORS âTIED-HOUSEâ Unlawful Inducements Interest in Retail License § 6.26 Indirect interest. Industry member interest in retail licenses includes any interest acquired by corporate...

  10. 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... OF THE TREASURY LIQUORS âTIED-HOUSEâ Unlawful Inducements Interest in Retail Property § 6.32 Indirect interest. Industry member interest in retail property includes any interest acquired by corporate...

  11. 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

    Chikeka, Ijeuru; Matute, Armando J; Dumler, J Stephen; Woods, Christopher W; Mayorga, Orlando; Reller, Megan E

    2016-06-01

    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

  12. The Clinical Significance of the Dense Fine Speckled Immunofluorescence Pattern on HEp-2 Cells for the Diagnosis of Systemic Autoimmune Diseases

    PubMed Central

    Mahler, Michael; Fritzler, Marvin J.

    2012-01-01

    Antinuclear antibodies (ANAs) are a serological hallmark in the diagnosis of systemic autoimmune rheumatic diseases (SARD). The indirect immunofluorescence (IIF) assay on HEp-2 cells is a commonly used test for the detection of ANA and has been recently recommended as the screening test of choice by a task force of the American College of Rheumatology. However, up to 20% of apparently healthy individuals (HI) have been reported to have a positive IIF ANA test, primarily related to autoantibodies that target the dense fine speckles 70 (DFS70) antigen. Even more important, the DFS IIF pattern has been reported in up to 33% of ANA positive HI, but not in ANA positive SARD sera. Since the intended use of the ANA HEp-2 test is to aid in the diagnosis and classification of SARD, the detection and reporting of anti-DFS70 antibodies and their associated pattern (DFS) as a positive test significantly reduce the specificity and the positive likelihood of the ANA test. This has significant implications for medical management and diagnostic algorithms involving the detection of ANA. Recently, a novel immunoadsorption method has been developed that specifically blocks anti-DFS70 antibodies and, therefore, significantly increases the specificity of the ANA test for SARD. This immunoadsorption method has the potential to overcome a significant limitation of the ANA HEp-2 assay. The present paper summarizes the current knowledge about anti-DFS70 antibodies and their clinical impact on ANA testing. PMID:23304189

  13. Combined autoradiography and immunofluorescence for estimation of single cell activity by ammonium-oxidizing bacteria

    SciTech Connect

    Ward, B.B.

    1984-03-01

    Immunofluorescence and /sup 14/CO/sub 2/ autoradiography were used for simultaneously enumerating and assaying the autotrophic activity of ammonium-oxidizing bacteria in seawater. Relative activity (/sup 14/CO/sub 2/ assimilation as measured by autoradiography) and abundance were measured in simulated in situ incubations at seven stations in the primary NO/sub 2//sup -/ maximum region of the Northeast Pacific Ocean. More than 10/sup 4/ cells-liter/sup -1/ were present; relative activity often showed a peak near the surface and an increase in the NO/sub 2//sup -/ max region below the photic zone. The method permits assessment of individual cell activity; most cells at all depths were active in CO/sub 2/ assimilation, usually at low and quite variable levels. Relative activity was positively correlated with the abundance of ammonium-oxidizing bacteria, temperature, total dark CO/sub 2/ assimilation and phenopigment concentration.

  14. Skeletal Muscle Tissue Clearing for LacZ and Fluorescent Reporters, and Immunofluorescence Staining.

    PubMed

    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

  15. Reduction in nonfluorescence state of quantum dots on an immunofluorescence staining

    SciTech Connect

    Li-Shishido, Songhua; Watanabe, Tomonobu M.; Tada, Hiroshi; Higuchi, Hideo . E-mail: higuchi@tubero.tohoku.ac.jp; Ohuchi, Noriaki

    2006-12-08

    Fluorescence quantum dots are widely used in immunofluorescence staining because of their intense and stable fluorescence. However, the nonfluorescence state of the quantum dots is their disadvantage. Here, the nonfluorescence state of the dots labeled to cells and tissues was suppressed. Cells and tissues where the receptor HER2 had been overexpressed were fixed and then labeled with anti-HER2 crosslinked with the dots. The intensity of the dots increased with the illumination time. The majority of the single dots were in the nonfluorescence state at beginning of the illumination period and the number of fluorescence dots observed increased with the illumination time. Living cells were also labeled with the anti-HER2-Qdots. Blinking and bleaching of the Qdots was effectively suppressed by adding {beta}-mercaptoethanol and glutathione. Therefore, the movement of the Qdots bound to cell membrane could be observed for long periods of time.

  16. 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. PMID:21370034

  17. Immunofluorescence Tomography of Mouse Ocular Surface Epithelial Stem Cells and Their Niche Microenvironment

    PubMed Central

    Parfitt, Geraint J.; Kavianpour, Behdad; Wu, Karen L.; Xie, Yilu; Brown, Donald J.; Jester, James V.

    2015-01-01

    Purpose Currently, there are no definitive immunomarkers for epithelial stem cells (corneal and conjunctival) or their poorly understood niche microenvironment. The H2B-GFP/K5tTA mouse enables visualization of label-retaining cells (LRCs), which exhibit the functional marker of stem cell quiescence. We used immunofluorescence tomography to evaluate putative stem cell markers and LRCs of the mouse ocular surface. Methods H2B-GFP/K5tTA mice were pulsed for 56 days and then chased with doxycycline to label LRCs. Limbus and eyelid tissue was 3-dimensionally (3-D) reconstructed using immunofluorescence tomography to identify and characterize LRCs using the putative stem cell markers sox9, keratin 19, lrig1, blimp1, and abcb5. Results After 28 days of chase, LRCs were localized to the entire limbus epithelium and, infrequently, the anterior limbal stroma. Label-retaining cells comprised 3% of limbal epithelial cells after 56 days of chase. Conjunctival LRCs were localized to the fornix and comprised 4% of the total fornix epithelial cells. No stem cell immunomarker was specific for ocular surface LRCs; however, blimp1 enriched for limbal basal epithelial cells and 100% of green fluorescent protein-positive (GFP+) cells at the limbus and fornix were found to be lrig1-positive. Conclusions Label-retaining cells represent a larger population of the mouse limbus than previously thought. They decrease in number with increased doxycycline chase, suggesting that LRC populations with different cell cycle lengths exist at the limbus. We conclude that current immunomarkers are unable to colocalize with the functional marker of epithelial stem cell quiescence; however, blimp1 may enrich for limbal epithelial basal cells. PMID:26559480

  18. 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. PMID:25789548

  19. Double immunofluorescence shows coexpression of Bcl-x with GFAP in a variety of glial lesions.

    PubMed

    Tan, Kong-Bing; Magdalene Koh, Hui-Keng; Tan, Soo-Yong

    2006-12-01

    Bcl-x is an important member of the bcl-2 family of proteins that has been shown to be expressed by both native nervous system tissue and several nervous system tumors. Its anti-apoptotic activity is believed to contribute to nervous system tumorigenesis. We seek to compare the staining characteristics of Bcl-x and GFAP in various neuronal and glial lesions, both neoplastic and non-neoplastic. We also use a double immunofluorescence technique to assess for coexpression of Bcl-x and GFAP by the same lesional cells. Forty cases of brain tumors and reactive brain conditions were reviewed. The former included astrocytomas, GBMs, ependymomas, oligodendrogliomas, gangliogliomas, subependymomas and neurocytomas. The latter included cases of gliosis, cerebritis and mesial temporal sclerosis. Immunohistochemistry for Bcl-x and GFAP was performed. Double immunofluorescent labeling using antibodies to both GFAP and Bcl-x was also carried out. Expression of Bcl-x closely follows that of GFAP with strong expression in both reactive astrocytes and astrocytomas. There is more focal expression in other gliomas. Immunostaining for Bcl-x is generally more intense and distinct, compared to that for GFAP. Expression of both GFAP and Bcl-x is more focal in oligodendrogliomas, with staining of mainly intervening astrocytic processes. Double immunolabelling confirms the coexpression of Bcl-x and GFAP in various gliomas and reactive brain conditions. As immunostaining for Bcl-x is generally more distinct and intense than that for GFAP, it may serve as a useful alternative to help highlight glial cells in selected diagnostic settings. PMID:16773221

  20. Evolution of spite through indirect reciprocity.

    PubMed Central

    Johnstone, Rufus A.; Bshary, Redouan

    2004-01-01

    How can cooperation persist in the face of a temptation to 'cheat'? Several recent papers have suggested that the answer may lie in indirect reciprocity. Altruistic individuals may benefit by eliciting altruism from observers, rather than (as in direct reciprocity) from the recipient of the aid they provide. Here, we point out that indirect reciprocity need not always favour cooperation; by contrast, it may support spiteful behaviour, which is costly for the both actor and recipient. Existing theory suggests spite is unlikely to persist, but we demonstrate that it may do so when spiteful individuals are less likely to incur aggression from observers (a negative form of indirect reciprocity). PMID:15347514

  1. 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)

  2. 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

  3. 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... general practice in the insurance industry....

  4. NIH Seeks Reduction in "Indirect Costs."

    ERIC Educational Resources Information Center

    Culliton, Barbara J.

    1983-01-01

    The National Institute of Health (NIH) is currently seeking a reduction in indirect costs associated with the awarding of research funds. Various issues related to these costs, which are causing tension between university administrators and academic researchers, are discussed. (JN)

  5. Indirect Costs of Federally Financed Projects

    ERIC Educational Resources Information Center

    Simmons, William

    1970-01-01

    Describes how indirect costs, which have been incurred by a school district or an educational agency to support Federally financed projects, may be reimbursed in accordance with 1969 amendments to the ESEA. (JF)

  6. 19 CFR 10.3024 - Indirect materials.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...-originating rubber gloves for use by workers in the production of good C. Good C is subject to a tariff shift... to be considered originating, the rubber gloves do not because they are indirect materials and...

  7. 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...

  8. 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...

  9. 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...

  10. 40 CFR 35.940-4 - Indirect costs.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Indirect costs. Indirect costs shall be allowable in accordance with an indirect cost agreement negotiated... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Indirect costs. 35.940-4 Section 35.940... the same as the actual indirect costs....

  11. 40 CFR 35.940-4 - Indirect costs.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Indirect costs. Indirect costs shall be allowable in accordance with an indirect cost agreement negotiated... 40 Protection of Environment 1 2014-07-01 2014-07-01 false Indirect costs. 35.940-4 Section 35.940... the same as the actual indirect costs....

  12. 40 CFR 35.940-4 - Indirect costs.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Indirect costs. Indirect costs shall be allowable in accordance with an indirect cost agreement negotiated... 40 Protection of Environment 1 2011-07-01 2011-07-01 false Indirect costs. 35.940-4 Section 35.940... the same as the actual indirect costs....

  13. 34 CFR 76.560 - General indirect cost rates; exceptions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... organizations, at 34 CFR 80.22. (b) A grantee must have a current indirect cost rate agreement to charge indirect costs to a grant. To obtain an indirect cost rate, a grantee must submit an indirect cost proposal... rates; exceptions. (a) The differences between direct and indirect costs and the principles...

  14. 34 CFR 76.560 - General indirect cost rates; exceptions.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... organizations, at 34 CFR 80.22. (b) A grantee must have a current indirect cost rate agreement to charge indirect costs to a grant. To obtain an indirect cost rate, a grantee must submit an indirect cost proposal... rates; exceptions. (a) The differences between direct and indirect costs and the principles...

  15. 40 CFR 35.940-4 - Indirect costs.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Indirect costs. Indirect costs shall be allowable in accordance with an indirect cost agreement negotiated... 40 Protection of Environment 1 2012-07-01 2012-07-01 false Indirect costs. 35.940-4 Section 35.940... the same as the actual indirect costs....

  16. 48 CFR 2452.242-70 - Indirect costs.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Indirect costs. As prescribed in 2442.705-70, insert the following clause in cost-reimbursement type... provisional indirect cost rates pending establishment of final indirect cost rates. Indirect Costs (APR 1984... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Indirect costs....

  17. 40 CFR 35.940-4 - Indirect costs.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Indirect costs. Indirect costs shall be allowable in accordance with an indirect cost agreement negotiated... 40 Protection of Environment 1 2013-07-01 2013-07-01 false Indirect costs. 35.940-4 Section 35.940... the same as the actual indirect costs....

  18. 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...

  19. 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. PMID:25313189

  20. 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.

  1. 48 CFR 242.705 - Final indirect cost rates.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 3 2011-10-01 2011-10-01 false Final indirect cost rates..., DEPARTMENT OF DEFENSE CONTRACT MANAGEMENT CONTRACT ADMINISTRATION AND AUDIT SERVICES Indirect Cost Rates 242.705 Final indirect cost rates....

