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Sample records for double staining antigens

  1. Two-colour immunoenzymatic technique using sequential staining by APAAP to evaluate two cell antigens.

    PubMed Central

    Burgess, R.; Hyde, K.; Maguire, P. J.; Kelsey, P. R.; Yin, J. A.; Geary, C. G.

    1992-01-01

    AIMS: To extend the alkaline phosphatase-antialkaline phosphatase (APAAP) immunoenzyme single stain method to a more generally applicable double stain technique. This will allow two primary antibodies of the same isotype of IgG and specifically the nuclear antigen bromodeoxyuridine (BRdU) to be evaluated with a cell surface antigen identifier. METHOD: Sequential applications of the APAAP method showed two antigen sites by different dye couplings to a common alkaline phosphatase substrate, producing blue and red reaction products on the same slide. Antigens on different cell populations as well as those in different compartments of the same cell were analysed. The method allowed a surface antigen monoclonal to be revealed first, using an optimal fixative, before alcohol/gluteraldehyde fixation was used to start the second (BRdU) staining sequence. RESULTS: An analysis of double staining of T lymphocyte subsets (CD4 and CD8) showed no significant difference in the order of application of the primaries (n = 10) and no significant difference from their corresponding single stain results (n = 50), confirming the validity of the technique where antigens are exclusively distributed. Other examples, including antigens distributed in different compartments of the same cell, displayed discrete staining which implied validity. CONCLUSION: Double staining by APAAP with this technique seems to be applicable to those cases where antigens are exclusively distributed and includes cases where different compartments of the same cell are stained. It is especially useful in revealing antigens that require different fixation and preparation--that is DNA incorporated BRdU with a surface antigen. But it does seem to have a limited ability to produce a dual colour at a common site. Images PMID:1372917

  2. Protein Stains to Detect Antigen on Membranes.

    PubMed

    Dsouza, Anil; Scofield, R Hal

    2015-01-01

    Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after western blotting. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. "Detection" is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical. PMID:26139252

  3. Reaction of goat's milk with stained antigens in the detection of brucellosis

    PubMed Central

    Alivisatos, G. P.; Edipides, T.

    1953-01-01

    Testing for brucellosis in goats by the reaction of milk with stained antigens was carried out on 206 milk-samples and the results compared with those of sero-agglutination tests on the same animals. The authors find that in most cases the milk reactions correspond to the sero-agglutination results, and that it is the agglutination of the stained antigen which is the principal sign of a positive reaction rather than ring formation, which is very irregular. Haematoxylin antigen is generally more reliable than Bendtsen antigen, but, with either, the results should be read after 1-2 hours' incubation at 37°C. The authors conclude that the stained antigen test can be used for the detection of infection in herds of goats, but that it is not yet possible to say whether it is also valuable for detecting individual infection. PMID:13141139

  4. Ultrastructural study of Chlamydia trachomatis surface antigens by immunogold staining with monoclonal antibodies.

    PubMed Central

    Kuo, C C; Chi, E Y

    1987-01-01

    Surface antigens of Chlamydia trachomatis were studied by immunogold staining with monoclonal antibodies and by electron microscopy. The serovar- and subspecies-specific epitopes were the most surface accessible. The species- and genus-specific epitopes were the least surface exposed. Similar serological specificity as that in the microimmunofluorescence test was demonstrated by immunogold staining. Images PMID:2437035

  5. Cell-cycle-related staining patterns of anti-proliferating cell nuclear antigen monoclonal antibodies. Comparison with BrdUrd labeling and Ki-67 staining.

    PubMed Central

    van Dierendonck, J. H.; Wijsman, J. H.; Keijzer, R.; van de Velde, C. J.; Cornelisse, C. J.

    1991-01-01

    Monoclonal antibodies (MAbs) to nuclear antigens are increasingly used as tools to obtain valuable information concerning the proliferative characteristics of various types of cancer. Prerequisite for the application of these MAbs in surgical pathology is establishment of the level of expression and/or cellular distribution of the antigens in relation to distinct cell-cycle compartments. In this study the topologic distribution of proliferating cell nuclear antigen (PCNA), an auxiliary protein of DNA polymerase delta, as recognized by human autoantiserum (AK) and two recently developed MAbs (19A2 and 19F4), was evaluated. Using cultured human cancer cells as a model system, and providing optimal fixation/permeation procedures are applied, these antibodies display a high affinity for PCNA bound to nuclear replicon clusters, resulting in distinct granular staining patterns. A more diffuse nucleoplasmic PCNA staining was mainly restricted to non-S-phase cells; in methanol-fixed cells, staining intensity of this form relative to the replicon-bound form appeared higher after staining with 19A2 than with 19F4 or AK. Comparing PCNA expression (detected with 19A2) with the expression of the Ki-67 antigen, PCNA-negative cells are also Ki-67 negative. In MCF-7 human breast cancer cells treated with 10(-6) mol/l (molar) tamoxifen, the fraction of nuclei showing replication patterns decreased from 42% to 8% within 8 days, but PCNA and Ki-67 antigens remained detectable in most cells during this interval, indicating a relatively slow decrease of antigen expression in cells that have entered a quiescent state. Treatment of MCF-7 cells with 10(-6) mol/l methotrexate resulted in a rapid accumulation of cells with an early S-phase DNA content; PCNA replication patterns showing a frequency distribution reflecting this DNA content were observed up to 48 hours after treatment. This indicates that the presence of replication patterns as visualized with anti-PCNAs is not a measure of

  6. [Development of a novel Francisella tularensis antigen stained with tetrazolium-blue for tularemia microagglutination test].

    PubMed

    Celebi, Bekir; Kılıç, Selçuk

    2013-07-01

    The isolation of Francisella tularensis in cultures is the reference method for the laboratory diagnosis of tularemia. However, due to the limitations such as the low sensitivity and need for high safety level and equipped laboratories, serologic methods are frequently used as diagnostic tools. F.tularensis-specific antibodies may be demonstrated by several methods, however microagglutination test (MA) remains the most common method with its high sensitivity and specificity. The aim of this study was to develop a novel MA test antigen prepared from whole F.tularensis cells and stained with tetrazolium-blue for more clear and easier evaluation. F.tularensis NCTC 10857 strain was cultured on the cysteine heart agar supplemented with 9% sheep blood and bacterial cells were harvested by scraping, collected in physiological saline (PS) and centrifuged at 1500 rpm for 10 minutes. For preparing stock antigen suspension cell concentration was adjusted to OD600=1.5, spectrophotometrically. Tetrazolium-blue solution (BTC [3,3'-(3,3'-Dimethoxy[1,1'-biphenyl]-4,4'-diyl) bis [2,5-diphenyl-2H-tetrazolium dichloride], T4375 Sigma-Aldrich) at the final concentration of 1% was added to cell suspension and incubated at 37°C for 5 hours for absorption. Then, the living cells were chemically inactivated by formaldehyde. Repeating centrifugations were performed to discard excess dye and formaline, then 0.4% formaline saline was added on the sediment. Optimum concentration of this novel antigen (BTC-Ag) for MA test was determined by plate titration method by using standard serum sample with a known MA titer (1/128). The performance of BTC-Ag in MA test was evaluated by using 100 patient sera positive for F.tularensis antibodies, and 100 tularemia negative patient sera (of them 50 were seropositive for brucellosis). All of the results were compared with standard MA test in which safranin-O stained antigen (SO-Ag) was used. There was 100% agreement between the two tests performed with

  7. In Situ Peptide-MHC-II Tetramer Staining of Antigen-Specific CD4+ T Cells in Tissues.

    PubMed

    Dileepan, Thamotharampillai; Kim, Hyeon O; Cleary, P Patrick; Skinner, Pamela J

    2015-01-01

    The invention of peptide-MHC-tetramer technology to label antigen-specific T cells has led to an enhanced understanding of T lymphocyte biology. Here we describe the development of an in situ pMHC-II tetramer staining method to visualize antigen-specific CD4+ T cells in tissues. This method complements other methods developed that similarly use MHC class II reagents to stain antigen-specific CD4+ T cells in situ. In this study, we used group A streptococcus (GAS) expressing a surrogate peptide (2W) to inoculate C57BL/6 mice, and used fresh nasal-associated lymphoid tissues (NALT) in optimizing the in situ staining of 2W:I-Ab specific CD4+ T cells. The results showed 2W:I-Ab tetramer-binding CD4+ T cells in GAS-2W but not GAS infected mice. This method holds promise to be broadly applicable to study the localization, abundance, and phenotype of antigen-specific CD4+ T cells in undisrupted tissues. PMID:26067103

  8. 9 CFR 147.3 - The stained-antigen, rapid, whole-blood test. 3

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... medicine dropper whose tip is adjusted to deliver 0.05 cc. is used to measure the antigen. A glass plate... still clearly visible clumps of antigen surrounded by spaces only partially clear. Between this...

  9. 9 CFR 147.3 - The stained-antigen, rapid, whole-blood test. 3

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... medicine dropper whose tip is adjusted to deliver 0.05 cc. is used to measure the antigen. A glass plate... still clearly visible clumps of antigen surrounded by spaces only partially clear. Between this...

  10. 9 CFR 147.3 - The stained-antigen, rapid, whole-blood test. 3

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... medicine dropper whose tip is adjusted to deliver 0.05 cc. is used to measure the antigen. A glass plate... still clearly visible clumps of antigen surrounded by spaces only partially clear. Between this...

  11. Patterns of MiB-1 staining of brain tumors: microwave-antigen retrieval as a tool in diagnostic pathology.

    PubMed

    Meera, G; Jo, H; Ravi, R

    1995-11-01

    MiB-1 antibody staining can be carried out on formalin fixed, paraffin embedded tissue only if microwave antigen retrieval is performed. This method was used to identify proliferating cells in archival paraffin sections of 137 cases of human brain tumors. The tumors concerned were astrocytomas (57), glioblastomas (16), oligodendrogliomas (11), mixed gliomas (8), ependymomas (9), subependymoma (2), medulloblastomas (4) and meningiomas (30). For the tumors which were histologically graded (the astrocytomas, oligodendrogliomas and ependymomas), generally the scores of MiB-1 staining were correlated with the tumor grade, although there were exceptions to this rule, for example, a few low grade tumors displayed high MiB-1 counts while some highly malignant brain tumors failed to display any positive staining for MiB-1, despite having high mitotic counts. This phenomenon was probably due to loss of proliferating antigens. In the MiB-1 positive sections, hot spots with a high mitotic activity could be identified with ease and the abnormal morphology of the tumor nuclei and of the atypical mitoses were highlighted by the contrasting intensily brown staining. In addition, the dividing cell type could be easily identified and differences in mitotic behavior of invasive and necrotic parts of tumors could be observed. PMID:8835134

  12. A double filtering method for measuring the translational velocity of fluorescently stained cells

    SciTech Connect

    Yasokawa, Toshiki; Ishimaru, Ichirou; Kuriyama, Shigeki; Masaki, Tsutomu; Takegawa, Kaoru; Tanaka, Naotaka

    2007-09-24

    The authors propose a double filtering method to measure translational velocity for tracking fluorescently stained cells. This method employs two diffraction gratings installed in the infinity space through which the parallel pencil beam of the fluorescence passes. With this method, the change in light intensity whose period is proportional to the translational velocity of the sample can be obtained at the imaging surface. By using a sample that has a random distribution of fluorescence intensity, the authors verified that translational velocity measurements could be achieved using the proposed method.

  13. 9 CFR 147.3 - The stained-antigen, rapid, whole-blood test. 3

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... medicine dropper whose tip is adjusted to deliver 0.05 cc. is used to measure the antigen. A glass plate about 15 inches square, providing space for 48 tests, has proved satisfactory for this work. The use...

  14. 9 CFR 147.3 - The stained-antigen, rapid, whole-blood test. 3

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... medicine dropper whose tip is adjusted to deliver 0.05 cc. is used to measure the antigen. A glass plate about 15 inches square, providing space for 48 tests, has proved satisfactory for this work. The use...

  15. Flow cytometric two-color staining technique for simultaneous determination of human erythrocyte membrane antigen and intracellular malarial DNA.