  2. 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

    2015-10-01

    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. PMID:26339978

  3. Characterization of the bone marrow immunofluorescence test in childhood autoimmune neutropenia.

    PubMed

    Lane, S W; Hassell, P; Kennedy, G A; Fung, Y L; Williams, B A

    2009-10-01

    The bone marrow immunofluorescenece test (BMIFT) demonstrates autoantibodies to granulocytes and their precursors on fresh-frozen bone marrow slides. It may be used to differentiate childhood autoimmune neutropenia (AIN) from other causes of childhood neutropenia, even when circulating neutrophil counts are low. We sought to characterize the diagnostic utility of the BMIFT in childhood AIN. All BMIFT requests for investigation of children with neutropenia between January 1998 and May 2007 were reviewed. Patients were classified as AIN or nonautoimmune causes. Baseline demographic data, results of BMIFT, granulocyte immunofluorescence testing and bone marrow findings were collected from clinical records and the institutional laboratory database. Seventy-six children had BMIFT performed for investigation of neutropenia. There were 45 patients diagnosed with AIN, 28 with nonimmune neutropenia and three failed tests. The median age of children with AIN was 1.2 years (range 0.3-15.3), compared with 3.6 years (range 0.1-15.7) in the nonautoimmune group. The median neutrophil count in AIN was 0.3 x 10(9)/l (0.9 x 10(9)/l in nonautoimmune). BMIFT was positive in 24 of 45 patients with AIN and 0 of 28 with nonautoimmune neutropenia (sensitivity 53%, specificity 100%, positive predictive value (PPV) 100%, negative predictive value 57%). Ten patients had other autoimmune diatheses at diagnosis. The BMIFT is a simple, highly specific test with excellent PPV and thus is a clinically useful test to confirm AIN in children. PMID:18637806

  4. 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

  5. Optimization of spermatozoa detection using immunofluorescent staining and laser micro-dissection.

    PubMed

    Ping, Yueh Shyang; Chan, Xavier Liang Shun; Goh, Sze Kae; Syn, Christopher Kiu Choong

    2015-10-01

    The present study evaluated the use of an immunofluorescence-based assay for the microscopic detection of human spermatozoa, following which the fluorescence-labelled spermatozoa could be excised with a laser micro-dissection system. The Sperm Hy-Liter™ PI kit was able to detect spermatozoa from as little as 20nL of semen. No interference or non-specificity were observed when the kit was used on semen mixed with various body fluids such as blood and urine, as well as when semen was spiked onto different types of fabric. Good results could also be obtained with rectal samples which contain auto-fluorescent fecal materials through the use of dual FITC/PI filters. We also developed a method for concurrent testing of two protein biomarkers of semen (semenogelin and prostate-specific antigen) and detection of spermatozoa. This approach would maximize the evidential value from a single piece of sexual assault exhibit. The results also showed that staining by Sperm Hy-Liter™ PI does not interfere with DNA recovery, facilitating the generation of clear male DNA profiles from dissected spermatozoa, thereby making profile interpretation less complex. In summary, Sperm Hy-Liter™ PI staining was demonstrated to be sensitive, robust and specific. PMID:26338669

  6. Immunofluorescence detection of the cytoskeleton and extracellular matrix in tissue and cultured cells.

    PubMed

    Smith-Clerc, Josiane; Hinz, Boris

    2010-01-01

    "A picture is worth a thousand words" goes the proverb. A poor picture however can be worse than saying nothing at all. This is particularly true for immunofluorescence pictures that in addition to their informative character bear an esthetic component. We here provide a panel of straightforward methods to process tissue sections and cultured cells for immunostaining of cytoskeletal elements, primarily those associated with actin filaments. We want to emphasize to the reader the fact that the choice of the processing method will have an important influence on the outcome of the immunostaining and thus on the interpretation of the results. Fixation of cultured cells with cross-linking reagents such as paraformaldehyde efficiently preserves structural elements at the expense of reduced antigenicity. The degree and timing of cell permeabilization with detergents, along with chemical cross-linking, contributes to the clarity and resolution of distinct structures but can also lead to loss of information. Fixation with organic solvents like methanol will, in most cases, better preserve antigens but will produce a higher background and impact on structural integrity. Therefore, it is recommended to test different protocols for a "new" protein or epitope - the results will pay back your investment. PMID:19960321

  7. Combined autoradiography and immunofluorescence for estimation of single cell activity by ammonium-oxidizing bacteria

    SciTech Connect

    Ward, B.B.

    1984-03-01

    Immunofluorescence and /sup 14/CO/sub 2/ autoradiography were used for simultaneously enumerating and assaying the autotrophic activity of ammonium-oxidizing bacteria in seawater. Relative activity (/sup 14/CO/sub 2/ assimilation as measured by autoradiography) and abundance were measured in simulated in situ incubations at seven stations in the primary NO/sub 2//sup -/ maximum region of the Northeast Pacific Ocean. More than 10/sup 4/ cells liter/sup -1/ were present; relative activity often showed a peak near the surface and an increase in the NO/sub 2//sup -/ max region below the photic zone. The method permits assessment of individual cell activity; most cells at all depths were active in CO/sub 2/ assimilation, usually at low and quite variable levels. There were no differences in relative activity between samples incubated under simulated in situ conditions and in the dark. Relative activity was positively correlated with the abundance of ammonium-oxidizing bacteria, temperature, total dark CO/sub 2/ assimilation (as measured by liquid scintillation counting of replicate samples), and pheopigment concentration, and negatively correlated with oxygen concentration.

  8. Sensitive Immunofluorescent Staining of Cells via Generation of Fluorescent Nanoscale Polymer Films in Response to Biorecognition

    PubMed Central

    Avens, Heather J.; Berron, Brad J.; May, Allison M.; Voigt, Katerina R.; Seedorf, Gregory J.; Balasubramaniam, Vivek; Bowman, Christopher N.

    2011-01-01

    Immunofluorescent staining is central to nearly all cell-based research, yet only a few fluorescent signal amplification approaches for cell staining exist, each with distinct limitations. Here, the authors present a novel, fluorescent polymerization-based amplification (FPBA) method that is shown to enable similar signal intensities as the highly sensitive, enzyme-based tyramide signal amplification (TSA) approach. Being non-enzymatic, FPBA is not expected to suffer from nonspecific staining of endogenous enzymes, as occurs with enzyme-based approaches. FPBA employs probes labeled with photopolymerization initiators, which lead to the controlled formation of fluorescent polymer films only at targeted biorecognition sites. Nuclear pore complex proteins (NPCs; in membranes), vimentin (in filaments), and von Willebrand factor (in granules) were all successfully immunostained by FPBA. Also, FPBA was demonstrated to be capable of multicolor immunostaining of multiple antigens. To assess relative sensitivity, decreasing concentrations of anti-NPC antibody were used, indicating that both FPBA and TSA stained NPC down to a 1:100,000 dilution. Nonspecific, cytoplasmic signal resulting from NPC staining was found to be reduced up to 5.5-fold in FPBA as compared to TSA, demonstrating better signal localization with FPBA. FPBA’s unique approach affords a combination of preferred attributes, including high sensitivity and specificity not otherwise available with current techniques. PMID:21339175

  9. Immunofluorescence detection and localization of B[a]P and TCDD in earthworm tissues.

    PubMed

    Sforzini, Susanna; Moore, Michael N; Boeri, Marta; Benfenati, Emilio; Colombo, Andrea; Viarengo, Aldo

    2014-07-01

    An immunohistochemical method using antibodies against polycyclic aromatic hydrocarbons (PAHs) and dioxins was developed on frozen tissue sections of the earthworm Eisenia andrei exposed to environmentally relevant concentrations of benzo[a]pyrene (B[a]P) (0.1, 10, 50 ppm) and 2,3,7,8-tetrachloro-dibenzo-para-dioxin (TCDD) (0.01, 0.1, 2 ppb) in spiked standard soils. The concentrations of B[a]P and TCDD in E. andrei exposed to the same conditions were also measured using analytical chemical procedures. The results demonstrated that tissues of worms exposed to even minimal amount of B[a]P and TCDD reacted positively and specifically to anti-PAHs and -dioxins antibody. Immunofluorescence revealed a much more intense staining for the gut compared to the body wall; moreover, positively immunoreactive amoeboid coelomocytes were also observed, i.e. cells in which we have previously demonstrated the occurrence of genotoxic damage. The double immunolabelling with antibodies against B[a]P/TCDD and the lysosomal enzyme cathepsin D demonstrated the lysosomal accumulation of the organic xenobiotic compounds, in particular in the cells of the chloragogenous tissue as well as in coelomocytes, involved into detoxification and protection of animals against toxic chemicals. The method described is timesaving, not expensive and easily applicable. PMID:24412505

  10. An Immunofluorescent Method for Characterization of Barrett’s Esophagus Cells

    PubMed Central

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

    2014-01-01

    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. PMID:25079877

  11. 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.

  12. 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. PMID:21462312

  13. [Direct immunofluorescence assay performance in diagnosis of the Influenza A(H1N1) virus].

    PubMed

    Pianciola, Luis; González, Gladys; Mazzeo, Melina; Navello, Mariano; Quidel, Natalia; Bulgheroni, María Fernanda

    2010-06-01

    By 25 April 2009, less than one month after the first human with Influenza A(H1N1) virus was detected in Mexico, the disease had already spread to more than 40 countries, with over 10,000 cases reported. Due to its unpredictability, this type of virus requires appropriate, reliable, and safe diagnostic methods that are also accessible to clinical laboratories. Through the analysis of 291 samples taken from patients with suspected Influenza A(H1N1) virus infection in Neuquén, Argentina, this study compares the two diagnostic methods used simultaneously: direct immunofluorescence assay (DFA) and real-time polymerase chain reaction (RT-PCR). DFA had a sensitivity of 44.4%, a specificity of 99.6%, a positive predictive value of 95.2%, and a negative predictive value of 90.7%. Positive results obtained with this method can be considered true positives. A negative result does not rule out the presence of the virus. In this case, the sample should be examined by RT-PCR. Out of a total of 291 samples, there were 45 positive results with RT-PCR and 21 positive results with DFA. PMID:20721445

  14. Studies on Bovine Virus Diarrhea: Serum Neutralization, Complement-fixation and Immunofluorescence

    PubMed Central

    Ruckerbauer, Gerda M.; Girard, A.; Bannister, G. L.; Boulanger, P.

    1971-01-01

    The complement-fixation, the serum neutralization tests and the fluorescent-antibody technique were the serological methods applied in this laboratory for the detection of antigens for bovine virus diarrhea (BVD). As observed previously, the modified direct complement-fixation (MDCF) test was required to demonstrate antibodies against virus infections of cattle. At a certain stage of infection, the MDCF test was found to be as accurate and less time-consuming than the serum neutralization test for the detection of antibodies in bovine sera. The modified direct complement-fixing antibodies were detectable in the serum from approximately three weeks up to a few months after infection as compared to several years for the serum neutralization test. Thus, as in most other viral diseases, the MDCF test was of value for detecting recent infections while the serum neutralization test detects both recent and long-standing infections. The fluorescent antibody technique was of value to detect viral antigens of both cytopathogenic and noncytopathogenic strains of BVD in primary fetal kidney cell cultures inoculated with field specimens. In addition, the virus was detected in six of 220 fetuses collected at a local slaughter house for the preparation of primary cell cultures. The length of time required for the detection and identification of specific viral antigens by immunofluorescence was considerably reduced over that of the serum neutralization and virus interference tests. ImagesFig. 3. PMID:4254898

  15. 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.

  16. 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

  17. Procedures for the quantification of whole-tissue immunofluorescence images obtained at single-cell resolution during murine tubular organ development.

    PubMed

    Hirashima, Tsuyoshi; Adachi, Taiji

    2015-01-01

    Whole-tissue quantification at single-cell resolution has become an inevitable approach for further quantitative understanding of morphogenesis in organ development. The feasibility of the approach has been dramatically increased by recent technological improvements in optical tissue clearing and microscopy. However, the series of procedures required for this approach to lead to successful whole-tissue quantification is far from developed. To provide the appropriate procedure, we here show tips for each critical step of the entire process, including fixation for immunofluorescence, optical clearing, and digital image processing, using developing murine internal organs such as epididymis, kidney, and lung as an example. Through comparison of fixative solutions and of clearing methods, we found optimal conditions to achieve clearer deep-tissue imaging of specific immunolabeled targets and explain what methods result in vivid volume imaging. In addition, we demonstrated that three-dimensional digital image processing after optical clearing produces objective quantitative data for the whole-tissue analysis, focusing on the spatial distribution of mitotic cells in the epididymal tubule. The procedure for the whole-tissue quantification shown in this article should contribute to systematic measurements of cellular processes in developing organs, accelerating the further understanding of morphogenesis at the single cell level. PMID:26258587

  18. Membrane specific mapping and colocalization of malarial and host skeletal proteins in the Plasmodium falciparum infected erythrocyte by dual-color near-field scanning optical microscopy.

    PubMed

    Enderle, T; Ha, T; Ogletree, D F; Chemla, D S; Magowan, C; Weiss, S

    1997-01-21

    Accurate localization of proteins within the substructure of cells and cellular organelles enables better understanding of structure-function relationships, including elucidation of protein-protein interactions. We describe the use of a near-field scanning optical microscope (NSOM) to simultaneously map and detect colocalized proteins within a cell, with superresolution. The system we elected to study was that of human red blood cells invaded by the human malaria parasite Plasmodium falciparum. During intraerythrocytic growth, the parasite expresses proteins that are transported to the erythrocyte cell membrane. Association of parasite proteins with host skeletal proteins leads to modification of the erythrocyte membrane. We report on colocalization studies of parasite proteins with an erythrocyte skeletal protein. Host and parasite proteins were selectively labeled in indirect immunofluorescence antibody assays. Simultaneous dual-color excitation and detection with NSOM provided fluorescence maps together with topography of the cell membrane with subwavelength (100 nm) resolution. Colocalization studies with laser scanning confocal microscopy provided lower resolution (310 nm) fluorescence maps of cross sections through the cell. Because the two excitation colors shared the exact same near-field aperture, the two fluorescence images were acquired in perfect, pixel-by-pixel registry, free from chromatic aberrations, which contaminate laser scanning confocal microscopy measurements. Colocalization studies of the protein pairs of mature parasite-infected erythrocyte surface antigen (MESA) (parasite)/protein4.1(host) and P. falciparum histidine rich protein (PfHRP1) (parasite)/protein4.1(host) showed good real-space correlation for the MESA/protein4.1 pair, but relatively poor correlation for the PfHRP1/protein4.1 pair. These data imply that NSOM provides high resolution information on in situ interactions between proteins in biological membranes. This method of

  19. 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

  20. 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.

  1. A universal scheme for indirect quantum control

    NASA Astrophysics Data System (ADS)

    Layden, David; Martin-Martinez, Eduardo; Kempf, Achim

    The goal of indirect quantum control is to coherently steer a quantum system solely by acting on a quantum actuator to which it is coupled. This approach to quantum control is convenient in many physical settings, as it allows one to avoid direct addressing of the system--and any associated difficulties--altogether. While it is known in principle that control of the actuator typically yields universal control of the system, the practical details of how such indirect control can be achieved are less clear. This deficiency has led to a number of implementation- and model-specific indirect control schemes, in lieu of a general recipe applicable to any physical setting. Here, we present such a recipe, in the form of an open-loop control scheme which implements arbitrary unitary operations on the system by exploiting open dynamics in the actuator. arXiv:1506.06749.

  2. Indirect laminate veneer: a conservative novel approach.

    PubMed

    Prajapati, Paranjay; Sethuraman, Rajesh; Naveen, Y G; Patel, Jayanti R

    2013-01-01

    Various treatment options and materials are available for restoration of an endodontically treated tooth. Laminate veneer is conservative treatment usually employed for aesthetic correction or improvement. The indirect composite is available in a wide range of shades and specific characterisation is easily performed chair side in the operatory area, which makes it a quick procedure and time saving for both the patient and the dentist. The physical properties and optical properties are good enough to use it as indirect restorative material, so in this particular case it was the material of choice for fabrication of laminate veneer on anterior tooth. In this case, the endodontically treated tooth with a fractured incisal edge was restored with indirect composite material. PMID:23975914

  3. Advances in Urine Microscopy.

    PubMed

    Becker, Gavin J; Garigali, Giuseppe; Fogazzi, Giovanni B

    2016-06-01

    Urine microscopy is an important tool for the diagnosis and management of several conditions affecting the kidneys and urinary tract. In this review, we describe the automated instruments, based either on flow cytometry or digitized microscopy, that are currently in use in large clinical laboratories. These tools allow the examination of large numbers of samples in short periods. We also discuss manual urinary microscopy commonly performed by nephrologists, which we encourage. After discussing the advantages of phase contrast microscopy over bright field microscopy, we describe the advancements of urine microscopy in various clinical conditions. These include persistent isolated microscopic hematuria (which can be classified as glomerular or nonglomerular on the basis of urinary erythrocyte morphology), drug- and toxin-related cystalluria (which can be a clue for the diagnosis of acute kidney injury associated with intrarenal crystal precipitation), and some inherited conditions (eg, adenine phosphoribosyltransferase deficiency, which is associated with 2,8-dihydroxyadenine crystalluria, and Fabry disease, which is characterized by unique urinary lamellated fatty particles). Finally, we describe the utility of identifying "decoy cells" and atypical malignant cells, which can be easily done with phase contrast microscopy in unfixed samples. PMID:26806004

  4. 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

  5. L'Interrogation Indirecte (Indirect Interrogation). Montreal Working Papers in Linguistics, Vol. 4.

    ERIC Educational Resources Information Center

    Nieger, Monique; Paradis, Monique

    This study is divided into two sections: the first examines Standard French indirect interrogation, noting several distinct verb classes which are discussed in terms of permutations of WH-words, reduction, multiple WH-words, cleavage, semantic compatibility, and the "que-" completive; the second part focuses on indirect interrogation and relatives…

  6. 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.

  7. 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. PMID:19368959

  8. 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.

  9. Simplified CLARITY for visualizing immunofluorescence labeling in the developing rat brain.

    PubMed

    Zheng, Huiyuan; Rinaman, Linda

    2016-05-01

    CLARITY is an innovative technological advance in which intact biological tissue is transformed into a "nanoporous hydrogel-hybridized form" (Chung et al. 2013; Chung and Deisseroth 2013) with markedly improved chemical and optical accessibility, permitting fluorescent visualization and extraction of high-resolution structural data from mm-thick blocks of tissue. CLARITY affords an excellent but as yet unexploited opportunity to visualize the growth and maturation of phenotypically identified neurons and axonal processes in the developing brain. This brief report describes a moderately revised, simplified, and less expensive CLARITY protocol that effectively reveals the structure of chemically identified neurons in whole neonatal/juvenile rat brains and tissue slabs. Rats [postnatal day (P)0-24] were transcardially perfused with one of two fixative/hydrogel solutions, followed by hydrogel polymerization to generate brain hybrids. Whole brain hybrids or 2.0-mm-thick coronal slabs were passively cleared of lipid and then processed for dual immunofluorescence labeling, including labeling using tyramide signal amplification. After refractive index matching using 2,20-Thiodiethanol (60 % solution), a Leica confocal microscope equipped with a CLARITY objective was used to view the hypothalamus in whole brain hybrids or slabs. Collected image stacks revealed the distribution and three-dimensional structure of hypothalamic pro-oxyphysin (oxytocin)-, neuropeptide Y-, glucagon-like peptide-1-, and tyrosine hydroxylase-immunopositive neurons and processes within large tissue volumes. Outstanding structural preservation and immunolabeling quality demonstrates the efficacy of this approach for interrogating chemically defined neural circuits as they develop in postnatal rodent brain. PMID:25772507

  10. 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

  11. 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.

  12. Ultrafast scanning probe microscopy

    SciTech Connect

    Botkin, D.; Weiss, S.; Ogletree, D.F.; Salmeron, M.; Chemla, D.S.

    1994-01-01

    The authors have developed a general technique which combines the temporal resolution of ultrafast laser spectroscopy with the spatial resolution of scanned probe microscopy (SPM). Using this technique with scanning tunneling microscopy (STM), they have obtained simultaneous 2 ps time resolution and 50 {angstrom} spatial resolution. This improves the time resolution currently attainable with STM by nine orders of magnitude. The potential of this powerful technique for studying ultrafast dynamical phenomena on surfaces with atomic resolution is discussed.

  13. 48 CFR 342.705 - Final indirect cost rates.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Final indirect cost rates... MANAGEMENT CONTRACT ADMINISTRATION Indirect Cost Rates 342.705 Final indirect cost rates. (a) The Division of Cost Allocation, PSC, shall establish indirect cost rates, research patient care rates, and,...

  14. 2 CFR 200.19 - Cognizant agency for indirect costs.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 2 Grants and Agreements 1 2014-01-01 2014-01-01 false Cognizant agency for indirect costs. 200.19... for indirect costs. Cognizant agency for indirect costs means the Federal agency responsible for reviewing, negotiating, and approving cost allocation plans or indirect cost proposals developed under...

  15. 48 CFR 52.216-15 - Predetermined Indirect Cost Rates.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... indirect costs shall be obtained by applying final indirect cost rates established in accordance with the Allowable Cost and Payment clause. (g) Allowable indirect costs for the period from the beginning of... clause of this contract, the allowable indirect costs under this contract shall be obtained by...

  16. 48 CFR 842.705 - Final indirect cost rates.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... settlement of indirect costs for a specific contract. ... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false Final indirect cost rates... MANAGEMENT CONTRACT ADMINISTRATION AND AUDIT SERVICES Indirect Cost Rates 842.705 Final indirect cost...

  17. 48 CFR 52.216-15 - Predetermined Indirect Cost Rates.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... indirect costs shall be obtained by applying final indirect cost rates established in accordance with the Allowable Cost and Payment clause. (g) Allowable indirect costs for the period from the beginning of... clause of this contract, the allowable indirect costs under this contract shall be obtained by...

  18. 48 CFR 1542.705 - Final indirect cost rates.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 6 2011-10-01 2011-10-01 false Final indirect cost rates... CONTRACT MANAGEMENT CONTRACT ADMINISTRATION Indirect Cost Rates 1542.705 Final indirect cost rates. (a) The... shall be required to negotiate final indirect cost rates. (b) Contracting officers shall insert...

  19. 48 CFR 3442.705 - Final indirect cost rates.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false Final indirect cost rates... REGULATION CONTRACT MANAGEMENT CONTRACT ADMINISTRATION Indirect Cost Rates 3442.705 Final indirect cost rates... establish final indirect cost rates under FAR 42.705-1 and 42.705-2....

  20. 48 CFR 842.705 - Final indirect cost rates.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... settlement of indirect costs for a specific contract. ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false Final indirect cost rates... MANAGEMENT CONTRACT ADMINISTRATION AND AUDIT SERVICES Indirect Cost Rates 842.705 Final indirect cost...

  1. 48 CFR 842.705 - Final indirect cost rates.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... settlement of indirect costs for a specific contract. ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false Final indirect cost rates... MANAGEMENT CONTRACT ADMINISTRATION AND AUDIT SERVICES Indirect Cost Rates 842.705 Final indirect cost...

  2. 48 CFR 42.705 - Final indirect cost rates.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 1 2013-10-01 2013-10-01 false Final indirect cost rates... MANAGEMENT CONTRACT ADMINISTRATION AND AUDIT SERVICES Indirect Cost Rates 42.705 Final indirect cost rates. (a) Final indirect cost rates shall be established on the basis of— (1) Contracting...

  3. 48 CFR 52.216-15 - Predetermined Indirect Cost Rates.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... indirect costs shall be obtained by applying final indirect cost rates established in accordance with the Allowable Cost and Payment clause. (g) Allowable indirect costs for the period from the beginning of... clause of this contract, the allowable indirect costs under this contract shall be obtained by...

  4. 48 CFR 42.705 - Final indirect cost rates.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 1 2011-10-01 2011-10-01 false Final indirect cost rates... MANAGEMENT CONTRACT ADMINISTRATION AND AUDIT SERVICES Indirect Cost Rates 42.705 Final indirect cost rates. (a) Final indirect cost rates shall be established on the basis of— (1) Contracting...

  5. 48 CFR 842.705 - Final indirect cost rates.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... settlement of indirect costs for a specific contract. ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Final indirect cost rates... MANAGEMENT CONTRACT ADMINISTRATION AND AUDIT SERVICES Indirect Cost Rates 842.705 Final indirect cost...

  6. 48 CFR 1542.705 - Final indirect cost rates.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 6 2012-10-01 2012-10-01 false Final indirect cost rates... CONTRACT MANAGEMENT CONTRACT ADMINISTRATION Indirect Cost Rates 1542.705 Final indirect cost rates. (a) The... shall be required to negotiate final indirect cost rates. (b) Contracting officers shall insert...

  7. 2 CFR 200.414 - Indirect (F&A) costs.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... indirect costs. If an extension is granted the non-Federal entity may not request a rate review until the... REQUIREMENTS FOR FEDERAL AWARDS Cost Principles Direct and Indirect (f&a) Costs § 200.414 Indirect (F&A) costs... the types of cost which may be classified as indirect (F&A) cost in all situations....

  8. Astrophysical Reaction Rates Obtained By Indirect Techniques

    SciTech Connect

    Tribble, R. E.; Al-Abdullah, T.; Alharbi, A.; Banu, A.; Chen, X.; Clark, H. L.; Fu, C.; Gagliardi, C. A.; Hardy, J. C.; Iacob, V. E.; Lui, Y.-W.; McCleskey, M.; Mukhamedzhanov, A.; Nica, N.; Park, H. I.; Roeder, B.; Simmons, E.; Tabacaru, G.; Tokimoto, Y.; Trache, L.

    2010-08-12

    Indirect techniques have been used to obtain information about reaction rates for several proton capture reactions that occur on short-lived nuclei. The techniques used to carry out the measurements are reviewed and the results obtained are presented. Also future prospects for further measurements with a new facility, T-REX are discussed.

  9. 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...

  10. 48 CFR 31.203 - Indirect costs.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... coverage, the contractor shall follow the criteria and guidance in 48 CFR 9904.406 for selecting the cost... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Indirect costs. 31.203... REQUIREMENTS CONTRACT COST PRINCIPLES AND PROCEDURES Contracts With Commercial Organizations 31.203...

  11. 48 CFR 2131.203 - Indirect costs.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 6 2011-10-01 2011-10-01 false Indirect costs. 2131.203 Section 2131.203 Federal Acquisition Regulations System OFFICE OF PERSONNEL MANAGEMENT, FEDERAL EMPLOYEES GROUP LIFE INSURANCE FEDERAL ACQUISITION REGULATION GENERAL CONTRACTING REQUIREMENTS CONTRACT...

  12. 48 CFR 1631.203 - Indirect costs.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 6 2011-10-01 2011-10-01 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...

  13. 48 CFR 2131.203 - 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. 2131.203 Section 2131.203 Federal Acquisition Regulations System OFFICE OF PERSONNEL MANAGEMENT, FEDERAL EMPLOYEES GROUP LIFE INSURANCE FEDERAL ACQUISITION REGULATION GENERAL CONTRACTING REQUIREMENTS CONTRACT...

  14. Indirect Costs: Daily Bread, Cake, or Cracker.

    ERIC Educational Resources Information Center

    Lucas, Robert A.

    1988-01-01

    The tradition of negotiating indirect costs in grants should be abandoned, and research administrators should instead offer different levels of service depending on what the sponsor wants to spend. Three levels of overhead rate are suggested (super, regular, and economy) and their corresponding levels of service are defined. (MSE)

  15. 24 CFR 576.109 - Indirect costs.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 24 Housing and Urban Development 3 2012-04-01 2012-04-01 false Indirect costs. 576.109 Section 576.109 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF ASSISTANT SECRETARY FOR COMMUNITY PLANNING AND DEVELOPMENT, DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT COMMUNITY FACILITIES...

  16. 7 CFR 3430.54 - Indirect costs.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 15 2012-01-01 2012-01-01 false Indirect costs. 3430.54 Section 3430.54 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND AGRICULTURE COMPETITIVE AND NONCOMPETITIVE NON-FORMULA FEDERAL ASSISTANCE PROGRAMS-GENERAL AWARD ADMINISTRATIVE PROVISIONS Post-Award and Closeout §...

  17. Teaching Indirect Speech: Deixis Points the Way.

    ERIC Educational Resources Information Center

    Harman, Ian P.

    1990-01-01

    Suggests an alternative approach to the teaching of indirect or reported speech. Deixis is proposed as a means of clarifying the anomalies of reported speech. The problem is assessed from a grammatical and semantic point of view in the reporting of statements (as opposed to the reporting of questions or commands). (GLR)

  18. 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...

  19. 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...