    PubMed

    Pattanapanyasat, K; Webster, H K; Udomsangpetch, R; Wanachiwanawin, W; Yongvanitchit, K

    1992-01-01

    A novel fixative and permeabilization method is described which allows simultaneous flow cytometric detection of red blood cell membrane antigen and intracellular malaria parasites. To illustrate the method, red blood cells from patients with paroxysmal nocturnal hemoglobinuria were infected with Plasmodium falciparum and maintained in synchronous red blood cell culture. The infected red blood cells were immunolabeled with antibodies directed to the complement regulatory protein decay-accelerating factor (DAF) followed by subsequent fixations in paraformaldehyde and then glutaraldehyde in phosphate-buffered saline. Finally, DNA of the intraerythrocytic parasites was stained with propidium iodide. Using this technique, cellular morphology was well preserved, no cell aggregation was observed, and high-quality indirect immunofluorescence and parasite DNA staining were obtained with negligible nonspecific labelling. Simultaneous measurement of parasite DNA and red blood cell membrane determinants makes possible the investigation of alterations of red cell membrane proteins in association with development of intracellular malaria parasites. PMID:1372210

  16. Unexpected interference in cell surface staining by monoclonal antibodies to unrelated antigens.

    PubMed

    De Vita, Martina; Catzola, Valentina; Buzzonetti, Alexia; Fossati, Marco; Battaglia, Alessandra; Zamai, Loris; Fattorossi, Andrea

    2014-10-01

    Background: The possible occurrence of an erroneous immunophenotyping due to interference between monoclonal antibodies (MoAbs) is often overlooked when the epitopes are assumed to be not close to each other. This is particularly important when exploring immune cell populations whose identification is still investigational. The commonly held view is that myeloid derived suppressor cells (MDSC) can be identified as either HLA-DR(neg) (/dim) cells or interleukin-4 receptor-α (CD124)(+) cells among peripheral blood monocytes. We made the serendipitous observation that the fluorescence signal provided by the PE-CD124 MoAb was attenuated when the PE-CF594-HLA-DR MoAb was added to the staining tube. Methods: Peripheral blood mononuclear cells (PBMC) from healthy donors were stained with the PE-CD124 MoAb and, as control, PE -CD40, -CD4 and -CD14, and either the PE-CF594-HLA-DR MoAb or its unlabeled form. B cells, which also express CD124, were analyzed for comparison. Results: The PE-CF594-HLA-DR MoAb but not its unlabeled form reduced PE-CD124 MoAb staining on monocytes and B cells. No other monocyte and B cell surface marker staining was affected by the PE-CF594-HLA-DR MoAb. The PE-CF594-HLA-DR MoAb interfered with the PE-CD124 MoAb likely because of steric hindrance by bulky fluorochromes, although a quenching due to fluorescence resonance energy transfer might also cooperate to the PE-CD124 MoAb staining attenuation. Conclusions: Present observations highlight the importance of interference between MoAbs as a source of error when analyzing multicolor flow cytometry data. © 2014 Clinical Cytometry Society. PMID:25270399

  17. A novel assay for typing Rh antigens in blood-stains using a lectin specific to the bisecting N-acetyl-D-glucosamine side chain of glycoprotein.

    PubMed

    Matsubara, K; Tanabe, K; Akane, A; Nakamura, H; Takahashi, S; Kimura, K

    1994-08-01

    A unique sandwich enzyme-linked immunosorbent assay for the determination of Rh antigens in blood stains has been developed using Rh antisera and phaseolus vulgaris E4/peroxidase conjugate (PHAE4/PO). The appropriate antiserum for detecting Rh C, c, D, E or e was coated on the inner surface of microplate wells, and the sample antigens from blood stains, solubilized with n-octyl-beta-D-glucopyranoside, were then placed in the wells. After washing the wells repeatedly, PHAE4/PO was added. Bound PHAE4/PO was detected by the development of colors using o-phenylenediamine/H2O2. All Rh antigens corresponding to the antisera were clearly detected using this technique. The detection limit expressed by sample dilution was more than 2 x 10(5) times (volume/dried blood weight) for the various antigens from the fresh 5 x 5 mm2 blood stain. Even when the blood stain samples were left beside a sunny window at room temperature for 2 months, Rh antigens were still detected. When the ABH, MN, P1, Kidd, Duffy and Lewis blood grouping systems were tested with similar ELISA procedures PHAE4 did not recognize any antigen. Since PHAE4 specifically recognizes and combines with the bisecting N-acetyl-D-glucosamine side chain, it was concluded that the glycoprotein was a component of all Rh antigens immune complexes. PMID:8046252

  18. A double fluorescence staining protocol to determine the cross-sectional area of myofibers using image analysis

    NASA Technical Reports Server (NTRS)

    Mozdziak, P. E.; Fassel, T. A.; Schultz, E.; Greaser, M. L.; Cassens, R. G.

    1996-01-01

    A double fluorescence staining protocol was developed to facilitate computer based image analysis. Myofibers from experimentally treated (irradiated) and control growing turkey skeletal muscle were labeled with the anti-myosin antibody MF-20 and detected using fluorescein-5-isothiocyanate (FITC). Extracellular material was stained with concanavalin A (ConA)-Texas red. The cross-sectional area of the myofibers was determined by calculating the number of pixels (0.83 mu m(2)) overlying each myofiber after subtracting the ConA-Texas red image from the MF-20-FITC image for each region of interest. As expected, myofibers in the irradiated muscle were smaller (P < 0.05) than those in the non-irradiated muscle. This double fluorescence staining protocol combined with image analysis is accurate and less labor-intensive than classical procedures for determining the cross-sectional area of myofibers.

  19. Comparison of Fecal Antigen Detection Using Enzyme Linked Immunosorbent Assay With the Auramine Phenol Staining Method for Diagnosis of Human Cryptosporidiosis

    PubMed Central

    Jafari, Rasool; Maghsood, Amir Hossein; Safari, Marzieh; Latifi, Milad; Fallah, Mohammad

    2015-01-01

    Background: Fecal antigen detection using the enzyme linked immunosorbent assay (ELISA) and oocyst detection using auramine phenol (AP) staining methods, are told to be more sensitive compared to other conventional methods, for diagnosis of cryptosporidiosis. Objectives: The aim of the present study was to evaluate the antigen-detection capacity in the stool specimens using ELISA and oocyst detection by AP staining methods, for the diagnosis of human cryptosporidiosis. Materials and Methods: A total of 228 fecal samples were collected from residents of rural areas of Hamadan, West of Iran. Each fecal sample was divided into two parts, one kept frozen at -20˚C for Ag-capture ELISA and the other in 10% formalin for the AP staining method. Cryptosporidium Ag-detection ELISA procedure was performed according to the manual of the manufacturer. The preserved samples concentrated using the formalin-ether concentration technique were stained with AP and then investigated under florescent microscopy. Results: Eight (3.5%) and three (1.3%) out of 228 fecal samples were positive for Cryptosporidium infection by ELISA and AP staining methods, respectively. Cryptosporidium Ag-detection using ELISA showed an increased frequency of the infection, compared to the AP staining method (P = 0.062). Conclusions: For epidemiological studies and diagnostic purposes of the Cryptosporidium infection, especially in asymptomatic individuals, Ag-detection ELISA is an easy to perform and accurate method, compared to other conventional microscopic methods. PMID:25825642

  20. Comparing two methods of plastination and glycerin preservation to study skeletal system after Alizarin red-Alcian blue double staining

    PubMed Central

    Mohsen, Setayesh M.; Esfandiari, Ebrahim; Rabiei, Abbas A.; Hanaei, Mahsa S.; Rashidi, Bahman

    2013-01-01

    Background: Plastination is a new method of preserving tissue samples for a long time. This study aimed to compare the new plastination technique with the conventional preservative method in glycerin for fetus skeleton tissues and young rats dyed by Alizarin red- Alcian blue double staining. Materials and Methods: In this study, 4 groups of 1-day, 3-day, 12-day and mature rats were selected and, after being anesthetized and slaughtered, their skin was completely removed. In Alizarin red- Alcian blue double staining method, first the samples were fixed in 95% ethanol and then their cartilages were dyed by 0.225% Alcian blue solution; after that, they were cleared in 1% KOH. Then, the bones were dyed in 0.003% Alizarin red solution and finally the tissue was decolorized in 95% ethanol. In each group, half of the samples were preserved by the conventional method in a glycerin container and the other half were plastinated. Results: In the present study, the samples preserved by plastination technique were dry, odorless, indecomposable and tangible. Quality of coloring had an inverse relationship with rats’ age. Transparency of the plastinated samples had also an inverse relationship with rats’ age. Therefore, skeletal tissue of younger rats had higher quality and transparency in both preservation methods (glycerin and plastination). Conclusion: This study showed that plastination technique was an appropriate method in comparison with glycerin preservation, which conserved skeletal tissue of fetus and young rats colored by Alizarin red- Alcian blue double staining. And the final result was that plastination technique can generate dry, odorless, indecomposable and tangible samples. PMID:23930264

  1. Proliferation assessment in breast carcinomas using digital image analysis based on virtual Ki67/cytokeratin double staining.

    PubMed

    Røge, Rasmus; Riber-Hansen, Rikke; Nielsen, Søren; Vyberg, Mogens

    2016-07-01

    Manual estimation of Ki67 Proliferation Index (PI) in breast carcinoma classification is labor intensive and prone to intra- and interobserver variation. Standard Digital Image Analysis (DIA) has limitations due to issues with tumor cell identification. Recently, a computer algorithm, DIA based on Virtual Double Staining (VDS), segmenting Ki67-positive and -negative tumor cells using digitally fused parallel cytokeratin (CK) and Ki67-stained slides has been introduced. In this study, we compare VDS with manual stereological counting of Ki67-positive and -negative cells and examine the impact of the physical distance of the parallel slides on the alignment of slides. TMAs, containing 140 cores of consecutively obtained breast carcinomas, were stained for CK and Ki67 using optimized staining protocols. By means of stereological principles, Ki67-positive and -negative cell profiles were counted in sampled areas and used for the estimation of PIs of the whole tissue core. The VDS principle was applied to both the same sampled areas and the whole tissue core. Additionally, five neighboring slides were stained for CK in order to examine the alignment algorithm. Correlation between manual counting and VDS in both sampled areas and whole core was almost perfect (correlation coefficients above 0.97). Bland-Altman plots did not reveal any skewness in any data ranges. There was a good agreement in alignment (>85 %) in neighboring slides, whereas agreement decreased in non-neighboring slides. VDS gave similar results compared with manual counting using stereological principles. Introduction of this method in clinical and research practice may improve accuracy and reproducibility of Ki67 PI. PMID:27283833

  2. Gram stain

    MedlinePlus

    A Gram stain is a test used to identify bacteria. It is one of the most common ways to ... Urethral discharge Gram stain; Feces Gram stain; Stool Gram stain; Joint fluid Gram stain; Pericardial fluid Gram stain; Gram stain of ...

  3. Localization of hepatitis B surface antigen in conventional paraffin sections of the liver. Comparison of immunofluorescence, immunoperoxidase, and orcein staining methods with regard to their specificity and reliability as antigen marker.

    PubMed Central

    Nayak, N. C.; Sachdeva, R.

    1975-01-01

    Hepatitis B antigen (HBAg) has been demonstrated in conventional formalin-fixed paraffin-embedded liver tissue by peroxidase and fluorescent immunostaining as well as by orcein. Complete locational and morphologic identity is seen between material stained by specific immunologic methods and by orcein. The antigen is restricted to the cytoplasm and is generally observed in the hepatocyte; it is present in three morphologic forms. Certain morphologic forms can even be identified in hematoxylin and eosin-stained tissue. Results of immunostaining procedures indicate that the antigen demonstrated in this study consists entirely of surface coat of hepatitis B virus (HBsAg). This seems to be the only component revealed by orcein staining. The latter is considered to be a good marker of the surface antigen and to have certain advantages over immunostaining. It is suggested that suitability of conventional paraffin sections for the detection of HBAg has wide and important implications. Images Figures 1-5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 PMID:55076

  4. DNA Double-Strand Breaks and Telomeres Play Important Roles in Trypanosoma brucei Antigenic Variation

    PubMed Central

    2015-01-01

    Human-infecting microbial pathogens all face a serious problem of elimination by the host immune response. Antigenic variation is an effective immune evasion mechanism where the pathogen regularly switches its major surface antigen. In many cases, the major surface antigen is encoded by genes from the same gene family, and its expression is strictly monoallelic. Among pathogens that undergo antigenic variation, Trypanosoma brucei (a kinetoplastid), which causes human African trypanosomiasis, Plasmodium falciparum (an apicomplexan), which causes malaria, Pneumocystis jirovecii (a fungus), which causes pneumonia, and Borrelia burgdorferi (a bacterium), which causes Lyme disease, also express their major surface antigens from loci next to the telomere. Except for Plasmodium, DNA recombination-mediated gene conversion is a major pathway for surface antigen switching in these pathogens. In the last decade, more sophisticated molecular and genetic tools have been developed in T. brucei, and our knowledge of functions of DNA recombination in antigenic variation has been greatly advanced. VSG is the major surface antigen in T. brucei. In subtelomeric VSG expression sites (ESs), VSG genes invariably are flanked by a long stretch of upstream 70-bp repeats. Recent studies have shown that DNA double-strand breaks (DSBs), particularly those in 70-bp repeats in the active ES, are a natural potent trigger for antigenic variation in T. brucei. In addition, telomere proteins can influence VSG switching by reducing the DSB amount at subtelomeric regions. These findings will be summarized and their implications will be discussed in this review. PMID:25576484

  5. [Definitive ability of Stamp-staining, antigen-ELISA, PCR and cell culture for the detection of Coxiella burnetii].