  20. 48 CFR 2131.203 - 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. 2131.203 Section 2131.203 Federal Acquisition Regulations System OFFICE OF PERSONNEL MANAGEMENT, FEDERAL EMPLOYEES GROUP LIFE INSURANCE FEDERAL ACQUISITION REGULATION GENERAL CONTRACTING REQUIREMENTS CONTRACT...

  1. 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...

  2. 48 CFR 2131.203 - 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. 2131.203 Section 2131.203 Federal Acquisition Regulations System OFFICE OF PERSONNEL MANAGEMENT, FEDERAL EMPLOYEES GROUP LIFE INSURANCE FEDERAL ACQUISITION REGULATION GENERAL CONTRACTING REQUIREMENTS CONTRACT...

  3. 48 CFR 2131.203 - 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. 2131.203 Section 2131.203 Federal Acquisition Regulations System OFFICE OF PERSONNEL MANAGEMENT, FEDERAL EMPLOYEES GROUP LIFE INSURANCE FEDERAL ACQUISITION REGULATION GENERAL CONTRACTING REQUIREMENTS CONTRACT...

  4. Model Intercomparison of Indirect Aerosol Effects

    NASA Technical Reports Server (NTRS)

    Penner, J. E.; Quaas, J.; Storelvmo, T.; Takemura, T.; Boucher, O.; Guo, H.; Kirkevag, A.; Kristjansson, J. E.; Seland, O.

    2006-01-01

    Modeled differences in predicted effects are increasingly used to help quantify the uncertainty of these effects. Here, we examine modeled differences in the aerosol indirect effect in a series of experiments that help to quantify how and why model-predicted aerosol indirect forcing varies between models. The experiments start with an experiment in which aerosol concentrations, the parameterization of droplet concentrations and the autoconversion scheme are all specified and end with an experiment that examines the predicted aerosol indirect forcing when only aerosol sources are specified. Although there are large differences in the predicted liquid water path among the models, the predicted aerosol first indirect effect for the first experiment is rather similar, about -0.6 W/sq m to -0.7 W/sq m. Changes to the autoconversion scheme can lead to large changes in the liquid water path of the models and to the response of the liquid water path to changes in aerosols. Adding an autoconversion scheme that depends on the droplet concentration caused a larger (negative) change in net outgoing shortwave radiation compared to the 1st indirect effect, and the increase varied from only 22% to more than a factor of three. The change in net shortwave forcing in the models due to varying the autoconversion scheme depends on the liquid water content of the clouds as well as their predicted droplet concentrations, and both increases and decreases in the net shortwave forcing can occur when autoconversion schemes are changed. The parameterization of cloud fraction within models is not sensitive to the aerosol concentration, and, therefore, the response of the modeled cloud fraction within the present models appears to be smaller than that which would be associated with model "noise". The prediction of aerosol concentrations, given a fixed set of sources, leads to some of the largest differences in the predicted aerosol indirect radiative forcing among the models, with values of

  5. Two distinct neural mechanisms underlying indirect reciprocity.

    PubMed

    Watanabe, Takamitsu; Takezawa, Masanori; Nakawake, Yo; Kunimatsu, Akira; Yamasue, Hidenori; Nakamura, Mitsuhiro; Miyashita, Yasushi; Masuda, Naoki

    2014-03-18

    Cooperation is a hallmark of human society. Humans often cooperate with strangers even if they will not meet each other again. This so-called indirect reciprocity enables large-scale cooperation among nonkin and can occur based on a reputation mechanism or as a succession of pay-it-forward behavior. Here, we provide the functional and anatomical neural evidence for two distinct mechanisms governing the two types of indirect reciprocity. Cooperation occurring as reputation-based reciprocity specifically recruited the precuneus, a region associated with self-centered cognition. During such cooperative behavior, the precuneus was functionally connected with the caudate, a region linking rewards to behavior. Furthermore, the precuneus of a cooperative subject had a strong resting-state functional connectivity (rsFC) with the caudate and a large gray matter volume. In contrast, pay-it-forward reciprocity recruited the anterior insula (AI), a brain region associated with affective empathy. The AI was functionally connected with the caudate during cooperation occurring as pay-it-forward reciprocity, and its gray matter volume and rsFC with the caudate predicted the tendency of such cooperation. The revealed difference is consistent with the existing results of evolutionary game theory: although reputation-based indirect reciprocity robustly evolves as a self-interested behavior in theory, pay-it-forward indirect reciprocity does not on its own. The present study provides neural mechanisms underlying indirect reciprocity and suggests that pay-it-forward reciprocity may not occur as myopic profit maximization but elicit emotional rewards. PMID:24591599

  6. Two distinct neural mechanisms underlying indirect reciprocity

    PubMed Central

    Watanabe, Takamitsu; Takezawa, Masanori; Nakawake, Yo; Kunimatsu, Akira; Yamasue, Hidenori; Nakamura, Mitsuhiro; Miyashita, Yasushi; Masuda, Naoki

    2014-01-01

    Cooperation is a hallmark of human society. Humans often cooperate with strangers even if they will not meet each other again. This so-called indirect reciprocity enables large-scale cooperation among nonkin and can occur based on a reputation mechanism or as a succession of pay-it-forward behavior. Here, we provide the functional and anatomical neural evidence for two distinct mechanisms governing the two types of indirect reciprocity. Cooperation occurring as reputation-based reciprocity specifically recruited the precuneus, a region associated with self-centered cognition. During such cooperative behavior, the precuneus was functionally connected with the caudate, a region linking rewards to behavior. Furthermore, the precuneus of a cooperative subject had a strong resting-state functional connectivity (rsFC) with the caudate and a large gray matter volume. In contrast, pay-it-forward reciprocity recruited the anterior insula (AI), a brain region associated with affective empathy. The AI was functionally connected with the caudate during cooperation occurring as pay-it-forward reciprocity, and its gray matter volume and rsFC with the caudate predicted the tendency of such cooperation. The revealed difference is consistent with the existing results of evolutionary game theory: although reputation-based indirect reciprocity robustly evolves as a self-interested behavior in theory, pay-it-forward indirect reciprocity does not on its own. The present study provides neural mechanisms underlying indirect reciprocity and suggests that pay-it-forward reciprocity may not occur as myopic profit maximization but elicit emotional rewards. PMID:24591599

  7. Development of passive CLARITY and immunofluorescent labelling of multiple proteins in human cerebellum: understanding mechanisms of neurodegeneration in mitochondrial disease

    PubMed Central

    Phillips, Jonathan; Laude, Alex; Lightowlers, Robert; Morris, Chris M.; Turnbull, Doug M.; Lax, Nichola Z.

    2016-01-01

    CLARITY enables immunofluorescent labelling and imaging of large volumes of tissue to provide a better insight into the three dimensional relationship between cellular morphology and spatial interactions between different cell types. In the current study, we optimise passive CLARITY and immunofluorescent labelling of neurons and mitochondrial proteins in mouse and human brain tissues to gain further insights into mechanisms of neurodegeneration occurring in mitochondrial disease. This is the first study to utilise human cerebellum fixed in paraformaldehyde and cryoprotected in conjunction with formalin-fixed tissues opening up further avenues for use of archived tissue. We optimised hydrogel-embedding and passive clearance of lipids from both mouse (n = 5) and human (n = 9) cerebellum as well as developing an immunofluorescent protocol that consistently labels different neuronal domains as well as blood vessels. In addition to visualising large structures, we were able to visualise mitochondrial proteins in passively cleared tissues to reveal respiratory chain deficiency associated with mitochondrial disease. We also demonstrate multiple use of tissues by stripping antibodies and re-probing the cerebellum. This technique allows interrogation of large volumes intact brain samples for better understanding of the complex pathological changes taking place in mitochondrial disease. PMID:27181107

  8. 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

  9. Robust tumor morphometry in multispectral fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Tabesh, Ali; Vengrenyuk, Yevgen; Teverovskiy, Mikhail; Khan, Faisal M.; Sapir, Marina; Powell, Douglas; Mesa-Tejada, Ricardo; Donovan, Michael J.; Fernandez, Gerardo

    2009-02-01

    Morphological and architectural characteristics of primary tissue compartments, such as epithelial nuclei (EN) and cytoplasm, provide important cues for cancer diagnosis, prognosis, and therapeutic response prediction. We propose two feature sets for the robust quantification of these characteristics in multiplex immunofluorescence (IF) microscopy images of prostate biopsy specimens. To enable feature extraction, EN and cytoplasm regions were first segmented from the IF images. Then, feature sets consisting of the characteristics of the minimum spanning tree (MST) connecting the EN and the fractal dimension (FD) of gland boundaries were obtained from the segmented compartments. We demonstrated the utility of the proposed features in prostate cancer recurrence prediction on a multi-institution cohort of 1027 patients. Univariate analysis revealed that both FD and one of the MST features were highly effective for predicting cancer recurrence (p <= 0.0001). In multivariate analysis, an MST feature was selected for a model incorporating clinical and image features. The model achieved a concordance index (CI) of 0.73 on the validation set, which was significantly higher than the CI of 0.69 for the standard multivariate model based solely on clinical features currently used in clinical practice (p < 0.0001). The contributions of this work are twofold. First, it is the first demonstration of the utility of the proposed features in morphometric analysis of IF images. Second, this is the largest scale study of the efficacy and robustness of the proposed features in prostate cancer prognosis.

  10. Interferometric synthetic aperture microscopy

    NASA Astrophysics Data System (ADS)

    Ralston, Tyler S.; Marks, Daniel L.; Scott Carney, P.; Boppart, Stephen A.

    2007-02-01

    State-of-the-art methods in high-resolution three-dimensional optical microscopy require that the focus be scanned through the entire region of interest. However, an analysis of the physics of the light-sample interaction reveals that the Fourier-space coverage is independent of depth. Here we show that, by solving the inverse scattering problem for interference microscopy, computed reconstruction yields volumes with a resolution in all planes that is equivalent to the resolution achieved only at the focal plane for conventional high-resolution microscopy. In short, the entire illuminated volume has spatially invariant resolution, thus eliminating the compromise between resolution and depth of field. We describe and demonstrate a novel computational image-formation technique called interferometric synthetic aperture microscopy (ISAM). ISAM has the potential to broadly impact real-time three-dimensional microscopy and analysis in the fields of cell and tumour biology, as well as in clinical diagnosis where in vivo imaging is preferable to biopsy.

  11. Nonlinear vibrational microscopy

    DOEpatents

    Holtom, Gary R.; Xie, Xiaoliang Sunney; Zumbusch, Andreas

    2000-01-01

    The present invention is a method and apparatus for microscopic vibrational imaging using coherent Anti-Stokes Raman Scattering or Sum Frequency Generation. Microscopic imaging with a vibrational spectroscopic contrast is achieved by generating signals in a nonlinear optical process and spatially resolved detection of the signals. The spatial resolution is attained by minimizing the spot size of the optical interrogation beams on the sample. Minimizing the spot size relies upon a. directing at least two substantially co-axial laser beams (interrogation beams) through a microscope objective providing a focal spot on the sample; b. collecting a signal beam together with a residual beam from the at least two co-axial laser beams after passing through the sample; c. removing the residual beam; and d. detecting the signal beam thereby creating said pixel. The method has significantly higher spatial resolution then IR microscopy and higher sensitivity than spontaneous Raman microscopy with much lower average excitation powers. CARS and SFG microscopy does not rely on the presence of fluorophores, but retains the resolution and three-dimensional sectioning capability of confocal and two-photon fluorescence microscopy. Complementary to these techniques, CARS and SFG microscopy provides a contrast mechanism based on vibrational spectroscopy. This vibrational contrast mechanism, combined with an unprecedented high sensitivity at a tolerable laser power level, provides a new approach for microscopic investigations of chemical and biological samples.

  12. A rapid method combining immunofluorescence and flow cytometry for improved understanding of competitive interactions between lactic acid bacteria (LAB) and methicillin-resistant S. aureus (MRSA) in mixed culture.

    PubMed

    Schellenberg, John; Smoragiewicz, Wanda; Karska-Wysocki, Barbara

    2006-04-01

    The increasing frequency of methicillin-resistant Staphylococcus aureus (MRSA) infections in hospital and community settings highlights the need for effective anti-MRSA agents that will not contribute to the growing problem of antibiotic resistance. Lactic acid bacteria (LAB) are known to exclude various pathogens through multiple mechanisms. In vitro models studying interactions of pathogens and LAB in mixed cultures use selective agar plates to quantify changes in target populations. We applied commercially available S. aureus-specific polyclonal antibodies conjugated with fluorescein isothiocyanate (FITC) for this purpose, producing a bright green signal that clearly differentiates S. aureus from LAB species when mixed cultures are analyzed by flow cytometry and fluorescent microscopy. Flow cytometry of mixed cultures revealed a much larger population of MRSA cells than was detectable using selective agar plates. To our knowledge, this is the first time immunofluorescent flow cytometry has been applied to the study of competitive exclusion in mixed bacterial populations over time. PMID:16154216

  13. Imaging interferometric microscopy.

    PubMed

    Schwarz, Christian J; Kuznetsova, Yuliya; Brueck, S R J

    2003-08-15

    We introduce and demonstrate a new microscopy concept: imaging interferometric microscopy (IIM), which is related to holography, synthetic-aperture imaging, and off-axis-dark-field illumination techniques. IIM is a wavelength-division multiplex approach to image formation that combines multiple images covering different spatial-frequency regions to form a composite image with a resolution much greater than that permitted by the same optical system using conventional techniques. This new type of microscopy involves both off-axis coherent illumination and reinjection of appropriate zero-order reference beams. Images demonstrate high resolution, comparable with that of a high-numerical-aperture (NA) objective, while they retain the long working distance, the large depth of field, and the large field of view of a low-NA objective. A Fourier-optics model of IIM is in good agreement with the experiment. PMID:12943079

  14. Multiphoton microscopy in neuroscience

    NASA Astrophysics Data System (ADS)

    Denk, Winfried

    2002-06-01

    The study of the nervous system requires to an exceptional extent observation of and experimentation on intact tissue. There, in particular, high-resolution optical microscopy benefits from the inherent advantages of multi-photon fluorescence excitation. Several cases will be presented from a number of different tissues and organisms, where multi-photon excited laser scanning fluorescence microscopy has been an essential experimental tool. Those examples include the discovery of biochemical coincidence detection in synaptic spines and the clarification of the underlying mechanism; the observation of sensory evoked dendritic signaling in intact animals and the observation of light induced calcium signals in the intact retina. Recently a fiber coupled two-photon microscopy has been developed that allows the imaging in moving animal.

  15. Controllable tomography phase microscopy

    NASA Astrophysics Data System (ADS)

    Xiu, Peng; Zhou, Xin; Kuang, Cuifang; Xu, Yingke; Liu, Xu

    2015-03-01

    Tomography phase microscopy (TPM) is a new microscopic method that can quantitatively yield the volumetric 3D distribution of a sample's refractive index (RI), which is significant for cell biology research. In this paper, a controllable TPM system is introduced. In this system a circulatory phase-shifting method and piezoelectric ceramic are used which enable the TPM system to record the 3D RI distribution at a more controllable speed, from 1 to 40 fps, than in the other TPM systems reported. The resolution of the RI distribution obtained by this controllable TPM is much better than that in images recorded by phase contrast microscopy and interference tomography microscopy. The realization of controllable TPM not only allows for the application of TPM to the measurement of kinds of RI sample, but also contributes to academic and technological support for the practical use of TPM.

  16. Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

    PubMed Central

    Watanabe, Shigeki; Richards, Jackson; Hollopeter, Gunther; Hobson, Robert J.; Davis, Wayne M.; Jorgensen, Erik M.

    2012-01-01

    Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated 1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated 4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot 8-10. We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged

  17. [Direct and indirect mucosal wave imaging techniques].

    PubMed

    Krasnodębska, Paulina; Szkiełkowska, Agata

    2016-04-01

    The vocal folds play a key role in the process of phonation. Cyclical movements of the vocal folds model a space called glottis, what leads to voice formation. The space contains surface between the vocal folds and the inner surface of the arytenoid cartilages. The best indicator of the vocal folds vibratory function is the mucosal wave. The presence and size of the mucosal wave is widely recognized as an indicator of tension and plasticity of vocal folds. It is also essential in the process of creating a proper, resonant voice. In the article, current knowledge of mucosal wave imaging techniques is given. Imaging can be carried out directly and indirectly. Among the direct methods, the following are distinguished: laryngostroboscopy, laryngovideostroboscopy, videokymography and high-speed digital imaging. Indirect methods include: electroglottography, photoglottography and ultrasonography. PMID:27137829

  18. Universal scheme for indirect quantum control

    NASA Astrophysics Data System (ADS)

    Layden, David; Martín-Martínez, Eduardo; Kempf, Achim

    2016-04-01

    We consider a bipartite quantum object, composed of a quantum system and a quantum actuator which is periodically reset. We show that the reduced dynamics of the system approaches unitarity as the reset frequency of the actuator is increased. This phenomenon arises because quantum systems interacting for a short time can impact each other faster than they can become significantly entangled. In the high reset-frequency limit, the effective Hamiltonian describing the system's unitary evolution depends on the state to which the actuator is reset. This makes it possible to indirectly implement a continuous family of effective Hamiltonians on one part of a bipartite quantum object, thereby reducing the problem of indirect control (via a quantum actuator) to the well-studied one of direct quantum control.

  19. Scalar dark matter: direct vs. indirect detection

    NASA Astrophysics Data System (ADS)

    Duerr, Michael; Pérez, Pavel Fileviez; Smirnov, Juri

    2016-06-01

    We revisit the simplest model for dark matter. In this context the dark matter candidate is a real scalar field which interacts with the Standard Model particles through the Higgs portal. We discuss the relic density constraints as well as the predictions for direct and indirect detection. The final state radiation processes are investigated in order to understand the visibility of the gamma lines from dark matter annihilation. We find two regions where one could observe the gamma lines at gamma-ray telescopes. We point out that the region where the dark matter mass is between 92 and 300 GeV can be tested in the near future at direct and indirect detection experiments.

  20. Indirect Acquisition of Information in Quantum Mechanics

    NASA Astrophysics Data System (ADS)

    Ballesteros, M.; Fraas, M.; Fröhlich, J.; Schubnel, B.

    2016-02-01

    Long sequences of successive direct (projective) measurements or observations of just a few "uninteresting" physical quantities pertaining to a quantum system, such as clicks of some detectors, may reveal indirect, but precise and unambiguous information on the values of some very "interesting" observables of the system. In this paper, the mathematics underlying this claim is developed; i.e., we attempt to contribute to a mathematical theory of indirect and, in particular, non-demolition observations and measurements in quantum mechanics. Our attempt leads us to make some novel uses of classical notions and results of probability theory, such as the "algebra of functions measurable at infinity", the Central Limit Theorem, results concerning relative entropy and its role in the theory of large deviations, etc.

  1. Real medical benefit assessed by indirect comparison.

    PubMed

    Falissard, Bruno; Zylberman, Myriam; Cucherat, Michel; Izard, Valérie; Meyer, François

    2009-01-01

    Frequently, in data packages submitted for Marketing Approval to the CHMP, there is a lack of relevant head-to-head comparisons of medicinal products that could enable national authorities responsible for the approval of reimbursement to assess the Added Therapeutic Value (ASMR) of new clinical entities or line extensions of existing therapies.Indirect or mixed treatment comparisons (MTC) are methods stemming from the field of meta-analysis that have been designed to tackle this problem. Adjusted indirect comparisons, meta-regressions, mixed models, Bayesian network analyses pool results of randomised controlled trials (RCTs), enabling a quantitative synthesis.The REAL procedure, recently developed by the HAS (French National Authority for Health), is a mixture of an MTC and effect model based on expert opinions. It is intended to translate the efficacy observed in the trials into effectiveness expected in day-to-day clinical practice in France. PMID:19671436

  2. Indirect techniques for astrophysical reaction rates determinations

    NASA Astrophysics Data System (ADS)

    Hammache, F.; Oulebsir, N.; Benamara, S.; De Séréville, N.; Coc, A.; Laird, A.; Stefan, I.; Roussel, P.

    2016-05-01

    Direct measurements of nuclear reactions of astrophysical interest can be challenging. Alternative experimental techniques such as transfer reactions and inelastic scattering reactions offer the possibility to study these reactions by using stable beams. In this context, I will present recent results that were obtained in Orsay using indirect techniques. The examples will concern various astrophysical sites, from the Big-Bang nucleo synthesis to the production of radioisotopes in massive stars.

  3. Color indirect effects on melatonin regulation

    NASA Astrophysics Data System (ADS)

    Mian, Tian; Liu, Timon C.; Li, Yan

    2002-04-01

    Color indirect effect (CIE) is referred to as the physiological and psychological effects of color resulting from color vision. In previous papers, we have studied CIE from the viewpoints of the integrated western and Chinese traditional medicine, put forward the color-autonomic- nervous-subsystem model (CAM), and provided its time-theory foundation. In this paper, we applied it to study light effects on melatonin regulation in humans, and suggested that it is CIE that mediates light effects on melatonin suppression.

  4. A portable monocular indirect ophthalmoscopic technique.

    PubMed

    Sislowitz, M J

    1975-01-01

    A simple, inexpensive, and portable method of monocular indirect ophthalmoscopy is used as the physician's examining eye is aligned in the axis of the patient's eye, a flashlight is held against the examiner's malar area beneath the dominant eye, and the beam of the flashlight is directed to the patient's eye. A plus 30 lens is then interposed 1 or 2 inches in front of the patient's eye and the fundus viewed. PMID:1110195

  5. Indirect hemagglutination test for chlamydial antibodies.

    PubMed

    Lewis, V J; Thacker, W L; Engelman, H M

    1972-07-01

    An indirect hemagglutination (IHA) test is described for chlamydial antibodies in psittacosis diagnostic sera; for this test tanned sheep erythrocytes sensitized with a deoxycholate extract of Chlamydia psittaci grown in Vero cell monolayers were used. Adaptation of the IHA test to the Microtiter system decreased sensitivity; nevertheless, the Microtiter-IHA test was more sensitive than the complement fixation test. Lymphogranuloma venereum antibodies also were detected by using antigen extracted from C. psittaci. PMID:4626906

  6. Gall insects and indirect plant defenses

    PubMed Central

    De Moraes, Consuelo M

    2008-01-01

    Many plants can defend themselves against insect herbivory by attracting natural enemies that kill feeding herbivores and limit the damage they inflict. Such “indirect defenses” can be induced by insects feeding on different plant tissues and using a variety of feeding styles. However, we have recently shown that gall-inducing insect species can avoid the indirect defenses of their host plant species and even alter volatile emissions following subsequent herbivory. One of the species we studied, Eurosta solidaginis, induces galls on goldenrod (Solidago altissima) and appears to exert a unique influence over the indirect defenses of its host plant that is not readily explained by levels of defense-related phytohormones, gall formation or resource depletion. Our evidence suggests that this gall-insect species may be able to manipulate its host plant species to avoid and/or modify its defensive responses. The results also provide insight into gall induction because the gall-insect species that we screened did not increase levels of jasmonic acid, which, in addition to triggering volatile emissions, is a powerful growth regulator that could prevent the cell growth and division that leads to gall formation. PMID:19704500

  7. Indirect Comprehensive Review Board (ICRB). Final Report

    SciTech Connect

    1996-12-01

    Lockheed Martin Idaho Technologies Company (LMITCO) used a systems engineering approach to take the first step toward defining a requirements baseline for all indirect work at the Idaho National Engineering Laboratory. The intent of this effort was to define the requirements for indirect work, identify the activities necessary to meet the requirements, and to produce defensible cost estimates for the work. The result of this effort is a scrubbed-down, defensible budget for all indirect work in FY 1997. Buying power for each dollar of direct work was increased by $.02. Recommendations are identified for improvements to this process in FY 1998. The purpose of this report is twofold. First is to report the final results of the 1996 ICRB process, and second is to document the process used such that incremental improvements may be made in future years. Objectives, processes, and approaches are described to provide a trail for future boards. Appendices contain copies of board composition, documentation of the process, as well as the actual training materials.

  8. 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.

  9. Video Telescope Operating Microscopy.

    PubMed

    Divers, Stephen J

    2015-09-01

    Exotic pet veterinarians frequently have to operate on small animals, and magnification is commonly used. Existing endoscopy equipment can be used with a mechanical arm and telescope to enable video telescope operating microscopy. The additional equipment items and their specifics are described, and several case examples are provided. PMID:26117519

  10. Photoacoustic computed microscopy

    NASA Astrophysics Data System (ADS)

    Yao, Lei; Xi, Lei; Jiang, Huabei

    2014-05-01

    Photoacoustic microscopy (PAM) is emerging as a powerful technique for imaging microvasculature at depths beyond the ~1 mm depth limit associated with confocal microscopy, two-photon microscopy and optical coherence tomography. PAM, however, is currently qualitative in nature and cannot quantitatively measure important functional parameters including oxyhemoglobin (HbO2), deoxyhemoglobin (HbR), oxygen saturation (sO2), blood flow (BF) and rate of oxygen metabolism (MRO2). Here we describe a new photoacoustic microscopic method, termed photoacoustic computed microscopy (PACM) that combines current PAM technique with a model-based inverse reconstruction algorithm. We evaluate the PACM approach using tissue-mimicking phantoms and demonstrate its in vivo imaging ability of quantifying HbO2, HbR, sO2, cerebral BF and cerebral MRO2 at the small vessel level in a rodent model. This new technique provides a unique tool for neuroscience research and for visualizing microvasculature dynamics involved in tumor angiogenesis and in inflammatory joint diseases.

  11. Interferometric synthetic aperture microscopy

    NASA Astrophysics Data System (ADS)

    Ralston, Tyler S.

    State-of-the-art interferometric microscopies have problems representing objects that lie outside of the focus because the defocus and diffraction effects are not accounted for in the processing. These problems occur because of the lack of comprehensive models to include the scattering effects in the processing. In this dissertation, a new modality in three-dimensional (3D) optical microscopy, Interferometric Synthetic Aperture Microscopy (ISAM), is introduced to account for the scattering effects. Comprehensive models for interferometric microscopy, such as optical coherence tomography (OCT) are developed, for which forward, adjoint, normal, and inverse operators are formulated. Using an accurate model for the probe beam, the resulting algorithms demonstrate accurate linear estimation of the susceptibility of an object from the interferometric data. Using the regularized least squares solution, an ISAM reconstruction of underlying object structure having spatially invariant resolution is obtained from simulated and experimental interferometric data, even in regions outside of the focal plane of the lens. Two-dimensional (2D) and 3D interferometric data is used to resolve objects outside of the confocal region with minimal loss of resolution, unlike in OCT. Therefore, high-resolution details are recovered from outside of the confocal region. Models and solutions are presented for the planar-scanned, the rotationally scanned, and the full-field illuminated geometry. The models and algorithms presented account for the effects of a finite beam width, the source spectrum, the illumination and collection fields, as well as defocus, diffraction and dispersion effects.