    PubMed

    Henning, Klaus; Sting, Reinhard

    2002-01-01

    Many assays are used for the detection of the aetiological agent of Q fever, Coxiella burnetii, i.e. staining according to the method of Stamp, capture ELISA, PCR or isolation by cell culture. In this study the results of these four assays are compared for their sensitivity and specificity. Staining smears according to the method of Stamp gave many false positive or false negative results. The capture ELISA seems to be a very sensitive assay for the detection of Coxiella burnetii but it has a lack in specificity. It is a useful test system for the screening of large scales of samples. Positive ELISA results should be confirmed by PCR, a very sensitive and specific method for the detection of Coxiella burnetii but more time consumptive than the ELISA. Isolation of the agent using cell cultures was not completely satisfactory because of its lack in sensitivity. Therefore it should only be used in special cases. PMID:12357676

  6. Double-antigen sandwich ELISA for the detection of anti-hepatitis C virus antibodies.

    PubMed

    He, Jing; Xiu, Bingshui; Wang, Guohua; Chen, Kun; Feng, Xiaoyan; Song, Xiaoguo; Zhu, Cuixia; Ling, Shigan; Zhang, Heqiu

    2011-01-01

    A double-antigen sandwich ELISA was developed a detection of HCV antibodies by a recombinant multi-epitope HCV antigen and a biotin-streptavidin amplification system. Three plasma specimens from 1708 individuals who were suspected previously to be HCV-positive using an HCV antibody diagnostic kit (Chuangxin, Xiamen, China) displayed negative results when using the ELISA. These results were validated by a recombinant immunoblotting assay (two were negative, and one was indeterminate). Among 889 blood specimens donated for clinical evaluation, 246 were positive and 630 were negative using the ELISA. The sensitivity and specificity of the ELISA were 98.7% and 100%, respectively. In 43 donors and 14 patients with chronic hepatitis C, the detectable rates for HCV IgM by both ELISA and the HCV anti-IgM detection reagents (Huimin, Shenyang, China) were 100%, and the detectable rate for HCV IgG using an indirect HCV-antibody detection kit (GWK, Beijing, China) was 98.3%. Thus, the double-antigen sandwich ELISA exhibits strong specificity and sensitivity and has been approved by the China State Food and Drug Administration (SFDA). The performance of the double-antigen sandwich ELISA was similar to the Ortho ELISA 3.0. It did not give false-negative results otherwise IgM was undetectable using an indirect HCV-antibody detection kit. This ELISA provides another method for the detection of HCV antibodies. PMID:21029749

  7. Genomic organization and evolution of double minutes/homogeneously staining regions with MYC amplification in human cancer.

    PubMed

    L'Abbate, Alberto; Macchia, Gemma; D'Addabbo, Pietro; Lonoce, Angelo; Tolomeo, Doron; Trombetta, Domenico; Kok, Klaas; Bartenhagen, Christoph; Whelan, Christopher W; Palumbo, Orazio; Severgnini, Marco; Cifola, Ingrid; Dugas, Martin; Carella, Massimo; De Bellis, Gianluca; Rocchi, Mariano; Carbone, Lucia; Storlazzi, Clelia Tiziana

    2014-08-01

    The mechanism for generating double minutes chromosomes (dmin) and homogeneously staining regions (hsr) in cancer is still poorly understood. Through an integrated approach combining next-generation sequencing, single nucleotide polymorphism array, fluorescent in situ hybridization and polymerase chain reaction-based techniques, we inferred the fine structure of MYC-containing dmin/hsr amplicons harboring sequences from several different chromosomes in seven tumor cell lines, and characterized an unprecedented number of hsr insertion sites. Local chromosome shattering involving a single-step catastrophic event (chromothripsis) was recently proposed to explain clustered chromosomal rearrangements and genomic amplifications in cancer. Our bioinformatics analyses based on the listed criteria to define chromothripsis led us to exclude it as the driving force underlying amplicon genesis in our samples. Instead, the finding of coexisting heterogeneous amplicons, differing in their complexity and chromosome content, in cell lines derived from the same tumor indicated the occurrence of a multi-step evolutionary process in the genesis of dmin/hsr. Our integrated approach allowed us to gather a complete view of the complex chromosome rearrangements occurring within MYC amplicons, suggesting that more than one model may be invoked to explain the origin of dmin/hsr in cancer. Finally, we identified PVT1 as a target of fusion events, confirming its role as breakpoint hotspot in MYC amplification. PMID:25034695

  8. Rapid detection of total and viable Legionella pneumophila in tap water by immunomagnetic separation, double fluorescent staining and flow cytometry.

    PubMed

    Keserue, Hans-Anton; Baumgartner, Andreas; Felleisen, Richard; Egli, Thomas

    2012-11-01

    We developed a rapid detection method for Legionella pneumophila (Lp) by filtration, immunomagnetic separation, double fluorescent staining, and flow cytometry (IMS-FCM method). The method requires 120 min and can discriminate 'viable' and 'membrane-damaged' cells. The recovery is over 85% of spiked Lp SG 1 cells in 1 l of tap water and detection limits are around 50 and 15 cells per litre for total and viable Lp, respectively. The method was compared using water samples from house installations in a blind study with three environmental laboratories performing the ISO 11731 plating method. In 53% of the water samples from different taps and showers significantly higher concentrations of Lp were detected by flow cytometry. No correlation to the plate culture method was found. Since also 'viable but not culturable' (VNBC) cells are detected by our method, this result was expected. The IMS-FCM method is limited by the specificity of the used antibodies; in the presented case they target Lp serogroups 1-12. This and the fact that no Lp-containing amoebae are detected may explain why in 21% of all samples higher counts were observed using the plate culture method. Though the IMS-FCM method is not yet fit to completely displace the established plating method (ISO 11731) for routine Lp monitoring, it has major advantages to plating and can quickly provide important insights into the ecology of this pathogen in water distribution systems. PMID:23062200

  9. Negative staining and immunoelectron microscopy of adhesion-deficient mutants of Streptococcus salivarius reveal that the adhesive protein antigens are separate classes of cell surface fibril.

    PubMed Central

    Weerkamp, A H; Handley, P S; Baars, A; Slot, J W

    1986-01-01

    The subcellular distribution of the cell wall-associated protein antigens of Streptococcus salivarius HB, which are involved in specific adhesive properties of the cells, was studied. Mutants which had lost the adhesive properties and lacked the antigens at the cell surface were compared with the parent strain. Immunoelectron microscopy of cryosections of cells labeled with affinity-purified, specific antisera and colloidal gold-protein A complexes was used to locate the antigens. Antigen C (AgC), a glycoprotein involved in attachment to host surfaces, was mainly located in the fibrillar layer outside the cell wall. A smaller amount of label was also found throughout the cytoplasmic area in the form of small clusters of gold particles, which suggests a macromolecular association. Mutant HB-7, which lacks the wall-associated AgC, accumulated AgC reactivity intracellularly. Intracellular AgC was often found associated with isolated areas of increased electron density, but sometimes seemed to fill the entire interior of the cell. Antigen B (AgB), a protein responsible for interbacterial coaggregation, was also located in the fibrillar layer, although its distribution differed from that of the wall-associated AgC since AgB was found predominantly in the peripheral areas. A very small amount of label was also found in the cytoplasmic area as discrete gold particles. Mutant HB-V5, which lacks wall-associated AgB, was not labeled in the fibrillar coat, but showed the same weak intracellular label as the parent strain. Immunolabeling with serum against AgD, another wall-associated protein but of unknown function, demonstrated its presence in the fibrillar layer of strain HB. Negatively stained preparations of whole cells of wild-type S. salivarius and mutants that had lost wall-associated AgB or AgC revealed that two classes of short fibrils are carried on the cell surface at the same time. AgB and AgC are probably located on separate classes of short, protease

  10. Variable pulse frequency-doubled Nd:YAG laser versus flashlamp-pumped pulsed dye laser in the treatment of port wine stains.

    PubMed

    Lorenz, Susanne; Scherer, Kathrin; Wimmershoff, Monika Beatrix; Landthaler, Michael; Hohenleutner, Ulrich

    2003-01-01

    The flashlamp-pumped pulsed dye laser (FPDL) is regarded as the gold standard in the treatment of port wine stains. The purpose of this prospective, intra-individual, comparative clinical study was to investigate whether a frequency-doubled variable pulsed Nd:YAG laser (frequency-doubled Nd:YAG) is equally as safe and effective as established lasers. Forty-three patients with port wine stains were included in the study. Test treatments were performed using the frequency-doubled Nd:YAG laser (532 nm; 4 mm psi; 5-50 ms; 5.5 to 15 J/cm2) versus the FPDL (585 nm; 450 micros; 7 mm psi; 6 J/cm2). After 6 weeks, a full lesional treatment was performed using the device and the parameters showing the best clearance and the fewest side effects. The clearance of the lesions was generally good to fair. With the exception of poor results at 5 ms and 5.5 J/cm2 with the frequency-doubled Nd:YAG laser, there were no significant differences between the two laser devices. Scar formation, nevertheless, occurred in only 3% of the FPDL-treated sites versus up to 18% of the frequency-doubled Nd:YAG sites, increasing with pulse duration. In port wine stains, the FPDL remains the therapy of choice because of the somewhat better results and a lower frequency of side effects, especially scarring. PMID:12816158

  11. Antigen

    MedlinePlus

    An antigen is any substance that causes your immune system to produce antibodies against it. This means your immune ... and is trying to fight it off. An antigen may be a substance from the environment, such ...

  12. Decreased antigenicity profiles of immune-escaped and drug-resistant hepatitis B surface antigen (HBsAg) double mutants

    PubMed Central

    2013-01-01

    Background Selective pressure from either the immune response or the use of nucleoside analogs in antiviral therapy could be driving the emergence of HBV mutants. Because of the overlap of the open reading frame (ORF) S for the HBsAg and ORF P for viral polymerase, rtM204I and rtM204V mutations in the polymerase would produce sI195M and sW196S in the HBsAg. The combined effects of immune-escaped mutations (sT118M, sG145K, sG145R) and drug-resistant mutations (rtM204I, rtM204V) on the antigenicity profiles of HBsAg has not been widely explored. Methods To determine the combined effects of immune-escaped and drug-resistant mutants on the antigenicity profiles of HBsAg, recombinant plasmids encoding HBsAg double mutants were constructed using site-directed mutagenesis. The supernatant from each plasmid transfection was analyzed for HBsAg in the western-blotting and five of the most commonly used commercial ELISA kits in China. Results Western-blotting assay showed the successful expression of each HBsAg mutant. All five ELISA kits manifested similar avidity, which were demonstrated by the slope of the curves, for the sT118M mutant, and sT118M-rtM204I (sT118M-sI195M) and sT118M-rtM204V (sT118M-sW196S) double mutants, suggesting that drug-resistant YMDD mutants caused negligible losses in the antigenicity of immune-escaped sT118M HBsAg. In contrast, the presence of the rtM204I (sI195M) mutation, but not rtM204V (sW196S) in combination with the sG145K mutation significantly reduced the avidity of sG145K HBsAg. The rtM204I (sI195M) mutation also decreased the antigenicity profiles for sG145R HBsAg. Conclusions Drug-resistant mutations rtM204I (sI195M) and rtM204V (sW196S) caused significant reduction in antigenicity for the immune-escaped HBsAg mutants sG145K and sG145R, which may hamper HBV diagnosis and disease control from HBV blood-transfusion transmissions in China. The development of ELISA kits with a greater sensitivity for drug-resistant and immune-escaped HBs

  13. 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

  14. Identification of 5-bromo-2'-deoxyuridine-labeled cells during mouse spermatogenesis by heat-induced antigen retrieval in lectin staining and immunohistochemistry.

    PubMed

    Wakayama, Tomohiko; Nakata, Hiroki; Kumchantuek, Tewarat; Gewaily, Mahmoud Saad; Iseki, Shoichi

    2015-03-01

    DNA replication occurs during S-phase in spermatogonia and preleptotene spermatocytes during spermatogenesis. 5-Bromo-2'-deoxyuridine (BrdU) is incorporated into synthesized DNA and is detectable in the nucleus by immunohistochemistry (IHC). To identify BrdU-labeled spermatogenic cells, the spermatogenic stages must be determined by visualizing acrosomes and detecting cell type-specific marker molecules in the seminiferous tubules. However, the antibody reaction with BrdU routinely requires denaturation of the DNA, which is achieved by pretreating tissue sections with hydrochloric acid; however, this commonly interferes with further histochemical approaches. Therefore, we examined optimal methods for pretreating paraffin sections of the mouse testis to detect incorporated BrdU by an antibody and, at the same time, visualize acrosomes with peanut agglutinin (PNA) or detect several marker molecules with antibodies. We found that the use of heat-induced antigen retrieval (HIAR), which consisted of heating at 95C in 20 mM Tris-HCl buffer (pH 9.0) for 15 min, was superior to the use of 2 N hydrochloric acid for 90 min at room temperature in terms of the quality of subsequent PNA-lectin histochemistry with double IHC for BrdU and an appropriate stage marker protein. With this method, we identified BrdU-labeled spermatogenic cells during mouse spermatogenesis as A1 spermatogonia through to preleptotene spermatocytes. PMID:25479790

  15. Visualisation of Microglia with the use of Immunohistochemical Double Staining Method for CD-68 and Iba-1 of Cerebral Tissue Samples in Cases of Brain Contusions.

    PubMed

    Stankov, Aleksandar; Belakaposka-Srpanova, Viktorija; Bitoljanu, Natasa; Cakar, Ljupco; Cakar, Zdravko; Rosoklija, Gorazd

    2015-01-01

    In the recent years it has been confirmed that the main component of the immune response in an injury of the nerve cell comes from microglia and macrophages. The main challenge in the field of microglia research is to detect the different stages of cellular activation by visualization of the cell morphology. The existing visualization techniques are based on surface molecules expression in resting and activated microglia cells. For visualization of the microglial cells and their functional state we used double labeling method for cd-68 and iba1 in brain contusions with different survival time. Microglia are stained brown with Iba-1, whereas microglia impregnated with black, grainy color, represents activated microglia stained with CD 68. We had significantly positive results, and we were able to observe changes in the morphology of the microglia that correlated with the survival time. Using double labeling with Iba-1 and cd68 we were able to determine their physiological state based on the morphology and immunoreactivity. PMID:27442380

  16. Wood stains

    MedlinePlus

    The harmful substances in wood stains are hydrocarbons, or substances that contain only carbon and hydrogen. Other harmful ingredients may include: Alcohol Alkanes Cyclo alkanes Glycol ether Corrosives, such as sodium ...

  17. Gram Stain

    MedlinePlus

    ... definitively identify the cause of infection. Fungi , including yeast, may also be detected with a Gram stain. ^ ... white blood cells Fungi (in the form of yeasts or molds) may be seen on a Gram ...

  18. Double immunofluorescent staining of rat macrophages in formalin-fixed paraffin-embedded tissue using two monoclonal mouse antibodies.

    PubMed

    Isidro, Raymond A; Isidro, Angel A; Cruz, Myrella L; Hernandez, Siomara; Appleyard, Caroline B

    2015-12-01

    The conventional approach of double immunostaining to visualize more than one protein in tissues or cells using antibodies from two different host species is not always feasible due to limitations with antibody availability. Previously reported methodologies for performing multiple immunostains on the same tissue or cells with antibodies originating from the same species are varied in their complexity, sensitivity, and approach to prevent unwanted interactions between antibodies. In the ever-expanding field of macrophage biology, much more is known about mouse and human macrophages than their rat counterparts. The limited availability of validated and well-characterized monoclonal antibodies from different species is one factor responsible for preventing advances in rat macrophage biology. Here we describe an immunostaining method for identifying and examining rat macrophages that is sufficiently sensitive for use in formalin-fixed paraffin-embedded tissue and that uses only commercially available reagents and antibodies. This method can be used to help characterize both physiological and pathophysiological processes in rat macrophages and can be adapted for use with any two antibodies from the same species of origin as long as one of the antibodies is biotinylated. PMID:26403093

  19. Detection of Foreign Antigen-specific CD4+Foxp3+ Regulatory T Cells by MHC Class II Tetramer and Intracellular CD154 Staining

    PubMed Central

    Choi, Jin Young

    2013-01-01

    The unrestricted population of CD4+Foxp3+ regulatory T (Treg) cells, which have been known to control the expression of autoimmune diseases and protective immunity to inflammatory reactions, has led to greater appreciation of functional plasticity. Detecting and/or isolating Ag-specific CD4+Foxp3+ Tregs at the single cell level are required to study their function and plasticity. In this study, we established and compared both MHC class II tetramer and intracellular CD154 staining, in order to detect CD4+Foxp3+ Treg specific for foreign Ag in acute and chronic infections with lymphocytic choriomeningitis virus (LCMV). Our results revealed that MHC class II tetramer staining showed a lower detection rate of LCMV GP66-77-specific CD4+ T cells because most of MHC class II tetramers were unbound and unstable when combined staining was performed with intracellular cytokines. In contrast, intracellular CD154 staining was revealed to be easier and simple for detecting LCMV GP66-77-specific CD4+ T cells, compared to MHC class II tetramer staining. Subsequently, we employed intracellular CD154 staining to detect LCMV GP66-77-specific CD4+Foxp3+ Tregs using Foxp3GFP knock-in mouse, and found that LCMV GP66-77-specific CD4+Foxp3+ Tregs and polyclonal CD4+Foxp3+ Tregs showed differential expansion in mice infected with LCMV Arms or Cl13 at acute (8 and 13 days pi) and chronic phases (35 days pi). Therefore, our results provide insight into the valuable use of intracellular CD154 staining to detect and characterize foreign Ag-specific CD4+Foxp3+ Treg in various models. PMID:24385945

  20. Resolution of Viable and Membrane-Compromised Bacteria in Freshwater and Marine Waters Based on Analytical Flow Cytometry and Nucleic Acid Double Staining

    PubMed Central

    Grégori, Gérald; Citterio, Sandra; Ghiani, Alessandra; Labra, Massimo; Sgorbati, Sergio; Brown, Spencer; Denis, Michel

    2001-01-01

    The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid double-staining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214–218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes. PMID:11571170

  1. New epifluorescence microscope providing pairs of specific fluorescence images of double-stained cells for simultaneous visual perception and for quantification

    NASA Astrophysics Data System (ADS)

    Heiden, Thomas; Tribukait, Bernhard

    1996-05-01

    A new epi-fluorescence microscope for analysis of cells stained with two fluorochromes which can be spectrally isolated is described. The system makes it possible to perform independent and specific spectral selection of each dye (e.g. DAPI and CY3) while perceiving the two specific images simultaneously by eye. The optics uses splitting of the primary excitation and emission light beams, independent modification of the separated beams, and their reunification. Modifications in the separated beams comprise: (1) isolation of specific wavelengths (365 nm and 546 nm in the excitation light path, 435 - 500 nm and 590 - 750 nm in the emission light beams), (2) wavelength switching without image displacement and blur by means of a light chopper alternating between ultraviolet-excitation/blue-detection and green-excitation/red-detection at frequencies of up to 140 Hz for observation by eye without image flicker, and (3) the possible separate positioning of lenses for compensation of chromatic aberrations. The system demonstrates a good transmission of the chosen wavelengths. A high specificity of double fluorescence analysis with minimal effects of spectral overlap was attained with good temporal resolution. It has been shown that it is feasible to obtain separate chromatic compensations for the use of a microscope objective in spectral regions outside the range for which the objective is corrected. Quantitative and independent measurements of the two fluorescence images by a CCD camera synchronized with the light chopper are feasible. In conclusion, this imaging system is outlined for highly specific visual analysis and exact quantitative measurement of double fluorescence labeled specimens in cytology and histology.

  2. Quantification and characterization of specific T-cells by antigen-specific cytokine production using ELISPOT assay or intracellular cytokine staining.

    PubMed

    Letsch, Anne; Scheibenbogen, Carmen

    2003-10-01

    The novel generation of sensitive T-cell assays facilitates the direct quantitation and characterization of specific T-cell responses. Functional T-cell assays such as the ELISPOT assay and the intracellular cytokine cytometry (ICC) employ the antigen-specific induction of cytokines to detect specific T-cells on a single cell level. ICC has the advantage that the simultaneous phenotypic characterization of the antigen-specific T-cells is possible. There is evidence now from clinical cancer vaccination trials, that there is a relationship between the detection of vaccine-induced T-cells by cytokine-based assays and clinical responses. As these assays become increasingly relevant in clinical practice to suggest issues of assay validation and quality control become of major importance. PMID:12957572

  3. Leishmania chagasi T-cell antigens identified through a double library screen.

    PubMed

    Martins, Daniella R A; Jeronimo, Selma M B; Donelson, John E; Wilson, Mary E

    2006-12-01

    Control of human visceral leishmaniasis in regions where it is endemic is hampered in part by limited accessibility to medical care and emerging drug resistance. There is no available protective vaccine. Leishmania spp. protozoa express multiple antigens recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the cause of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T-cell antigens and T-dependent B-cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide by screening with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second-step screen for their ability to cause proliferation and gamma interferon responses in T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The corresponding antigens were derived from glutamine synthetase, a transitional endoplasmic reticulum ATPase, elongation factor 1gamma, kinesin K39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L. chagasi mounted both cellular and antibody responses to these proteins. Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines. PMID:17000724

  4. A dual-targeting triplebody mediates preferential redirected lysis of antigen double-positive over single-positive leukemic cells

    PubMed Central

    Schubert, Ingo; Saul, Domenica; Nowecki, Stefanie; Mackensen, Andreas; Fey, Georg H; Oduncu, Fuat S

    2014-01-01

    The single-chain triplebody HLA-ds16-hu19 consists of three single-chain Fv (scFv) antibody fragments connected in a single polypeptide chain. This protein with dual-targeting capacity mediated preferential lysis of antigen double-positive (dp) over single-positive (sp) leukemic cells by recruitment of natural killer (NK) cells as effectors. The two distal scFv modules were specific for the histocompatibility protein HLA-DR and the lymphoid antigen CD19, the central one for the Fc gamma receptor CD16. In antibody-dependent cellular cytotoxicity (ADCC) experiments with a mixture of leukemic target cells comprising both HLA-DR sp HuT-78 or Kasumi-1 cells and (HLA-DR plus CD19) dp SEM cells, the triplebody mediated preferential lysis of the dp cells even when the sp cells were present in ≤20-fold numerical excess. The triplebody promoted equal lysis of SEM cells at 2.5-fold and 19.5-fold lower concentrations than the parental antibodies specific for HLA-DR and CD19, respectively. Finally, the triplebody also eliminated primary leukemic cells at lower concentrations than an equimolar mixture of bispecific single-chain Fv fragments (bsscFvs) separately addressing each target antigen (hu19-ds16 and HLA-ds16). The increased selectivity of targeting and the preferential lysis of dp over sp cells achieved by dual-targeting open attractive new perspectives for the use of dual-targeting agents in cancer therapy. PMID:24135631

  5. A double-antibody sandwich ELISA for the detection of Entamoeba histolytica antigen in stool samples of humans.

    PubMed

    Baumann, D; Gottstein, B

    1987-06-01

    A double-antibody sandwich ELISA was developed to detect detergent-solubilized antigens of Entamoeba histolytica in stool samples of humans. The test system was evaluated for its methodical and diagnostic sensitivity and specificity. In recovery experiments the lower limit of detection was 400 ng E. histolytica (HK9) protein/ml stool, corresponding to approximately 2000 amoebic trophozoites/ml stool. Samples of 97 patients with suspected intestinal amoebiasis were examined. Specific antigens were detected by ELISA (= positive reaction) in 14 (93%) out of 15 stool samples containing trophozoites of E. histolytica. In contrast, 68 (93%) of 73 samples with other protozoa, including Entamoeba coli, E. hartmanni, Endolimax nana, Iodamoeba buetschlii and Giardia lamblia, did not react in the test system (= negative reaction). The test was shown to detect only trophozoites of E. histolytica and not the cyst stage. This fact could facilitate the differentiation between cyst carriers and persons excreting trophozoites. The results of this preliminary study justify a further large scale evaluation of the test system. PMID:2888183

  6. A double diastereoselective Michael-type addition as an entry to conformationally restricted tn antigen mimics.

    PubMed

    Aydillo, Carlos; Navo, Claudio D; Busto, Jesús H; Corzana, Francisco; Zurbano, María M; Avenoza, Alberto; Peregrina, Jesús M

    2013-11-01

    A totally stereocontrolled C-Michael addition of serine-equivalent C-nucleophiles to tri-O-benzyl-2-nitro-d-galactal was used as the key step to synthesize several pyrano[3,2-b]pyrrole structures. These scaffolds could be regarded as conformationally restricted Tn antigen mimics, as we have demonstrated by biological assays. The pyranose rings retain their (4)C1 chair conformation, as shown by molecular modeling and NMR spectroscopy. The expected bioactivity was established by a competition-tailored enzyme-linked lectin assay using both soybean and Vicia villosa agglutinins as model lectins. The facile described synthetic route and the strategic combination of computational and experimental techniques to reveal conformational features and bioactivity demonstrate the prepared glycomimics to be promising candidates for further exploitation of this scaffold to give glycans for lectin blocking and vaccination. PMID:24083620

  7. Port-wine stain

    MedlinePlus

    ... wine stains, including freezing, surgery, radiation, and tattooing. Laser therapy is most successful in eliminating port-wine stains. ... Prognosis) Stains on the face respond better to laser therapy than those on the arms, legs, or middle ...

  8. Joint fluid Gram stain

    MedlinePlus

    Gram stain of joint fluid ... result means no bacteria are present on the Gram stain. Normal value ranges may vary slightly among ... Abnormal results mean bacteria were seen on the Gram stain. This may be a sign of a ...

  9. Port-Wine Stain

    MedlinePlus

    ... and rashes clinical tools newsletter | contact Share | Port-Wine Stain A parent's guide for infants and babies ... a three-month-old infant with a port-wine stain. Overview A port-wine stain is a ...

  10. Development and Application of a Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Porcine Circovirus 2

    PubMed Central

    Ge, Meng; Luo, Wei; Jiang, Daliang; Li, Runcheng; Zhao, Wenwei; Chen, Guoliang; Yang, Xingdong

    2012-01-01

    A double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) is described for detection of porcine circovirus 2 (PCV2) antibodies using the well-characterized recombinant PCV2 capsid protein. In a comparative test of 394 pig sera against an indirect immunofluorescence (IIF) test and a commercial ELISA kit (also based on the recombinant PCV2 capsid protein), the results showed that the diagnostic sensitivity, specificity, and accuracy of the assay were, respectively, 90.61, 94.02, and 91.62% compared with IIF and 94.38, 95.28, and 94.67% compared with the commercial ELISA kit. Assay of 12 PCV-free pigs over a 5-week period produced only PCV2-negative titers by all 3 methods. These results and the seroprofiles of 4 pig farms obtained by both the commercial ELISA kit and the double-antigen sandwich ELISA indicate that the sandwich ELISA is a reliable method for detection of antibodies to PCV2. Additionally, the method described here permits the use of undiluted test serum samples simultaneously loaded with horseradish peroxidase (HRP)-conjugated antigen into the test well, and the complete test procedure can be performed in less than 90 min. This double-antigen sandwich ELISA should be a useful tool to aid swine industry professionals in deciding the intervention strategies for the control of PCV2-associated diseases. PMID:22815145

  11. An ultrasensitive squamous cell carcinoma antigen biosensing platform utilizing double-antibody single-channel amplification strategy.