  12. Spatially indirect excitons in coupled quantum wells

    SciTech Connect

    Lai, Chih-Wei Eddy

    2004-03-01

    Microscopic quantum phenomena such as interference or phase coherence between different quantum states are rarely manifest in macroscopic systems due to a lack of significant correlation between different states. An exciton system is one candidate for observation of possible quantum collective effects. In the dilute limit, excitons in semiconductors behave as bosons and are expected to undergo Bose-Einstein condensation (BEC) at a temperature several orders of magnitude higher than for atomic BEC because of their light mass. Furthermore, well-developed modern semiconductor technologies offer flexible manipulations of an exciton system. Realization of BEC in solid-state systems can thus provide new opportunities for macroscopic quantum coherence research. In semiconductor coupled quantum wells (CQW) under across-well static electric field, excitons exist as separately confined electron-hole pairs. These spatially indirect excitons exhibit a radiative recombination time much longer than their thermal relaxation time a unique feature in direct band gap semiconductor based structures. Their mutual repulsive dipole interaction further stabilizes the exciton system at low temperature and screens in-plane disorder more effectively. All these features make indirect excitons in CQW a promising system to search for quantum collective effects. Properties of indirect excitons in CQW have been analyzed and investigated extensively. The experimental results based on time-integrated or time-resolved spatially-resolved photoluminescence (PL) spectroscopy and imaging are reported in two categories. (i) Generic indirect exciton systems: general properties of indirect excitons such as the dependence of exciton energy and lifetime on electric fields and densities were examined. (ii) Quasi-two-dimensional confined exciton systems: highly statistically degenerate exciton systems containing more than tens of thousands of excitons within areas as small as (10 micrometer){sup 2} were

  13. A Sensitive, Reproducible and Objective Immunofluorescence Analysis Method of Dystrophin in Individual Fibers in Samples from Patients with Duchenne Muscular Dystrophy

    PubMed Central

    Beekman, Chantal; Sipkens, Jessica A.; Testerink, Janwillem; Giannakopoulos, Stavros; Kreuger, Dyonne; van Deutekom, Judith C.; Campion, Giles V.; de Kimpe, Sjef J.; Lourbakos, Afrodite

    2014-01-01

    Duchenne muscular dystrophy (DMD) is characterized by the absence or reduced levels of dystrophin expression on the inner surface of the sarcolemmal membrane of muscle fibers. Clinical development of therapeutic approaches aiming to increase dystrophin levels requires sensitive and reproducible measurement of differences in dystrophin expression in muscle biopsies of treated patients with DMD. This, however, poses a technical challenge due to intra- and inter-donor variance in the occurrence of revertant fibers and low trace dystrophin expression throughout the biopsies. We have developed an immunofluorescence and semi-automated image analysis method that measures the sarcolemmal dystrophin intensity per individual fiber for the entire fiber population in a muscle biopsy. Cross-sections of muscle co-stained for dystrophin and spectrin have been imaged by confocal microscopy, and image analysis was performed using Definiens software. Dystrophin intensity has been measured in the sarcolemmal mask of spectrin for each individual muscle fiber and multiple membrane intensity parameters (mean, maximum, quantiles per fiber) were calculated. A histogram can depict the distribution of dystrophin intensities for the fiber population in the biopsy. This method was tested by measuring dystrophin in DMD, Becker muscular dystrophy, and healthy muscle samples. Analysis of duplicate or quadruplicate sections of DMD biopsies on the same or multiple days, by different operators, or using different antibodies, was shown to be objective and reproducible (inter-assay precision, CV 2–17% and intra-assay precision, CV 2–10%). Moreover, the method was sufficiently sensitive to detect consistently small differences in dystrophin between two biopsies from a patient with DMD before and after treatment with an investigational compound. PMID:25244123

  14. Scanning Probe Microscopy and Spectroscopy

    NASA Astrophysics Data System (ADS)

    Wiesendanger, Roland

    1994-09-01

    Preface; List of acronyms; Introduction; Part I. Experimental Methods and Theoretical Background of Scanning Probe Microscopy and Spectroscopy: 1. Scanning tunnelling microscopy; 2. Scanning force microscopy; 3. Related scanning probe techniques; Part II. Applications of Scanning Probe Microscopy and Spectroscopy: 4. Condensed matter physics; 5. Chemistry; 6. Organic materials; 7. Metrology and standards; 8. Nanotechnology; References; Index.

  15. Distribution of tetrodotoxin in the ribbon worm Lineus alborostratus (Takakura, 1898) (nemertea): Immunoelectron and immunofluorescence studies.

    PubMed

    Magarlamov, Timur Yu; Shokur, Olga A; Chernyshev, Alexey V

    2016-03-15

    Transmission electron and confocal laser scanning (CLSM) microscopies with monoclonal anti-tetrodotoxin antibodies were used to locate tetrodotoxin (TTX) in tissues and gland cells of the ribbon worm Lineus alborostratus. CLSM studies have shown that the toxin is primarily localized in the cutis (special subepidermal layer) of the body wall and in the glandular epithelium of the proboscis. Immunoelectron micrographs have shown that only subepidermal bacillary gland cells type I in cutis and pseudocnidae-containing and mucoid gland cells manifested TTX-gold labeling. TTX was associated with the nuclear envelope, endoplasmic reticulum membrane, and secretory granules of TTX-positive gland cells. These studies indicate that ТТХ is brought into the cytoplasm of the glandular cells of the cutis and proboscis epithelium, where it is associated with membrane-enclosed organelles involved in protein secretion and then concentrated in glandular granules. PMID:26821373

  16. Immunofluorescent test for simultaneous detection of Edwardsiella ictaluri and Flavobacterium columnare.

    PubMed

    Panangalal, Victor S; Shelby, Richard A; Shoemaker, Craig A; Klesius, Phillip H; Mitra, Amitava; Morrison, Edward E

    2006-03-01

    Enteric septicemia of catfish (ESC) and columnaris disease are 2 bacterial diseases significantly affecting the aquaculture industry, and thus rapid diagnosis of disease is imperative for making judicious management decisions. A rapid indirect fluorescent antibody (IFA) test with antibody conjugated fluorochromes having 2 different spectral properties (Alexa Fluor 488-emitting green fluorescence, and Alexa Fluor 594-emitting red fluorescence) was compared with bacteriological culture (accepted standard) for simultaneous detection of Edwardsiella ictaluri (EI) and Flavobacterium columnare (FC) in 3 groups of experimentally infected channel catfish (Ictalurus punctatus Rafinesque), and a fourth group that acquired an aquarium-infection with F. columnare. A total of 303 samples (derived from kidney, brain and nares) from 101 fish were concurrently examined by both tests. Fish in the 3 experimentally infected groups (I to III) were culture positive for the bacteria with which they were infected, and fish in Group IV, (the spontaneously infected fish) revealed F. columnare only. The IFA test compared favorably in sensitivity (EI= 80.7 %; FC = 87.2%) and specificity (EI = 83.9%; FC = 88.9%) with the standard bacteriological culture. The positive predictive value (EI = 96.2% Group I, 90.8% Group II, 93.7% Groups I and II combined; FC = 95.2% Group II, 95.3% Groups II, III and IV combined) was high, while the negative predictive value (EI = 66.7% Group I, 31.3% Group II, 59.5% Groups I and II combined; FC = 73.7% Group II, 72.7% Groups II, III and IV combined) was relatively low. The IFA test will serve as an efficient tool for rapid simultaneous detection of E. ictaluri and F. columnare in outbreaks of disease. PMID:16610585

  17. [Immunofluorescent study of the distribution of adult neuro-specific antigens in the chick embryo (author's transl)].

    PubMed

    Touzet, N; Jeanmaire-Zylberberg, R; Chaminade, M

    1977-06-01

    The adult neuro-specific antigens have been localized by immunofluorescence techniques in diencephalon and mesencephalon of chick embryo. This study has been made using fresh or fixed tissues from embryos 72, 48 or 36 h old. At 72 h of incubation the wall of diencephalon shows marked fluorescence; at 48 h of incubation the fluorescent cells are localized in an outer layer and an inner one. In the 48 h-old embryo the reaction is more distinct and intensive in fresh tissues than in fixed tissues. At 36 h of incubation no fluorescence has been detected either in fresh tissues or in fixed tissues. PMID:328815

  18. Monitoring Ubiquitin-Coated Bacteria via Confocal Microscopy.

    PubMed

    Lork, Marie; Delvaeye, Mieke; Gonçalves, Amanda; Van Hamme, Evelien; Beyaert, Rudi

    2016-01-01

    Salmonella is a gram-negative facultative intracellular pathogen that is capable of infecting a variety of hosts. Inside host cells, most Salmonella bacteria reside and replicate within Salmonella-containing vacuoles. They use virulence proteins to manipulate the host cell machinery for their own benefit and hijack the host cytoskeleton to travel toward the perinuclear area. However, a fraction of bacteria escapes into the cytosol where they get decorated with a dense layer of polyubiquitin, which labels the bacteria for clearance by autophagy. More specifically, autophagy receptor proteins recognize the ubiquitinated bacteria and deliver them to autophagosomes, which subsequently fuse to lysosomes. Here, we describe methods used to infect HeLa cells with Salmonella bacteria and to detect their ubiquitination via immunofluorescence and laser scanning confocal microscopy. PMID:27613040

  19. Indirect interactions in the High Arctic.

    PubMed

    Roslin, Tomas; Wirta, Helena; Hopkins, Tapani; Hardwick, Bess; Várkonyi, Gergely

    2013-01-01

    Indirect interactions as mediated by higher and lower trophic levels have been advanced as key forces structuring herbivorous arthropod communities around the globe. Here, we present a first quantification of the interaction structure of a herbivore-centered food web from the High Arctic. Targeting the Lepidoptera of Northeast Greenland, we introduce generalized overlap indices as a novel tool for comparing different types of indirect interactions. First, we quantify the scope for top-down-up interactions as the probability that a herbivore attacking plant species i itself fed as a larva on species j. Second, we gauge this herbivore overlap against the potential for bottom-up-down interactions, quantified as the probability that a parasitoid attacking herbivore species i itself developed as a larva on species j. Third, we assess the impact of interactions with other food web modules, by extending the core web around the key herbivore Sympistis nigrita to other predator guilds (birds and spiders). We find the host specificity of both herbivores and parasitoids to be variable, with broad generalists occurring in both trophic layers. Indirect links through shared resources and through shared natural enemies both emerge as forces with a potential for shaping the herbivore community. The structure of the host-parasitoid submodule of the food web suggests scope for classic apparent competition. Yet, based on predation experiments, we estimate that birds kill as many (8%) larvae of S. nigrita as do parasitoids (8%), and that spiders kill many more (38%). Interactions between these predator guilds may result in further complexities. Our results caution against broad generalizations from studies of limited food web modules, and show the potential for interactions within and between guilds of extended webs. They also add a data point from the northernmost insect communities on Earth, and describe the baseline structure of a food web facing imminent climate change. PMID

  20. Evaluating The Indirect Effect of Cirrus Clouds

    NASA Astrophysics Data System (ADS)

    Dobbie, S.; Jonas, P. R.

    What effect would an increase in nucleating aerosols have on the radiative and cloud properties? What error would be incurred by evaluating the indirect effect by taking an evolved cloud and fixing the integrated water content and vary the number of ice crystals? These questions will be addressed in this work. We will use the UK LES cloud resolving model to perform a sensitivity study for cirrus clouds to the indirect effect, and will evaluate approximate methods in the process. In this work, we will initialize the base (no increase of aerosol) cirrus clouds so that the double moment scheme is constrained to agree with observations through the ef- fective radius. Effective radius is calculated using the local concentration and the ice water content. We then perform a sensitivity experiment to investigate the dependence of the average IWC, effective size, and radiative properties (including heating rates) to variations in the nucleation rate. Conclusions will be draw as to the possible ef- fect of changes in aerosol amounts on cirrus. We will determine how sensitive the cloud and radiative properties are to various aerosol increases. We will also discuss the applicability of the Meyer et al. (1992) nucleation formulae for our simulations. It is important to stress that in this work we only change the nucleation rate for the newly forming cloud. By doing this, we are not fixing the total water content and redistributing the water amongst increased ice crystals. We increase the number of aerosols available to be nucleated and allow the model to evolve the size distributions. In this way, there is competition for the water vapour, the ice particles are evolved dynamically with different fall speeds, the conversion rates to other hydrometers (such as aggregates) are affected, and the heating rates are different due to the different size distributions that evolve. We will look at how the water content, the distribution of water, and the radiative properties are affected

  1. Indirect Interactions in the High Arctic

    PubMed Central

    Roslin, Tomas; Wirta, Helena; Hopkins, Tapani; Hardwick, Bess; Várkonyi, Gergely

    2013-01-01

    Indirect interactions as mediated by higher and lower trophic levels have been advanced as key forces structuring herbivorous arthropod communities around the globe. Here, we present a first quantification of the interaction structure of a herbivore-centered food web from the High Arctic. Targeting the Lepidoptera of Northeast Greenland, we introduce generalized overlap indices as a novel tool for comparing different types of indirect interactions. First, we quantify the scope for top-down-up interactions as the probability that a herbivore attacking plant species i itself fed as a larva on species j. Second, we gauge this herbivore overlap against the potential for bottom-up-down interactions, quantified as the probability that a parasitoid attacking herbivore species i itself developed as a larva on species j. Third, we assess the impact of interactions with other food web modules, by extending the core web around the key herbivore Sympistis nigrita to other predator guilds (birds and spiders). We find the host specificity of both herbivores and parasitoids to be variable, with broad generalists occurring in both trophic layers. Indirect links through shared resources and through shared natural enemies both emerge as forces with a potential for shaping the herbivore community. The structure of the host-parasitoid submodule of the food web suggests scope for classic apparent competition. Yet, based on predation experiments, we estimate that birds kill as many (8%) larvae of S. nigrita as do parasitoids (8%), and that spiders kill many more (38%). Interactions between these predator guilds may result in further complexities. Our results caution against broad generalizations from studies of limited food web modules, and show the potential for interactions within and between guilds of extended webs. They also add a data point from the northernmost insect communities on Earth, and describe the baseline structure of a food web facing imminent climate change. PMID

  2. Dynamic Transmission Electron Microscopy

    SciTech Connect

    Evans, James E.; Jungjohann, K. L.; Browning, Nigel D.

    2012-10-12

    Dynamic transmission electron microscopy (DTEM) combines the benefits of high spatial resolution electron microscopy with the high temporal resolution of ultrafast lasers. The incorporation of these two components into a single instrument provides a perfect platform for in situ observations of material processes. However, previous DTEM applications have focused on observing structural changes occurring in samples exposed to high vacuum. Therefore, in order to expand the pump-probe experimental regime to more natural environmental conditions, in situ gas and liquid chambers must be coupled with Dynamic TEM. This chapter describes the current and future applications of in situ liquid DTEM to permit time-resolved atomic scale observations in an aqueous environment, Although this chapter focuses mostly on in situ liquid imaging, the same research potential exists for in situ gas experiments and the successful integration of these techniques promises new insights for understanding nanoparticle, catalyst and biological protein dynamics with unprecedented spatiotemporal resolution.

  3. Quad stereo-microscopy

    NASA Astrophysics Data System (ADS)

    Hay, Rebecca F.; Gibson, Graham M.; Lee, Michael P.; Padgett, Miles J.; Phillips, David B.

    2014-09-01

    Stereo-microscopy is a technique that enables a sample to be imaged from two directions simultaneously, allowing the tracking of microscopic objects in three dimensions. This is achieved by illuminating the sample from different directions, each illumination direction producing an individual image. These images are superimposed in the image plane but can be easily separated using a diffractive optical element in the Fourier plane of the imaging arm. Therefore this enables 3-dimensional coordinates to be reconstructed using simple 2-dimensional image tracking and parallax. This is a powerful technique when combined with holographic optical tweezers (HOT), where multiple objects can be trapped and tracked simultaneously in three dimensions. In this work, we extend this concept to four different illumination directions: quad stereo-microscopy. This allows us to measure the accuracy of tracking in three dimensions, and to optimise the system.

  4. Multimodal Nonlinear Optical Microscopy

    PubMed Central

    Yue, Shuhua; Slipchenko, Mikhail N.; Cheng, Ji-Xin

    2013-01-01

    Because each nonlinear optical (NLO) imaging modality is sensitive to specific molecules or structures, multimodal NLO imaging capitalizes the potential of NLO microscopy for studies of complex biological tissues. The coupling of multiphoton fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering (CARS) has allowed investigation of a broad range of biological questions concerning lipid metabolism, cancer development, cardiovascular disease, and skin biology. Moreover, recent research shows the great potential of using CARS microscope as a platform to develop more advanced NLO modalities such as electronic-resonance-enhanced four-wave mixing, stimulated Raman scattering, and pump-probe microscopy. This article reviews the various approaches developed for realization of multimodal NLO imaging as well as developments of new NLO modalities on a CARS microscope. Applications to various aspects of biological and biomedical research are discussed. PMID:24353747

  5. Scanning Electrochemical Microscopy

    NASA Astrophysics Data System (ADS)

    Amemiya, Shigeru; Bard, Allen J.; Fan, Fu-Ren F.; Mirkin, Michael V.; Unwin, Patrick R.

    2008-07-01

    This review describes work done in scanning electrochemical microscopy (SECM) since 2000 with an emphasis on new applications and important trends, such as nanometer-sized tips. SECM has been adapted to investigate charge transport across liquid/liquid interfaces and to probe charge transport in thin films and membranes. It has been used in biological systems like single cells to study ion transport in channels, as well as cellular and enzyme activity. It is also a powerful and useful tool for the evaluation of the electrocatalytic activities of different materials for useful reactions, such as oxygen reduction and hydrogen oxidation. SECM has also been used as an electrochemical tool for studies of the local properties and reactivity of a wide variety of materials, including metals, insulators, and semiconductors. Finally, SECM has been combined with several other nonelectrochemical techniques, such as atomic force microscopy, to enhance and complement the information available from SECM alone.

  6. Expression of keratin 18 in the periderm cells of the lingual epithelium of fetal rats: visualization by fluorescence immunohistochemistry and differential interference contrast microscopy.

    PubMed

    Iwasaki, Shin-ichi; Aoyagi, Hidekazu; Asami, Tomoichiro

    2006-09-01

    We examined the expression of keratin 18 (K18), by immunofluorescence staining, while monitoring morphological changes in the periderm on the lingual epithelium of rats by laser-scanning microscopy of epoxy resin-embedded, semi-ultrathin sections. We also examined differential interference contrast (DIC) images of the same sections to define the histology and morphology of the cells. It is difficult to visualize histological details of the fetal lingual epithelium of the rat on semi-ultrathin sections by light microscopy after immunohistochemical staining, because the histological structures in such sections cannot be distinguished by standard counterstaining. To solve this problem and to visualize keratin 18 (K18), we used a combination of immunofluorescence staining of semi-ultrathin sections and corresponding differential contrast (DIC) images, obtained by laser-scanning microscopy. PMID:16998620

  7. Pure optical photoacoustic microscopy

    PubMed Central

    Xie, Zhixing; Chen, Sung-Liang; Ling, Tao; Guo, L. Jay; Carson, Paul L.; Wang, Xueding

    2011-01-01

    The concept of pure optical photoacoustic microscopy(POPAM) was proposed based on optical rastering of a focused excitation beam and optically sensing the photoacoustic signal using a microring resonator fabricated by a nanoimprinting technique. After the refinements of the microring’s working wavelength and in the resonator structure and mold fabrication, an ultrahigh Q factor of 3.0×105 was achieved which provided high sensitivity with a noise equivalent detectable pressure(NEDP) value of 29Pa. This NEDP is much lower than the hundreds of Pascals achieved with existing optical resonant structures such as etalons, fiber gratings and dielectric multilayer interference filters available for acoustic measurement. The featured high sensitivity allowed the microring resonator to detect the weak photoacoustic signals from micro- or submicroscale objects. The inherent superbroad bandwidth of the optical microring resonator combined with an optically focused scanning beam provided POPAM with high resolution in the axial as well as both lateral directions while the axial resolution of conventional photoacoustic microscopy (PAM) suffers from the limited bandwidth of PZT detectors. Furthermore, the broadband microring resonator showed similar sensitivity to that of our most sensitive PZT detector. The current POPAM system provides a lateral resolution of 5 μm and an axial resolution of 8 μm, comparable to that achieved by optical microscopy while presenting the unique contrast of optical absorption and functional information complementing other optical modalities. The 3D structure of microvasculature, including capillary networks, and even individual red blood cells have been discerned successfully in the proof-of-concept experiments on mouse bladders ex vivo and mouse ears in vivo. The potential of approximately GHz bandwidth of the microring resonator also might allow much higher resolution than shown here in microscopy of optical absorption and acoustic propagation

  8. Marginal integrity and microleakage of direct and indirect composite inlays: SEM and stereomicroscopic evaluation.

    PubMed

    Soares, Carlos José; Celiberto, Leonardo; Dechichi, Paula; Fonseca, Rodrigo Borges; Martins, Luis Roberto Marcondes

    2005-01-01

    The aim of this study was to evaluate the microleakage of direct and indirect composite inlays by stereomicroscopy and scanning electron microscopy (SEM). Thirty bovine incisors were ground to obtain an incisal platform, simulating the occlusal surface of a human molar. Each tooth received two 8 degrees proximal cavities with cervical finishing line prepared in dentine or enamel. One of the cavities was filled with Filtek Z250/Single Bond, using the direct technique, and the other was filled with with Solidex/Rely X ARC/Single Bond, using the indirect technique. The samples were stored in water at 37 degrees C for 24 hours and placed in a 50% silver nitrate solution for 6 hours in a dark container. Next, the samples were washed under running water, immersed in a developing solution and exposed to fluorescent light for 12 hours. The teeth were then severed and evaluated for dye penetration by stereomicroscopy and SEM. There were no significant differences between the direct and indirect techniques for the cervical finishing line in enamel, but for the finishing line in dentin, the indirect technique allowed less microleakage than the direct technique. SEM analysis showed leakage similar to that observed by stereomicroscopic analysis. The use of stereomicroscopic and SEM evaluations improves microleakage analysis. PMID:16491259

  9. Ion photon emission microscopy

    NASA Astrophysics Data System (ADS)

    Rossi, P.; Doyle, B. L.; Banks, J. C.; Battistella, A.; Gennaro, G.; McDaniel, F. D.; Mellon, M.; Vittone, E.; Vizkelethy, G.; Wing, N. D.

    2003-09-01

    A new ion-induced emission microscopy has been invented and demonstrated, which is called ion photon emission microscopy (IPEM). It employs a low current, broad ion beam impinging on a sample, previously coated or simply covered with a few microns of a fast, highly efficient phosphor layer. The light produced at the single ion impact point is collected with an optical microscope and projected at high magnification onto a single photon position sensitive detector (PSD). This allows maps of the ion strike effects to be produced, effectively removing the need for a microbeam. Irradiation in air and even the use of alpha particle sources with no accelerator are possible. Potential applications include ion beam induced charge collection studies of semiconducting and insulating materials, single event upset studies on microchips and even biological cells in radiobiological effectiveness experiments. We describe the IPEM setup, including a 60× OM-40 microscope with a 1.5 mm hole for the beam transmission and a Quantar PSD with 60 μm pixel. Bicron plastic scintillator blades of 10 μm were chosen as a phosphor for their nanosecond time resolution, homogeneity, utility and commercial availability. The results given in this paper are for a prototype IPEM system. They indicate a resolution of ˜12 μm, the presence of a spatial halo and a He-ion efficiency of ˜20%. This marks the first time that nuclear microscopy has been performed with a radioactive source.

  10. Dual-CARS microscopy

    NASA Astrophysics Data System (ADS)

    Enejder, Annika; Brackmann, Christian; Burkacky, Ondrej; Åkeson, Madeleine

    2007-02-01

    We present a new Coherent Anti-Stokes Raman Scattering (CARS) microscopy technique for label-free imaging of biomolecules in living cells; dual-CARS microscopy. The use of three synchronized laser pulses in a dual-pump/dualdetection configuration enables imaging of two species with different molecular vibrations simultaneously, as well as acquisition of images free of non-resonant background. We show the power of the method by imaging deuterated nonadecane slowly diffusing into a suspension of living yeast cells in medium, clearly distinguishing the medium and the lipid droplets in the cells by probing the CH II vibration from the D-nonadecane by probing the CD vibration. In addition, images of lipid stores in living C. elegans nematodes free of non-resonant background are shown. This results in a significant enhancement of the image contrast, allowing the visualization of emerging, low-density lipid stores in a dauer larva, difficult to distinguish in conventional CARS microscopy. The separation of the non-resonant background is shown to be beneficial also when monitoring molecules with weak vibrational modes. The improved sensitivity obtained is illustrated by probing the C=C vibration in polyunsaturated lipids extracted from fish. This enables the monitoring of the degree of unsaturation of lipids, a high value of which is reported in foods known to have positive effects on human health.

  11. 48 CFR 742.770 - Negotiated indirect cost rate agreement.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Negotiated indirect cost rate agreement. 742.770 Section 742.770 Federal Acquisition Regulations System AGENCY FOR INTERNATIONAL DEVELOPMENT CONTRACT MANAGEMENT CONTRACT ADMINISTRATION Indirect Cost Rates 742.770 Negotiated indirect cost rate agreement. Except for...

  12. 34 CFR 303.225 - Prohibition against supplanting; indirect costs.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... and 34 CFR part 76 of EDGAR. (3) In charging indirect costs under paragraph (c)(2)(i) and (c)(2)(ii... 34 Education 2 2013-07-01 2013-07-01 false Prohibition against supplanting; indirect costs. 303... Prohibition against supplanting; indirect costs. (a) Each application must provide satisfactory assurance...

  13. 48 CFR 1552.242-70 - Indirect costs.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Indirect costs. As prescribed in 1542.705-70, insert the following clause in all cost-reimbursement type... Government representative. Pursuant to the “Allowable Cost and Payment” clause, the allowable indirect costs... account of indirect costs in excess of the ceiling rates listed below: Cost center Period Rate Base...

  14. 7 CFR 550.14 - Indirect cost/tuition remission.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    .... Payment of indirect costs to State Cooperative Institutions in connection with non-assistance cooperative... of indirect costs to non-profit institutions in connection with USDA cooperative agreement, under the... organizations. With the exception of paragraphs (a)(1) and (2) of this section, payment of indirect costs...

  15. 48 CFR 1342.703-2 - Certificate of indirect costs.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... costs. 1342.703-2 Section 1342.703-2 Federal Acquisition Regulations System DEPARTMENT OF COMMERCE CONTRACT MANAGEMENT CONTRACT ADMINISTRATION Indirect Cost Rates 1342.703-2 Certificate of indirect costs... indirect cost rates is set forth in CAM 1301.70....

  16. 7 CFR 550.14 - Indirect cost/tuition remission.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    .... Payment of indirect costs to State Cooperative Institutions in connection with non-assistance cooperative... of indirect costs to non-profit institutions in connection with USDA cooperative agreement, under the... organizations. With the exception of paragraphs (a)(1) and (2) of this section, payment of indirect costs...

  17. 7 CFR 550.14 - Indirect cost/tuition remission.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    .... Payment of indirect costs to State Cooperative Institutions in connection with non-assistance cooperative... of indirect costs to non-profit institutions in connection with USDA cooperative agreement, under the... organizations. With the exception of paragraphs (a)(1) and (2) of this section, payment of indirect costs...

  18. 48 CFR 1552.242-70 - Indirect costs.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Indirect costs. As prescribed in 1542.705-70, insert the following clause in all cost-reimbursement type... Government representative. Pursuant to the “Allowable Cost and Payment” clause, the allowable indirect costs... account of indirect costs in excess of the ceiling rates listed below: Cost center Period Rate Base...

  19. 48 CFR 2442.705 - Final indirect cost rates.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Final indirect cost rates. 2442.705 Section 2442.705 Federal Acquisition Regulations System DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT CONTRACT MANAGEMENT CONTRACT ADMINISTRATION Indirect Cost Rates 2442.705 Final indirect cost rates....