    PubMed

    Ren, Xiang; Wu, Dan; Wang, Yuhuan; Zhang, Yunhui; Fan, Dawei; Pang, Xuehui; Li, Yueyun; Du, Bin; Wei, Qin

    2015-10-15

    A novel electrochemical immunosensor was developed for ultrasensitive detection of squamous cell carcinoma antigen (SCCA), which was based on the double-antibody single-channel amplification strategy. For the first time, human immunoglobulin antibody (anti-HIgG) was used as the supporting framework to amplify the loading quantity of SCCA antibody (anti-SCCA). In this strategy, SCCA can be detected without using mesoporous nanometers to amplify the signal. In addition, Pd icosahedrons were first used as the connecter to immobilize the antibodies and strengthen the sensitivity. Only one touch point exists under the limited condition between a sphere and another shape in geometry, thus the Pd icosahedron is an excellent candidate as the role of connecter. Gold nanoparticles (Au NPs) decorated with mercapto-functionalized graphene sheets (Au@GS) were synthesized as the transducing materials. The fabricated immunosensor exhibited an excellent detection limit of 2.8 pg/mL and wide linear range of 0.01-5 ng/mL. This kind of immunosensor would provide a potential application in clinical diagnosis. PMID:25982722

  12. Acid-fast stain

    MedlinePlus

    The acid-fast stain is a laboratory test that determines if a sample of tissue, blood, or other body ... dye. The slide is then washed with an acid solution and a different stain is applied. Bacteria ...

  13. Acid-fast stain

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003766.htm Acid-fast stain To use the sharing features on this page, please enable JavaScript. The acid-fast stain is a laboratory test that determines ...

  14. Port-wine stain

    MedlinePlus

    A port-wine stain is a birthmark in which swollen blood vessels create a reddish-purplish discoloration of the skin. ... Port-wine stains occur in about 3 out of 1,000 people. In rare cases, port-wine stains are ...

  15. Black stain - a review.

    PubMed

    Ronay, Valerie; Attin, Thomas

    2011-01-01

    The purpose of this review was to summarise the fundamentals about black stain, its diagnosis and possible differential diagnoses as well as its microbiology and therapy. In addition, various studies investigating the relationship between black stain and dental caries are examined. Many studies report lower caries prevalence in children with black stain, but this finding could not be confirmed by all authors. Also, a negative relation between degree of staining and caries severity has been described. Reasons for these results are not yet clear but it was speculated that they are related to the specific oral microflora described in black stain-affected individuals. PMID:21594205

  16. Gram stain of urethral discharge

    MedlinePlus

    Urethral discharge Gram stain ... microscope slide. A series of stains called a Gram stain is applied to the specimen. The stained ... culture ) should be performed in addition to the gram stain. More sophisticated diagnostic tests (such as PCR ...

  17. Expression of blood group antigens H-2, Le(y), and sialylated-Le(a) in human colorectal carcinoma. An immunohistochemical study using double-labeling techniques.

    PubMed Central

    Cooper, H. S.; Malecha, M. J.; Bass, C.; Fagel, P. L.; Steplewski, Z.

    1991-01-01

    In this study, double-labeling immunohistochemistry was used to gain insight into the coexpression or interrelationship between blood group antigens (BGA) that are differentiation antigens in the normal colon, and BGA that are sequential moieties in the same synthetic pathway. Paired-wise Sialylated-Le(a)/Le(y) and H-2/Le(y) was studied. The Sialylated-Le(a) and Le(y) are synthesized from type 1 and type 2 backbones, respectively. In the normal colon, the Le(y) and Sialylated-Le(a) are expressed by cells at the base and surface of the crypt, respectively, representing undifferentiated and differentiated enterocytes. The H-2 is considered oncofetal in nature, and is considered to be the immediate precursor in the synthesis of Le(y). In individual cancers. Sialylated-Lea and Le(y) were detected in different cancer cells within the same malignant glands, separately in different glands, and in different subcellular compartments of the same cell. Both H-2 and Le(y) were coexpressed in the same individual cells in 92% of cancers expressing both these BGA. In 50% of the cancers, the H-2 and Le(y) also were expressed separately in different malignant glands within individual tumors. These findings indicate that, in colorectal cancers, differentiation antigens (Sialylated Le(a) and Le(y)) are expressed by different individual cells within the same malignant gland somewhat, recapitulating the normal colon crypt. Antigens of different backbones occasionally may be expressed in the same cells but within different subcellular compartments. Precursor accumulation is common in cancers, and antigens in the same synthetic pathway are coexpressed in the same cell. The expression of H-2 and Le(y) in different glands (lack of coexpression) may be explained possibly by aberrant synthesis of Le(y) by an alternate pathway. Images Figure 1 PMID:1987759

  18. Dramatic Stained Glass.

    ERIC Educational Resources Information Center

    Prater, Michael

    2002-01-01

    Describes an art project that is appropriate for students in fifth through twelfth grade in which they create Gothic-style stained-glass windows. Discusses how college students majoring in elementary education created stained-glass windows. Addresses how to adapt this lesson for younger students. (CMK)

  19. Characterization of a double-CRD-mutated Gal-8 recombinant protein that retains co-stimulatory activity on antigen-specific T-cell response.

    PubMed

    Schroeder, Matías Nicolás; Tribulatti, María Virginia; Carabelli, Julieta; André-Leroux, Gwenaëlle; Caramelo, Julio Javier; Cattaneo, Valentina; Campetella, Oscar

    2016-04-01

    Galectins (Gals) constitute a family of mammalian lectins with affinity for β-galactosides, characterized by the presence of conserved CRDs (carbohydrate-recognition domains). We have found previously that Gal-8, from the tandem-repeat group with two linked CRDs, exerts two separate actions on CD4(+)T-cells: antigen-independent proliferation and, at lower concentration, antigen-specific co-stimulation. Whereas proliferation can be ascribed to the pro-inflammatory role of Gal-8, the co-stimulatory activity of borderline T-cell-specific responses allows the proposal of Gal-8 as an adjuvant in vaccination. To study the relevance of glycan-lectin interaction to these T-cell activities, we generated a double-mutated protein (Gal-8mut) by replacing canonical arginine residues on each CRD, so as to abolish sugar-binding capacity. As expected, Gal-8mut was unable to bind to lactosyl-Sepharose, confirming that lactose recognition was precluded; however, preservation of lectin activity was still evident since Gal-8mut displayed haemoagglutinatory effects and binding capacity to the T-cell surface. To search for glycan affinity, a glycan microarray analysis was conducted which revealed that Gal-8mut lost most low- and intermediate-, but retained high-, affinity interactions, mainly to polylactosamines and blood group antigens. These findings were supported further by molecular modelling. Regarding biological activity, Gal-8mut was unable to induce T-cell proliferation, but efficiently co-stimulated antigen-specific responses, bothin vitroandin vivo.Therefore Gal-8mut represents a useful tool to dissect the specificities of lectin-glycan interactions underlying distinctive Gal-8 activities on T-cell biology. Moreover, given its distinguishing properties, Gal-8mut could be used to enhance borderline immune responses without the non-specific pro-inflammatory activity or other potential adverse effects. PMID:26795039

  20. Pleural fluid Gram stain

    MedlinePlus

    Gram stain of pleural fluid ... lungs fill a person's chest with air. If fluid builds up in the space outside the lungs ... chest, it can cause many problems. Removing the fluid can relieve a person's breathing problems and help ...

  1. 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

  2. Phosphorylation of Simian Virus 40 T Antigen on Thr 124 Selectively Promotes Double-Hexamer Formation on Subfragments of the Viral Core Origin

    PubMed Central

    Barbaro, Brett A.; Sreekumar, K. R.; Winters, Danielle R.; Prack, Andrea E.; Bullock, Peter A.

    2000-01-01

    Cell cycle-dependent phosphorylation of simian virus 40 (SV40) large tumor antigen (T-ag) on threonine 124 is essential for the initiation of viral DNA replication. A T-ag molecule containing a Thr→Ala substitution at this position (T124A) was previously shown to bind to the SV40 core origin but to be defective in DNA unwinding and initiation of DNA replication. However, exactly what step in the initiation process is defective as a result of the T124A mutation has not been established. Therefore, to better understand the control of SV40 replication, we have reinvestigated the assembly of T124A molecules on the SV40 origin. Herein it is demonstrated that hexamer formation is unaffected by the phosphorylation state of Thr 124. In contrast, T124A molecules are defective in double-hexamer assembly on subfragments of the core origin containing single assembly units. We also report that T124A molecules are inhibitors of T-ag double hexamer formation. These and related studies indicate that phosphorylation of T-ag on Thr 124 is a necessary step for completing the assembly of functional double hexamers on the SV40 origin. The implications of these studies for the cell cycle control of SV40 DNA replication are discussed. PMID:10954562

  3. Apparatus Would Stain Microscope Slides

    NASA Technical Reports Server (NTRS)

    Breeding, James D.

    1993-01-01

    Proposed apparatus meters specific amounts of fluid out of containers at specific times to stain microscope slides. Intended specifically for semiautomated staining of microbiological and hematological samples in microgravity, leakproof apparatus used in other environments in which technicians have little time to allocate to staining procedures and/or exposure to toxic staining agents or to micro-organisms to be stained hazardous. Apparatus adapted to perform almost any staining procedure and accommodates multiple staining reagents, useful for small or remote clinical laboratories.

  4. Assessment of the cell viability of cultured Perkinsus marinus (Perkinsea), a parasitic protozoan of the Eastern oyster, Crassostrea virginica, using SYBRgreen-propidium iodide double staining and flow cytometry.

    PubMed

    Soudant, Philippe; Chu, Fu-Lin E; Lund, Eric D

    2005-01-01

    A flow cytometry (FCM) assay using SYBRgreen and propidium iodide double staining was tested to assess viability and morphological parameters of Perkinsus marinus under different cold- and heat-shock treatments and at different growth phases. P. marinus meront cells, cultivated at 28 degrees C, were incubated in triplicate for 30 min at -80 degrees C, -20 degrees C, 5 degrees C, and 20 degrees C for cold-shock treatments and at 32 degrees C, 36 degrees C, 40 degrees C, 44 degrees C, 48 degrees C, 52 degrees C, and 60 degrees C for heat-shock treatments. A slight and significant decrease in percentage of viable cells (PVC), from 93.6% to 92.7%, was observed at -20 degrees C and the lowest PVC was obtained at -80 degrees C (54.0%). After 30 min of heat shocks at 40 degrees C and 44 degrees C, PVC decreased slightly but significantly compared to cells maintained at 28 degrees C. When cells were heat shocked at 48 degrees C, 52 degrees C, and 60 degrees C heavy mortality occurred and PVC decreased to 33.8%, 8.0%, and 3.4%, respectively. No change in cell complexity and size was noted until cells were heat shocked at >or=44 degrees C. High cell mortality was detected at stationary phase of P. marinus cell culture. Cell viability dropped below 40% in 28-day-old cultures and ranged 11-25% in 38 to 47-day-old cultures. Results suggest that FCM could be a useful tool for determining viability of cultured P. marinus cells. PMID:16313441

  5. Posttreatment prostatic-specific antigen doubling time as a surrogate endpoint for prostate cancer-specific survival: An analysis of Radiation Therapy Oncology Group Protocol 92-02

    SciTech Connect

    Valicenti, Richard K. . E-mail: Richard.Valicenti@mail.tju.edu; DeSilvio, Michelle; Hanks, Gerald E.; Porter, Arthur; Brereton, Harmar; Rosenthal, Seth A.; Shipley, William U.; Sandler, Howard M.

    2006-11-15

    Purpose: We evaluated whether posttreatment prostatic-specific antigen doubling time (PSADT) was predictive of prostate cancer mortality by testing the Prentice requirements for a surrogate endpoint. Methods and Materials: We analyzed posttreatment PSA measurements in a cohort of 1,514 men with localized prostate cancer (T2c-4 and PSA level <150 ng/mL), treated and monitored prospectively on Radiation Therapy Oncology Group Protocol 92-02. From June 1992 to April 1995, men were randomized to neoadjuvant androgen deprivation and 65-70 Gy of radiation therapy (n = 761), or in combination with 24 months of adjuvant androgen deprivation (n = 753). Using an adjusted Cox proportional hazards model, we tested if PSADT was prognostic and independent of randomized treatment in this cohort. The endpoints were time to PSADT (assuming first-order kinetics for a minimum of 3 rising PSA measurements) and cancer-specific survival (CSS). Results: After a median follow-up time of 5.9 years, randomized treatment was a significant predictor for CSS (p{sub Cox} = 0.002), PSADT <6 months (p{sub Cox} < 0.001), PSADT <9 months (p{sub Cox} < 0.001), and PSADT <12 months (p{sub Cox} < 0.001) but not for PSADT <3 (p{sub Cox} = 0.4). The significant posttreatment PSADTs were also significant predictors of CSS (p{sub Cox}< 0.001). After adjusting for T stage, Gleason score and PSA, all of Prentice's requirements were not met, indicating that the effect of PSADT on CSS was not independent of the randomized treatment. Conclusions: Prostatic specific antigen doubling time is significantly associated with CSS, but did not meet all of Prentice's requirements for a surrogate endpoint of CSS. Thus, the risk of dying of prostate cancer is not fully explained by PSADT.

  6. Joint fluid Gram stain

    MedlinePlus

    Gram stain of joint fluid ... A sample of joint fluid is needed. The fluid sample is sent to a lab where a small drop is placed in a ... on how to prepare for the removal of joint fluid, see joint fluid aspiration .

  7. Fluorescein eye stain

    MedlinePlus

    ... eye. The health care provider then shines a blue light at your eye. Any problems on the surface of the cornea will be stained by the dye and appear green under the blue light. The provider can determine the location and ...

  8. Stained-Glass Pastels

    ERIC Educational Resources Information Center

    Laird, Shirley

    2009-01-01

    The author has always liked the look of stained-glass windows. Usually the designs are simplified and the shapes are easier for younger students to draw. This technique seemed to be the perfect place for her fifth-graders to try their hand at color mixing. The smaller spaces and simple shapes were just what she needed for this group. Her students…

  9. "Stained Glass" Landscape Windows

    ERIC Educational Resources Information Center

    Vannata, Janine

    2008-01-01

    Both adults and children alike marvel at the grand vivid stained-glass windows created by American artist Louis Comfort Tiffany. Today he is commonly recognized as one of America's most influential designers and artists throughout the last nineteenth and early twentieth century. In the lesson described in this article, students created their own…

  10. Port-Wine Stains

    MedlinePlus

    ... upsetting for kids, especially if they're large, dark, or on the face. And any birthmark can take a toll on a child's self-confidence, no matter how large or small the mark might be. The good news is that lasers (highly concentrated light energy) can make many kids' port-wine stains much ...

  11. Shimmering Stained Glass.

    ERIC Educational Resources Information Center

    Simon, Gail Murray

    1998-01-01

    Presents an art lesson for fifth- and sixth-graders where they create a translucent design of colored cellophane on black paper inspired by the stained-glass windows of the Middle Ages and the artwork of Lewis Comfort Tiffany. Enables the students to become crafts people rather than just observers of the past. (CMK)

  12. Elimination of iron pigments and background staining which mask immunoperoxidase reactions.

    PubMed

    Itoiz, M E; Orrea, S

    1983-01-01

    When immunohistochemical stainings are applied to demonstration of antigens in histopathological specimens, the ferrous pigments which may be present in the tissues usually mask the final precipitates of the reaction. These pigments can be eliminated with oxalic acid or sodium dithionite after the immunohistochemical staining. These treatments also help in the bleaching of unspecific background stain. PMID:6192670

  13. A phase II trial of personalized peptide vaccination in castration-resistant prostate cancer patients: prolongation of prostate-specific antigen doubling time

    PubMed Central

    2013-01-01

    Background Cancer vaccine is one of the attractive treatment modalities for patients with castration-resistant prostate cancer (CRPC). However, because of delayed immune responses, its clinical benefits, besides for overall survival (OS), are not well captured by the World Health Organization (WHO) and Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Several surrogate markers for evaluation of cancer vaccine, including prostate-specific antigen doubling time (PSADT), are currently sought. The purpose of this study was to assess prospectively the PSA kinetics and immune responses, as well as the efficacy, safety, and biomarkers of personalized peptide vaccination (PPV) in progressive CRPC. Methods One hundred patients with progressive CRPC were treated with PPV using 2–4 positive peptides from 31 candidate peptides determined by both human leukocyte antigen (HLA) class IA types and the levels of immunoglobulin G (IgG) against each peptide. The association between immune responses and PSADT as well as overall survival (OS) was studied. Results PPV was safe and well tolerated in all patients with a median survival time of 18.8 months. Peptide-specific IgG and T-cell responses strongly correlated with PSADT (p < 0.0001 and p = 0.0007, respectively), which in turn showed correlation with OS (p = 0.018). Positive IgG responses and prolongation of PSADT during PPV were also significantly associated with OS (p = 0.001 and p = 0.004) by multivariate analysis. Conclusions PSADT could be an appropriate surrogate marker for evaluation of the clinical benefit of cancer vaccine. Further randomized trials are needed to confirm these results. Trial registration UMIN000001850 PMID:24373380

  14. Cytokeratin 7 staining in mammary and extramammary Paget's disease.

    PubMed

    Smith, K J; Tuur, S; Corvette, D; Lupton, G P; Skelton, H G

    1997-11-01

    There are a variety of routine and immunohistochemical stains used to diagnose mammary and extramammary Paget's disease (MPD and EMPD). Most of the stains commonly used, however, show a positive reaction in the Paget's cells in all cases. We wanted to assess which immunohistochemical stain is the best for the diagnosis of MPD and EMPD, as well as the best stain for identifying small foci of tumors in evaluating tumor margins. We evaluated nine cases of MPD and nine cases of EMPD, which were randomly chosen, with a battery of immunohistochemical stains. These stains included cytokeratin 7, cytokeratin 20, carcinoembryonic antigen, Ber-EP4, and CAM 5.2. Cytokeratin 7 was the only immunohistochemical stain that stained all of the cases diffusely, and, in all of the cases, the staining of the Paget's cell was intense and specific within the epidermis. We concluded that cytokeratin 7 is the immunohistochemical stain of choice in the diagnosis of Paget's disease. Because cytokeratin 7 seems to identify single cells, it might also be valuable in evaluating surgical margins for small foci in a tumor such as EMPD, which might have a multifocal origin. PMID:9388055

  15. Blood stain pattern analysis.

    PubMed

    Peschel, O; Kunz, S N; Rothschild, M A; Mützel, E

    2011-09-01

    Bloodstain pattern analysis (BPA) refers to the collection, categorization and interpretation of the shape and distribution of bloodstains connected with a crime. These kinds of stains occur in a considerable proportion of homicide cases. They offer extensive information and are an important part of a functional, medically and scientifically based reconstruction of a crime. The following groups of patterns can essentially be distinguished: dripped and splashed blood, projected blood, impact patterns, cast-off stains, expirated and transferred bloodstains. A highly qualified analysis can help to estimate facts concerning the location, quality and intensity of an external force. A sequence of events may be recognized, and detailed questions connected with the reconstruction of the crime might be answered. In some cases, BPA helps to distinguish between accident, homicide and suicide or to identify bloodstains originating from a perpetrator. BPA is based on systematic training, a visit to the crime scene or alternatively good photographic documentation, and an understanding and knowledge of autopsy findings or statements made by the perpetrator and/or victim. A BPA working group has been established within the German Society of Legal Medicine aiming to put the knowledge and practical applications of this subdiscipline of forensic science on a wider basis. PMID:21069481

  16. Length of stain dosimeter

    NASA Technical Reports Server (NTRS)

    Lueck, Dale E. (Inventor)

    1994-01-01

    Payload customers for the Space Shuttle have recently expressed concerns about the possibility of their payloads at an adjacent pad being contaminated by plume effluents from a shuttle at an active pad as they await launch on an inactive pad. As part of a study to satisfy such concerns a ring of inexpensive dosimeters was deployed around the active pad at the inter-pad distance. However, following a launch, dosimeters cannot be read for several hours after the exposure. As a consequence factors such as different substrates, solvent systems, and possible volatilization of HCl from the badges were studied. This observation led to the length of stain (LOS) dosimeters of this invention. Commercial passive LOS dosimeters are sensitive only to the extent of being capable of sensing 2 ppm to 20 ppm if the exposure is 8 hours. To map and quantitate the HCl generated by Shuttle launches, and in the atmosphere within a radius of 1.5 miles from the active pad, a sensitivity of 2 ppm HCl in the atmospheric gases on an exposure of 5 minutes is required. A passive length of stain dosimeter has been developed having a sensitivity rendering it capable of detecting a gas in a concentration as low as 2 ppm on an exposure of five minutes.

  17. Several staining techniques to enhance the visibility of Acanthamoeba cysts.

    PubMed

    El-Sayed, Nagwa Mostafa; Hikal, Wafaa Mohamed

    2015-03-01

    Acanthamoeba is one of the most common free-living amoebae. It is widespread in the environment and can infect humans causing keratitis. Delayed diagnosis or misdiagnosis leads to extensive corneal inflammation and profound visual loss. Therefore, accurate and rapid diagnosis of Acanthamoeba keratitis is essential for successful treatment and good prognosis. This study was designed to use different staining techniques to facilitate the identification of Acanthamoeba cysts. Acanthamoeba cysts were isolated by cultivation of either corneal scraping specimens or tap water samples onto non-nutrient agar plates seeded with Escherichia coli. Subcultures were done from positive cultures until unique cysts were isolated. Acanthamoeba cysts were stained temporarily using iodine, eosin, methylene blue, and calcofluor white (CFW) stains and as permanent slides after processing for mounting using modified trichrome, Gimenez and Giemsa staining. These stains were compared on the basis of staining quality including clarity of morphological details, differentiation between cytoplasm and nuclei, color and contrast, and also other characteristics of the staining techniques, including ease of handling, time taken for the procedure, and cost effectiveness. The cysts of Acanthamoeba were recognized in the form of double-walled cysts: the outer wall (ectocyst) that was being differentiated from the variably stained surrounding background and the inner wall (endocyst) that was sometimes stellated, polygonal, round, or oval and visualized as separate from the spherical, sometimes irregular, outline of the ectocyst. Regarding the temporary stains, it was found that they were efficient for visualizing the morphological details of Acanthamoeba cysts. In CFW staining, Acanthamoeba cysts appeared as bluish-white or turquoise oval halos although the internal detail was not evident. On the other hand, the results of permanent-stained slides showed the most consistent stain for identification of

  18. Gram stain of tissue biopsy

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003453.htm Gram stain of tissue biopsy To use the sharing features on this page, please enable JavaScript. Gram stain of tissue biopsy test involves using crystal ...

  19. Use of immunohistochemical staining panel for characterisation of ovarian neoplasms.

    PubMed Central

    Ashorn, P; Helle, M; Helin, H; Ashorn, R; Krohn, K

    1988-01-01

    Eighty five ovarian epithelial and non-epithelial tumours were studied by peroxidase histochemical staining for their reactivity with six monoclonal human milk fat globule (HMFG) antibodies, peanut agglutinin (PNA) lectin, and a monoclonal cytokeratin antibody. HMFG IIIC12 and cytokeratin antibodies distinguished epithelial from non-epithelial tumours. The staining patterns of mucinous and serous tumours were essentially different from each other; poorly differentiated anaplastic carcinomas showed similar antigenic content to that of the serous cystadenocarcinomas. Furthermore, staining with PNA lectin and HMFG antibodies was useful in distinguishing clear cell carcinomas from other malignant epithelial tumours of the ovary. Images Fig 2 Fig 1 PMID:2449464

  20. Exposure to Agent Orange is a significant predictor of prostate-specific antigen (PSA)-based recurrence and a rapid PSA doubling time after radical prostatectomy

    PubMed Central

    Shah, Sagar R.; Freedland, Stephen J.; Aronson, William J.; Kane, Christopher J.; Presti, Joseph C.; Amling, Christopher L.; Terris, Martha K.

    2011-01-01

    OBJECTIVE To investigate and report the clinicopathological characteristics and outcomes after radical prostatectomy (RP) in patients with prostate cancer and previous exposure to Agent Orange (AO), particularly in relationship to race. PATIENTS AND METHODS In 1495 veterans who had undergone RP the clinicopathological characteristics, biochemical progression rates, and prostate-specific antigen (PSA) doubling time (DT) after recurrence between AO-exposed and unexposed men were compared using logistic and linear regression and Cox proportional hazards analyses, and stratified by race. RESULTS The 206 (14%) men with AO exposure were more likely to be black (P = 0.001), younger (P < 0.001), treated more recently (P < 0.001), have a higher body mass index (P = 0.001), have clinical stage T1 disease (P < 0.001), and have lower preoperative PSA levels (P = 0.001). After adjusting for several clinical characteristics, AO exposure was not significantly related to adverse pathological features but was significantly associated with biochemical progression risk (relative risk 1.55, 95% confidence interval 1.15–2.09, P = 0.004) and shorter PSADT (P < 0.001) after recurrence (8.2 vs 18.6 months). When stratified by race, these associations were present and similar in both races, with no significant interaction between race and AO exposure for predicting biochemical recurrence or mean adjusted PSADT (P interaction >0.20). CONCLUSIONS Patients with AO exposure and treated with RP were more likely to be black, present with lower risk features, have an increased risk of biochemical progression, and shorter PSADT after recurrence. When stratified by race, the association between AO exposure and poor outcomes was present in both races. These findings suggest that among selected men who choose RP, AO exposure might be associated with more aggressive prostate cancer. PMID:19298411

  1. Pasteurella pestis detection in fleas by fluorescent antibody staining*

    PubMed Central

    Hudson, Bruce W.; Kartman, Leo; Prince, Frank M.