  20. 10 CFR 605.16 - Indirect cost limitations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... PROGRAM § 605.16 Indirect cost limitations. Awards issued under this part for conferences and scientific/technical meetings will not include payment for indirect costs. ... 10 Energy 4 2011-01-01 2011-01-01 false Indirect cost limitations. 605.16 Section 605.16...

  1. 48 CFR 1342.703-2 - Certificate of indirect costs.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... costs. 1342.703-2 Section 1342.703-2 Federal Acquisition Regulations System DEPARTMENT OF COMMERCE CONTRACT MANAGEMENT CONTRACT ADMINISTRATION Indirect Cost Rates 1342.703-2 Certificate of indirect costs... indirect cost rates is set forth in CAM 1301.70....

  2. 34 CFR 303.225 - Prohibition against supplanting; indirect costs.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 34 Education 2 2014-07-01 2013-07-01 true Prohibition against supplanting; indirect costs. 303.225... against supplanting; indirect costs. (a) Each application must provide satisfactory assurance that the... charge indirect costs to its part C grant. (2) If approved by the lead agency's cognizant Federal...

  3. 10 CFR 605.16 - Indirect cost limitations.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... PROGRAM § 605.16 Indirect cost limitations. Awards issued under this part for conferences and scientific/technical meetings will not include payment for indirect costs. ... 10 Energy 4 2012-01-01 2012-01-01 false Indirect cost limitations. 605.16 Section 605.16...

  4. 48 CFR 1342.703-2 - Certificate of indirect costs.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false Certificate of indirect costs. 1342.703-2 Section 1342.703-2 Federal Acquisition Regulations System DEPARTMENT OF COMMERCE CONTRACT MANAGEMENT CONTRACT ADMINISTRATION Indirect Cost Rates 1342.703-2 Certificate of indirect costs. The designee authorized to waive...

  5. 48 CFR 49.303-4 - Adjustment of indirect costs.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Terminated for Convenience 49.303-4 Adjustment of indirect costs. (a) If the contract contains the clause at 52.216-7, Allowable Cost and Payment, and it appears that adjustment of indirect costs will unduly... agree with the contractor to— (1) Negotiate the amount of indirect costs for the contract period...

  6. 48 CFR 1552.242-70 - Indirect costs.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Government representative. Pursuant to the “Allowable Cost and Payment” clause, the allowable indirect costs... account of indirect costs in excess of the ceiling rates listed below: Cost center Period Rate Base (End... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Indirect costs....

  7. 10 CFR 605.16 - Indirect cost limitations.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... PROGRAM § 605.16 Indirect cost limitations. Awards issued under this part for conferences and scientific/technical meetings will not include payment for indirect costs. ... 10 Energy 4 2014-01-01 2014-01-01 false Indirect cost limitations. 605.16 Section 605.16...

  8. 48 CFR 1342.703-2 - Certificate of indirect costs.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... costs. 1342.703-2 Section 1342.703-2 Federal Acquisition Regulations System DEPARTMENT OF COMMERCE CONTRACT MANAGEMENT CONTRACT ADMINISTRATION Indirect Cost Rates 1342.703-2 Certificate of indirect costs... indirect cost rates is set forth in CAM 1301.70....

  9. 34 CFR 303.225 - Prohibition against supplanting; indirect costs.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... and 34 CFR part 76 of EDGAR. (3) In charging indirect costs under paragraph (c)(2)(i) and (c)(2)(ii... 34 Education 2 2012-07-01 2012-07-01 false Prohibition against supplanting; indirect costs. 303... Prohibition against supplanting; indirect costs. (a) Each application must provide satisfactory assurance...

  10. 48 CFR 2442.705 - Final indirect cost rates.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 6 2011-10-01 2011-10-01 false Final indirect cost rates. 2442.705 Section 2442.705 Federal Acquisition Regulations System DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT CONTRACT MANAGEMENT CONTRACT ADMINISTRATION Indirect Cost Rates 2442.705 Final indirect cost rates....

  11. 48 CFR 49.303-4 - Adjustment of indirect costs.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Terminated for Convenience 49.303-4 Adjustment of indirect costs. (a) If the contract contains the clause at 52.216-7, Allowable Cost and Payment, and it appears that adjustment of indirect costs will unduly... agree with the contractor to— (1) Negotiate the amount of indirect costs for the contract period...

  12. Indirect Costs: The Past, Present, and a Possible Institutional Response.

    ERIC Educational Resources Information Center

    Zyblut, Douglas J.

    1992-01-01

    Federal regulations concerning recovery of indirect costs of university research projects are changing. Institutions should explore possible alternatives for managing reporting of indirect costs for individual projects. One system, reporting indirect cost categories in the same format as direct costs, may give insight into expenditures and help…

  13. 48 CFR 2442.705 - Final indirect cost rates.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 6 2012-10-01 2012-10-01 false Final indirect cost rates. 2442.705 Section 2442.705 Federal Acquisition Regulations System DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT CONTRACT MANAGEMENT CONTRACT ADMINISTRATION Indirect Cost Rates 2442.705 Final indirect cost rates....

  14. 10 CFR 605.16 - Indirect cost limitations.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... PROGRAM § 605.16 Indirect cost limitations. Awards issued under this part for conferences and scientific/technical meetings will not include payment for indirect costs. ... 10 Energy 4 2013-01-01 2013-01-01 false Indirect cost limitations. 605.16 Section 605.16...

  15. 48 CFR 1552.242-70 - Indirect costs.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Indirect costs. As prescribed in 1542.705-70, insert the following clause in all cost-reimbursement type... Government representative. Pursuant to the “Allowable Cost and Payment” clause, the allowable indirect costs... account of indirect costs in excess of the ceiling rates listed below: Cost center Period Rate Base...

  16. 48 CFR 1342.703-2 - Certificate of indirect costs.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... costs. 1342.703-2 Section 1342.703-2 Federal Acquisition Regulations System DEPARTMENT OF COMMERCE CONTRACT MANAGEMENT CONTRACT ADMINISTRATION Indirect Cost Rates 1342.703-2 Certificate of indirect costs... indirect cost rates is set forth in CAM 1301.70....

  17. 7 CFR 550.14 - Indirect cost/tuition remission.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    .... Payment of indirect costs to State Cooperative Institutions in connection with non-assistance cooperative... of indirect costs to non-profit institutions in connection with USDA cooperative agreement, under the... organizations. With the exception of paragraphs (a)(1) and (2) of this section, payment of indirect costs...

  18. 10 CFR 605.16 - Indirect cost limitations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... PROGRAM § 605.16 Indirect cost limitations. Awards issued under this part for conferences and scientific/technical meetings will not include payment for indirect costs. ... 10 Energy 4 2010-01-01 2010-01-01 false Indirect cost limitations. 605.16 Section 605.16...

  19. 48 CFR 942.705 - Final indirect cost rates.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Final indirect cost rates. 942.705 Section 942.705 Federal Acquisition Regulations System DEPARTMENT OF ENERGY CONTRACT MANAGEMENT CONTRACT ADMINISTRATION AND AUDIT SERVICES Indirect Cost Rates 942.705 Final indirect cost rates....

  20. 48 CFR 952.216-15 - Predetermined indirect cost rates.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Predetermined indirect cost rates. Alternate (AUG 2009): As prescribed in 916.307(g), modify paragraph (c) of the clause at FAR 52.216-15, Predetermined Indirect Cost Rates, by deleting the words “Subpart 31.4... and predetermined indirect cost rates are to be used....

  1. 48 CFR 49.303-4 - Adjustment of indirect costs.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Terminated for Convenience 49.303-4 Adjustment of indirect costs. (a) If the contract contains the clause at 52.216-7, Allowable Cost and Payment, and it appears that adjustment of indirect costs will unduly... agree with the contractor to— (1) Negotiate the amount of indirect costs for the contract period...

  2. 48 CFR 1552.242-70 - Indirect costs.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Indirect costs. As prescribed in 1542.705-70, insert the following clause in all cost-reimbursement type... Government representative. Pursuant to the “Allowable Cost and Payment” clause, the allowable indirect costs... account of indirect costs in excess of the ceiling rates listed below: Cost center Period Rate Base...

  3. 49 CFR 30.9 - Citizenship: Direct or indirect control.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Citizenship: Direct or indirect control. 30.9... Citizenship: Direct or indirect control. A contractor, subcontractor, or person providing a service shall be considered to be a citizen or national of a foreign country, or controlled directly or indirectly by...

  4. 49 CFR 30.9 - Citizenship: Direct or indirect control.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 1 2013-10-01 2013-10-01 false Citizenship: Direct or indirect control. 30.9... Citizenship: Direct or indirect control. A contractor, subcontractor, or person providing a service shall be considered to be a citizen or national of a foreign country, or controlled directly or indirectly by...

  5. 49 CFR 30.9 - Citizenship: Direct or indirect control.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 1 2012-10-01 2012-10-01 false Citizenship: Direct or indirect control. 30.9... Citizenship: Direct or indirect control. A contractor, subcontractor, or person providing a service shall be considered to be a citizen or national of a foreign country, or controlled directly or indirectly by...

  6. 49 CFR 30.9 - Citizenship: Direct or indirect control.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 1 2014-10-01 2014-10-01 false Citizenship: Direct or indirect control. 30.9... Citizenship: Direct or indirect control. A contractor, subcontractor, or person providing a service shall be considered to be a citizen or national of a foreign country, or controlled directly or indirectly by...

  7. 49 CFR 30.9 - Citizenship: Direct or indirect control.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 1 2011-10-01 2011-10-01 false Citizenship: Direct or indirect control. 30.9... Citizenship: Direct or indirect control. A contractor, subcontractor, or person providing a service shall be considered to be a citizen or national of a foreign country, or controlled directly or indirectly by...

  8. 48 CFR 42.703-2 - Certificate of indirect costs.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 1 2013-10-01 2013-10-01 false Certificate of indirect costs. 42.703-2 Section 42.703-2 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION CONTRACT MANAGEMENT CONTRACT ADMINISTRATION AND AUDIT SERVICES Indirect Cost Rates 42.703-2 Certificate of indirect costs. (a) General. In...

  9. 21 CFR 870.3640 - Indirect pacemaker generator function analyzer.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Indirect pacemaker generator function analyzer. 870.3640 Section 870.3640 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Indirect pacemaker generator function analyzer. (a) Identification. An indirect pacemaker...

  10. 21 CFR 870.3640 - Indirect pacemaker generator function analyzer.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Indirect pacemaker generator function analyzer. 870.3640 Section 870.3640 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Indirect pacemaker generator function analyzer. (a) Identification. An indirect pacemaker...

  11. 21 CFR 870.3640 - Indirect pacemaker generator function analyzer.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Indirect pacemaker generator function analyzer. 870.3640 Section 870.3640 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Indirect pacemaker generator function analyzer. (a) Identification. An indirect pacemaker...

  12. 21 CFR 870.3640 - Indirect pacemaker generator function analyzer.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Indirect pacemaker generator function analyzer. 870.3640 Section 870.3640 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Indirect pacemaker generator function analyzer. (a) Identification. An indirect pacemaker...

  13. 21 CFR 870.3640 - Indirect pacemaker generator function analyzer.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Indirect pacemaker generator function analyzer. 870.3640 Section 870.3640 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Indirect pacemaker generator function analyzer. (a) Identification. An indirect pacemaker...

  14. 34 CFR 75.561 - Approval of indirect cost rates.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Approval of indirect cost rates. 75.561 Section 75.561 Education Office of the Secretary, Department of Education DIRECT GRANT PROGRAMS What Conditions Must Be Met by a Grantee? Indirect Cost Rates § 75.561 Approval of indirect cost rates. (a) If the Department...

  15. 34 CFR 76.561 - Approval of indirect cost rates.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 34 Education 1 2012-07-01 2012-07-01 false Approval of indirect cost rates. 76.561 Section 76.561 Education Office of the Secretary, Department of Education STATE-ADMINISTERED PROGRAMS What Conditions Must Be Met by the State and Its Subgrantees? Indirect Cost Rates § 76.561 Approval of indirect cost...

  16. 34 CFR 75.561 - Approval of indirect cost rates.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 1 2011-07-01 2011-07-01 false Approval of indirect cost rates. 75.561 Section 75.561 Education Office of the Secretary, Department of Education DIRECT GRANT PROGRAMS What Conditions Must Be Met by a Grantee? Indirect Cost Rates § 75.561 Approval of indirect cost rates. (a) If the Department...

  17. 34 CFR 75.561 - Approval of indirect cost rates.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 34 Education 1 2012-07-01 2012-07-01 false Approval of indirect cost rates. 75.561 Section 75.561 Education Office of the Secretary, Department of Education DIRECT GRANT PROGRAMS What Conditions Must Be Met by a Grantee? Indirect Cost Rates § 75.561 Approval of indirect cost rates. (a) If the Department...

  18. 34 CFR 75.561 - Approval of indirect cost rates.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 34 Education 1 2013-07-01 2013-07-01 false Approval of indirect cost rates. 75.561 Section 75.561 Education Office of the Secretary, Department of Education DIRECT GRANT PROGRAMS What Conditions Must Be Met by a Grantee? Indirect Cost Rates § 75.561 Approval of indirect cost rates. (a) If the Department...

  19. 34 CFR 76.561 - Approval of indirect cost rates.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Approval of indirect cost rates. 76.561 Section 76.561 Education Office of the Secretary, Department of Education STATE-ADMINISTERED PROGRAMS What Conditions Must Be Met by the State and Its Subgrantees? Indirect Cost Rates § 76.561 Approval of indirect cost...

  20. 34 CFR 76.561 - Approval of indirect cost rates.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 1 2011-07-01 2011-07-01 false Approval of indirect cost rates. 76.561 Section 76.561 Education Office of the Secretary, Department of Education STATE-ADMINISTERED PROGRAMS What Conditions Must Be Met by the State and Its Subgrantees? Indirect Cost Rates § 76.561 Approval of indirect cost...