    1966-01-01

    In an effort to develop a method for the rapid field identification of plague-infected fleas, the authors have studied the feasibility of direct fluorescent antibody staining of the midgut contents of fleas fed on mice infected with Pasteurella pestis. Fluorescent antibodies prepared from antisera derived from rabbits inoculated with the water-soluble P. pestis fraction 1b antigen, the somatic antigen of heat-killed P. pestis (Bryans strain), and live avirulent (strain A1122) or virulent (Yreka strain) plague vaccines were used used in this study. This direct staining method proved to be impracticable, but encouraging results were obtained by fluorescent antibody staining of broth cultures of macerates of infected fleas after 24-48 hours' incubation. The broth enrichment technique has not yet been evaluated in the field, but it is expected to be of value since it is relatively simple to perform and requires only material that can easily be transported to remote areas. PMID:5328902

  2. Demonstration of cytoplasmic and nuclear antigens in acute leukaemia using flow cytometry.

    PubMed Central

    Farahat, N; van der Plas, D; Praxedes, M; Morilla, R; Matutes, E; Catovsky, D

    1994-01-01

    AIMS--To detect cytoplasmic and nuclear antigens using flow cytometry in acute leukaemia and to use this technique for double marker combinations. METHODS--Cytoplasmic staining was carried out in samples from 40 cases of acute leukaemia with monoclonal antibodies against the myeloid antigen CD13, the lymphoid antigens CD3, CD22, mu chain and the enzymes terminal deoxynucleotidyl transferase (TdT) and myeloperoxidase (MPO). The cells were fixed with paraformaldehyde and permeabilised with Tween 20 and Becton Dickinson's FACS lysing solution. Flow cytometry results were compared in the same cases with immunocytochemistry results using the alkaline phosphatase anti-alkaline phosphatase method. RESULTS--The gentle permeabilisation induced by this method permitted preservation of the membrane antigens and the size and morphology of the cells. The results using flow cytometry were comparable with those obtained using immunocytochemistry, with nearly complete concordance in most cases. CONCLUSIONS--This technique is simple, rapid, sensitive and reproducible and it is suitable for double staining procedures, such as nuclear and cytoplasmic, nuclear and membrane, or cytoplasmic and membrane. It therefore provides a powerful tool for extending the use of immunophenotyping for the diagnosis and follow up of acute leukaemia. It could also be used for the investigation of minimal residual disease. PMID:7962655

  3. Double-Antigen Enzyme-Linked Immunosorbent Assay for Detection of Hepatitis E Virus-Specific Antibodies in Human or Swine Sera ▿

    PubMed Central

    Hu, Wei Ping; Lu, Yang; Precioso, Nestor Amadeo; Chen, Hsiao Ying; Howard, Teresa; Anderson, David; Guan, Ming

    2008-01-01

    A new double-antigen sandwich-based enzyme-linked immunosorbent assay (ELISA) for the detection of total antibodies (immunoglobulin G [IgG] and IgM) specific for hepatitis E virus (HEV) was developed by utilizing well-characterized recombinant protein ET2.1 and its peroxidase-labeled counterpart. Our study showed that the ELISA detected all the positive patient samples (n = 265) regardless of whether they contained IgM or IgG antibodies, or both, while it maintained an excellent specificity of 98.8% with samples from various patient or healthy control groups (total number of samples, 424). The test had a detection limit for anti-HEV IgG antibodies that was equivalent to 62 mIU/ml of the international reference. Compared with the serological status of the specimens determined on the basis of tests performed at the individual collection sites, the testing outcome generated by the new ELISA had a good agreement of 99.3%, with a kappa value of 0.985. The positive predictive value and the negative predictive value for the new test reached 98.1% and 100%, respectively. This ELISA had a positive delta value of 4.836 and a negative delta value of 3.314 (where delta is a measure of the number of standard deviations by which the cutoff is separated from the mean of the sample groups) (N. Crofts, W. Maskill, and I. D. Gust, J. Virol. Methods 22:51-59, 1988), indicating that it had an excellent ability to differentiate the infected and noninfected cohorts. Furthermore, the new design enables the detection of antibodies not only in human samples but also in pig samples. Our preliminary data showed that the ELISA could detect seroconversion in samples from pigs at as early as 14 days postinoculation. The potential utility of detecting specific antibodies in pigs will be an added advantage for managing the disease, with suggested zoonotic implications. PMID:18495846

  4. Further characterization of filarial antigens by SDS polyacrylamide gel electrophoresis

    PubMed Central

    Dissanayake, S.; Galahitiyawa, S. C.; Ismail, M. M.

    1983-01-01

    SDS (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis of an antigen isolated from sera of Wuchereria bancrofti-infected patients and Setaria digitata antigen SD2-4 is reported. Both antigens showed carbohydrate (glycoprotein) staining. The W. bancrofti antigen had an apparent relative molecular mass of 35 000 while the S. digitata antigen SD2-4 migrated at the marker dye position on SDS-polyacrylamide gel electrophoresis. SDS treatment of these antigens did not abolish the precipitation reaction with antibody. In the case of W. bancrofti antigen, SDS treatment probably exposed hitherto hidden antigen epitopes. PMID:6354508

  5. [A duplicate staining method for permanent specimen of Trichinella spiralis encapsulated larvae].

    PubMed

    Li, Dan; Yang, Ding; Pi, Ben-Wei; Niu, Li-Na; Zhang, Ying; Wang, Guo-Ying

    2012-04-30

    With single staining method, Trichinella spiralis encapsulated larvae specimens were fixed with formaldehyde alcohol acetic acid fixative solution, and stained with alcohol borax-carmine staining solution (4% borax solution 100 ml, carmine 1 g, and 70% alcohol 100 ml). With duplicate staining, the encapsulated larvae specimens were fixed with formaldehyde alcohol acetic acid fixative solution, and double stained with alcohol borax carmine staining solution and fast green staining solution (fast green 0.1 g, 95% alcohol 100 ml). The results showed that with single staining, it was not clear-cut between the cyst and muscle cells although the larva was differentiable, while with duplicate staining, the larva, cyst and muscle cells were distinguished more clearly. PMID:22908823

  6. Quantitative Detection of the Foot-And-Mouth Disease Virus Serotype O 146S Antigen for Vaccine Production Using a Double-Antibody Sandwich ELISA and Nonlinear Standard Curves.

    PubMed

    Feng, Xia; Ma, Jun-Wu; Sun, Shi-Qi; Guo, Hui-Chen; Yang, Ya-Min; Jin, Ye; Zhou, Guang-Qing; He, Ji-Jun; Guo, Jian-Hong; Qi, Shu-yun; Lin, Mi; Cai, Hu; Liu, Xiang-Tao

    2016-01-01

    The efficacy of an inactivated foot-and-mouth disease (FMD) vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV) particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG) analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA) is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS) ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R2 = 0.9215, P<0.01). In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 μg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings. PMID:26930597

  7. Quantitative Detection of the Foot-And-Mouth Disease Virus Serotype O 146S Antigen for Vaccine Production Using a Double-Antibody Sandwich ELISA and Nonlinear Standard Curves

    PubMed Central

    Feng, Xia; Ma, Jun-Wu; Sun, Shi-Qi; Guo, Hui-Chen; Yang, Ya-Min; Jin, Ye; Zhou, Guang-Qing; He, Ji-Jun; Guo, Jian-Hong; Qi, Shu-yun; Lin, Mi; Cai, Hu; Liu, Xiang-Tao

    2016-01-01

    The efficacy of an inactivated foot-and-mouth disease (FMD) vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV) particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG) analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA) is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS) ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R2 = 0.9215, P<0.01). In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 μg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings. PMID:26930597

  8. 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

  9. Array tomography: imaging stained arrays.

    PubMed

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

    2010-11-01

    Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time-consuming and require some practice to perfect. In this protocol, tissue arrays are imaged using conventional wide-field fluorescence microscopy. Images can be captured manually or, with the appropriate software and hardware, the process can be automated. PMID:21041399

  10. Somatic mutations in the variable regions of a human IgG anti-double-stranded DNA autoantibody suggest a role for antigen in the induction of systemic lupus erythematosus.

    PubMed

    van Es, J H; Gmelig Meyling, F H; van de Akker, W R; Aanstoot, H; Derksen, R H; Logtenberg, T

    1991-02-01

    The processes that govern the generation of pathogenic anti-DNA autoantibodies in human systemic lupus erythematosus (SLE) are largely unknown. Autoantibodies may arise as a consequence of polyclonal B cell activation and/or antigen-driven B cell activation and selection. The role of these processes in humoral autoimmunity may be studied by molecular genetic analysis of immunoglobulin (Ig) variable (V) regions of antibodies that are characteristic of SLE. We have analyzed the gene elements that encode a high affinity, IgG anti-double-stranded DNA autoantibody secreted by a monoclonal Epstein-Barr virus (EBV)-transformed cell line derived from a patient with active SLE. In addition, we have identified, cloned, and sequenced the germline counterparts of the VH and VL genes expressed in this autoantibody. The comparison of both sets of gene elements shows that the autoantibody VH and VL regions harbor numerous somatic mutations characteristic of an antigen-driven immune response. The light chain expressed in this autoantibody is a somatically mutated variant of the kv325 germline gene that is frequently associated with paraproteins having autoantibody activity and with Ig molecules produced by malignant B cells that express the CD5 antigen. Furthermore, the utilized DH segment has been repeatedly found in multireactive, low affinity IgM anti-DNA autoantibodies from SLE patients and healthy individuals. These results suggest that pathogenic IgG anti-DNA autoantibodies in human SLE may arise through antigen-driven selection of somatic mutations in the gene elements that frequently encode multireactive IgM autoantibodies. PMID:1899104

  11. Activated rat T cells synthesize and express functional major histocompatibility class II antigens.

    PubMed Central

    Broeren, C P; Wauben, M H; Lucassen, M A; Van Meurs, M; Van Kooten, P J; Boog, C J; Claassen, E; Van Eden, W

    1995-01-01

    In the present report, we studied the presence and functional significance of major histocompatibility complex (MHC) class II antigen on rat T cells. Most rat T-cell lines cultured in vitro were found to be MHC class II+. Also, these T-cell lines were shown to synthesize MHC class II molecules. Immunohistochemical and flow cytometric double stainings for T-cell receptor (TCR) and MHC class II showed that in vivo as well a large proportion of T cells was MHC class II+. The immunohistochemical staining of spleen sections enabled us to characterize the MHC class II+ and MHC class II- T cells. It was shown that resting T cells in vivo were MHC class II-. In contrast, activated T cells, as determined by their localization in the marginal zone of the spleen, proved to be MHC class II+. Finally, T-cell clones were found to be able to present peptidic antigens, but could only poorly present more complex exogenous antigens, probably due to inefficient uptake of such antigens. These features would endow activated rat T cells with the capacity to present cell-specific self-proteins, such as TCR, to regulatory CD4+ MHC class II-restricted T cells, as was described by our group elsewhere. Images Figure 2 Figure 5 PMID:7750994

  12. Distribution of viral antigen in uterus, placenta and foetus of cattle persistently infected with bovine virus diarrhoea virus.

    PubMed

    Fredriksen, B; Press, C M; Løken, T; Odegaard, S A

    1999-01-01

    The tissue distribution and cellular localisation of bovine virus diarrhoea virus (BVDV) was investigated in the uterus, placentomes, intercotyledonary foetal membranes and foetal organs of three persistently infected (PI) pregnant heifers. The uterus and ovaries of a non-pregnant PI heifer were also included in the study. Cryostat sections were examined using immunohistochemical techniques and monoclonal antibodies against BVDV. A double immunofluorescence technique was used to identify BVDV positive cells that also showed staining for either the leukocyte common antigen CD45 or the cytoskeletal filament vimentin. BVDV antigen was detected in all the organs examined, and was present in both epithelial and non-epithelial cells. In all organs many of the virus-positive cells also showed reactivity for vimentin. In the foetal liver and spleen a small, scattered population of virus-positive cells showed reactivity for CD45. A few cells showed reactivity both for BVDV antigen and for CD45 in the placentomes and intercotyledonary foetal membranes. In contrast to earlier reports, only scattered cells in the foetal part of the placentomes, the cotyledons, showed reactivity for BVDV antigen. However, in the chorion of the intercotyledonary foetal membranes, a larger proportion of the trophoblast cells showed reactivity for BVDV, especially the binuclear trophoblast cells. In the uterus, pregnancy appeared to favour virus replication, as the section from the pregnant heifers showed much stronger staining and a higher proportion of viral antigen-positive cells than sections from the non-pregnant PI heifer. PMID:10028166

  13. Automated single-slide staining system

    NASA Technical Reports Server (NTRS)

    Mills, S. M.; Wilkins, J. R.

    1974-01-01

    Apparatus developed to Gram-stain single slides automatically is flexible enough to accommodate other types of staining procedures. Method frees operator and eliminates necessity for subjective evaluations as to length of staining or decolorizing time.

  14. Whole Blood Cell Staining Device

    NASA Technical Reports Server (NTRS)

    Sams, Clarence F.; Clift, Vaughan L.; McDonald, Kelly E.

    2000-01-01

    An apparatus and method for staining particular cell markers is disclosed. The apparatus includes a flexible tube that is reversibly pinched into compartments with one or more clamps. Each compartment of the tube contains a separate reagent and is in selective fluid communication with adjoining compartments.

  15. Post-embedding Mammalian Tissue for Immunoelectron Microscopy: A Standardized Procedure Based on Heat-Induced Antigen Retrieval.

    PubMed

    Yamashita, Shuji

    2016-01-01

    We describe a standardized method of fixation, antigen retrieval, and image contrasting for post-embedding immunoelectron microscopy. Tissues are fixed with formaldehyde solutions containing Ca(2+) and Mg(2+) ions at pH 7.4 and then at pH 8.5. After dehydration with dimethylformamide, the specimens are embedded in LR-White resin. For antigen retrieval, ultrathin sections are heated in 0.5 M Tris-HCl, pH 9.0, for 1-2 h at 95 °C. After immunogold labeling, the sections are treated with a mixture of tannic acid and glutaraldehyde, with OsO4 solution, and then double-stained with uranyl acetate and lead citrate. The standardized method yields strong and reproducible immunoreactions for many antigens showing excellent image contrast without destruction of fine structures. PMID:27515088

  16. Methods for chromosome-specific staining

    DOEpatents

    Gray, Joe W.; Pinkel, Daniel

    1995-01-01

    Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogenous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include methods for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes.

  17. Salt stains from evaporating droplets.

    PubMed

    Shahidzadeh, Noushine; Schut, Marthe F L; Desarnaud, Julie; Prat, Marc; Bonn, Daniel

    2015-01-01

    The study of the behavior of sessile droplets on solid substrates is not only associated with common everyday phenomena, such as the coffee stain effect, limescale deposits on our bathroom walls , but also very important in many applications such as purification of pharmaceuticals, de-icing of airplanes, inkjet printing and coating applications. In many of these processes, a phase change happens within the drop because of solvent evaporation, temperature changes or chemical reactions, which consequently lead to liquid to solid transitions in the droplets. Here we show that crystallization patterns of evaporating of water drops containing dissolved salts are different from the stains reported for evaporating colloidal suspensions. This happens because during the solvent evaporation, the salts crystallize and grow during the drying. Our results show that the patterns of the resulting salt crystal stains are mainly governed by wetting properties of the emerging crystal as well as the pathway of nucleation and growth, and are independent of the evaporation rate and thermal conductivity of the substrates. PMID:26012481

  18. Salt stains from evaporating droplets

    PubMed Central

    Shahidzadeh, Noushine; Schut, Marthe F. L.; Desarnaud, Julie; Prat, Marc; Bonn, Daniel

    2015-01-01

    The study of the behavior of sessile droplets on solid substrates is not only associated with common everyday phenomena, such as the coffee stain effect, limescale deposits on our bathroom walls , but also very important in many applications such as purification of pharmaceuticals, de-icing of airplanes, inkjet printing and coating applications. In many of these processes, a phase change happens within the drop because of solvent evaporation, temperature changes or chemical reactions, which consequently lead to liquid to solid transitions in the droplets. Here we show that crystallization patterns of evaporating of water drops containing dissolved salts are different from the stains reported for evaporating colloidal suspensions. This happens because during the solvent evaporation, the salts crystallize and grow during the drying. Our results show that the patterns of the resulting salt crystal stains are mainly governed by wetting properties of the emerging crystal as well as the pathway of nucleation and growth, and are independent of the evaporation rate and thermal conductivity of the substrates. PMID:26012481

  19. Pasteurella pestis detection in Fleas by fluorescent antibody staining.

    PubMed

    Hudson, B W; Kartman, L; Prince, F M

    1966-01-01

    In an effort to develop a method for the rapid field identification of plague-infected fleas, the authors have studied the feasibility of direct fluorescent antibody staining of the midgut contents of fleas fed on mice infected with Pasteurella pestis. Fluorescent antibodies prepared from antisera derived from rabbits inoculated with the water-soluble P. pestis fraction 1b antigen, the somatic antigen of heat-killed P. pestis (Bryans strain), and live avirulent (strain A1122) or virulent (Yreka strain) plague vaccines were used used in this study.This direct staining method proved to be impracticable, but encouraging results were obtained by fluorescent antibody staining of broth cultures of macerates of infected fleas after 24-48 hours' incubation.The broth enrichment technique has not yet been evaluated in the field, but it is expected to be of value since it is relatively simple to perform and requires only material that can easily be transported to remote areas. PMID:5328902

  20. Semiquantitative immunohistochemical marker staining and localization in canine thyroid carcinoma and normal thyroid gland.

    PubMed

    Pessina, P; Castillo, V; Sartore, I; Borrego, J; Meikle, A

    2016-09-01

    Immunoreactive proteins in follicular cells, fibroblasts and endothelial cells were assessed in canine thyroid carcinomas and healthy thyroid glands. No differences were detected in thyrotropin receptor and thyroglobulin staining between cancer and normal tissues, but expression was higher in follicular cells than in fibroblasts. Fibroblast growth factor-2 staining was more intense in healthy follicular cells than in those of carcinomas. Follicular cells in carcinomas presented two- to three-fold greater staining intensity of thyroid transcription factor-1 and proliferating cell nuclear antigen, respectively, than healthy cells, and a similar trend was found for the latter antigen in fibroblasts. Vascular endothelial growth factor staining was more intense in the endothelial cells of tumours than in those of normal tissues. In conclusion, greater expression of factors related to proliferation and angiogenesis was demonstrated in several cell types within thyroid carcinomas compared to healthy tissues, which may represent mechanisms of tumour progression in this disease. PMID:25082554

  1. Histopathological Findings in Immunohistological Staining of the Granulomatous Tissue Reaction Associated with Tuberculosis

    PubMed Central

    Karimi, Shirin; Pourabdollah, Mihan; Sadr, Makan; Karbasi, Mehrdad; Bahadori, Moslem

    2014-01-01

    Purpose. The histological diagnosis of Mycobacterium tuberculosis (MTB) remains a diagnostic challenge despite different methods. Immunohistochemistry (IHC) not only could confirm granulomatous tissue involvement but also can demonstrate MTB antigen immunolocalization. This study tries to clarify the details of immunohistochemical staining for MTB with pAbBCG. Materials/Methods. Twenty-three confirmed TB granulomatous tissue samples were studied by Ziehl-Neelsen and immunohistochemistry (IHC) staining with pAbBCG. Samples were selected from the archive of the Department of Pathology, National Research Institute of Tuberculosis and Lung Disease, Tehran, Iran. Results. IHC staining was positive in all samples, whereas Ziehl-Neelsen was positive in 9 cases out of 23 (39.1%). Tissue types used were pleural tissue, lymph nodes, and lung tissue. IHC showed positive coarse granular cytoplasmic and round, fragmented bacillary staining. In this study, epithelioid cells clearly showed more positive staining at the periphery of the granuloma rather than the center of granuloma. There is also positive staining in endothelial cells, fibroblasts, plasma cells, lymphocytes, and macrophages outside the granuloma. Conclusion. Considering the criteria of positive immunohistochemical staining of TB granulomatous reactions, this stain not only highlights the presence of mycobacterial antigens for tissue diagnosis, but also could morphologically localize its distribution in different cells. PMID:24511393

  2. Heparin reduces nonspecific eosinophil staining artifacts in mass cytometry experiments.

    PubMed

    Rahman, Adeeb H; Tordesillas, Leticia; Berin, M Cecilia

    2016-06-01

    The analysis of heterogeneous cell samples by mass cytometry (CyTOF) relies on the assumption that metal labeled antibodies accurately bind to their target antigens. We report a previously unappreciated experimental artifact of non-specific antibody binding by eosinophils during intracellular CyTOF analysis of human whole blood samples. We hypothesized that this non-specific binding results from a charge-based interaction between the metal-labeled antibodies and highly cationic proteins found in eosinophillic granules and found that this non-specific staining artifact could be reduced to background levels with a simple blocking protocol using heparin as a competing anionic protein. This protocol eliminates a potential source of erroneous data interpretation in all experiments involving intracellular staining of human whole blood samples, and allows accurate assessment of dynamic changes in intracellular proteins in eosinophils by CyTOF. © 2016 International Society for Advancement of Cytometry. PMID:27061608

  3. The stain prevention efficacy of two tooth whitening dentifrices.

    PubMed

    Ayad, Farid; De Sciscio, Peter; Stewart, Bernal; De Vizio, William; Petrone, Margaret E; Volpe, Anthony R

    2002-08-01

    An 8-week randomized, double-blind, parallel group clinical study was conducted to assess the extrinsic stain prevention efficacy of three commercially available dentifrices: 1) a dentifrice containing 0.243% sodium fluoride with copolymer, tetrasodium pyrophosphate, and sodium tripolyphosphate in a silica base (Product 1); 2) a dentifrice containing 0.243% sodium fluoride, baking soda and peroxide, tetrasodium pyrophosphate, and sodium tripolyphosphate in a silica base (Product 2); and 3) a dentifrice containing 0.243% sodium fluoride in a silica base (Product 3). After the collection of baseline stain scores by a trained examiner and a subsequent oral prophylaxis, 126 volunteers were randomized to one of the three treatment groups (balanced for composite extrinsic stain scores). Throughout the 8-week treatment period, subjects brushed their teeth twice daily with their assigned dentifrice. At baseline, 4-, and 8-week evaluations, extrinsic dental stain was measured on the facial surfaces of the six maxillary anterior teeth and on the facial and lingual surfaces of the six mandibular anterior teeth using the Lobene Index. A total of 120 subjects completed the study. No adverse events were reported, and subjects who discontinued the study did so for reasons unrelated to the dentifrices. At the 4-week evaluation, composite stain scores were statistically significantly lower (P < .05) for both Product 1 (44.9%) and for Product 2 (34.6%) relative to Product 3. At the 8-week evaluation, composite stain scores were statistically significantly lower (P < .05) for both Product 1 (28.4%) and for Product 2 (29.6%) relative to Product 3. The results of this clinical study demonstrate that both dentifrices, one containing 0.234% sodium fluoride with copolymer, tetrasodium pyrophosphate, and sodium tripolyphosphate in a silica base; and one with 0.243% sodium fluoride with copolymer, tetrasodium pyrophosphate, and sodium tripolyphosphate in a silica base are more effective in

  4. Methods for chromosome-specific staining

    DOEpatents

    Gray, J.W.; Pinkel, D.

    1995-09-05

    Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogeneous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include ways for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes. 3 figs.

  5. Golgi-Cox Staining Step by Step

    PubMed Central

    Zaqout, Sami; Kaindl, Angela M.

    2016-01-01

    Golgi staining remains a key method to study neuronal morphology in vivo. Since most protocols delineating modifications of the original staining method lack details on critical steps, establishing this method in a laboratory can be time-consuming and frustrating. Here, we describe the Golgi-Cox staining in such detail that should turn the staining into an easily feasible method for all scientists working in the neuroscience field. PMID:27065817

  6. The effect of melanin bleaching on immunohistochemical staining in heavily pigmented melanocytic neoplasms.

    PubMed

    Orchard, G E; Calonje, E

    1998-08-01

    The accumulation of excessive amounts of melanin in melanocytic lesions can obscure cellular morphology and can further hinder immunocytochemical procedures. We have used a modification of the potassium permanganate/oxalic acid melanin-bleaching technique, involving much reduced bleaching times, in order to remove melanin granules prior to incubation with primary antibody. We have assessed a panel of antibodies applicable to the evaluation of melanocytic lesions and in addition have also assessed antibodies that may be more useful in research. The study attempts to determine which antigens may be affected by bleaching and which are not. Antigens S100, HMB 45, NKIC3, CD34, and L26 are relatively unaffected by this procedure. Factor-VIII-related antigen and vimentin and CD68 antigens produced enhanced staining. In contrast, antigens CD3, CD31, and CD45RO were abolished. In addition, smooth muscle actin and desmin antigens demonstrated considerable nonspecific background staining and were not reliable in this study. This technique demonstrates that a fairly wide range of antigens are preserved after bleaching and that distinction between melanocytes and melanophages can reliably be performed using the conventional immunocytochemical chromogen 3,3-diaminobenzidine and without the need for elaborate counterstaining. PMID:9700373

  7. 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

  8. Antibody Staining in Drosophila Germaria.

    PubMed

    Lie-Jensen, Anette; Haglund, Kaisa

    2016-01-01

    Drosophila oogenesis is a powerful model for studying a wide spectrum of cellular and developmental processes in vivo. Oogenesis starts in a specialized structure called the germarium, which harbors the stem cells for both germ and somatic cells. The germarium produces egg chambers, each of which will develop into an egg. Active areas of research in Drosophila germaria include stem cell self-renewal, division, and maintenance, cell cycle control and differentiation, oocyte specification, intercellular communication, and signaling, among others. The solid knowledge base, the genetic tractability of the Drosophila model, as well as the availability and fast development of tools and imaging techniques for oogenesis research ensure that studies in this model will keep being instrumental for novel discoveries within cell and developmental biology also in the future. This chapter focuses on antibody staining in Drosophila germaria and provides a protocol for immunostaining as well as an overview of commonly used antibodies for visualization of different cell types and cellular structures. The protocol is well-suited for subsequent confocal microscopy analyses, and in addition we present key adaptations of the protocol that are useful when performing structured illumination microscopy (SIM) super-resolution imaging. PMID:27557571

  9. Intracellular staining and detection of cytokines by fluorescence-activated flow cytometry.

    PubMed

    Freer, Giulia

    2014-01-01

    The detection of cytokines inside cells producing them has made a tremendous impact on the way immune reactivity is measured. Intracellular cytokine staining is the only immunological technique allowing determination of antigen-specific T cell function and phenotype at the same time; for this reason, it is one of the most popular methods to measure antigenicity in the evaluation of vaccine efficacy and in the study of infectious diseases. It is a flow cytometric technique based on staining of intracellular cytokines and cell markers (surface or cytoplasmic) with fluorescent antibodies after short term culture of stimulated immune cells in the presence of a protein secretion inhibitor, followed by fixation and permeabilization. Most experiments involve detection of five to ten different colors but many more can be detected by modern flow cytometers. Here, we discuss our experience using a standard protocol for intracellular cytokine staining. PMID:24908309

  10. Development of Cell Staining Technique for X-Ray Microscopy

    SciTech Connect

    Tseng, P. Y.; Shih, Y. T.; Liu, C. J.; Hsu, T.; Chien, C. C.; Leng, W. H.; Liang, K. S.; Yin, G. C.; Chen, F. R.; Je, J. H.; Margaritondo, G.; Hwu, Y.

    2007-01-19

    We report a technique for detection of sub-cellular organelles and proteins with hard x-ray microscopy. Several metals were used for enhancing contrast for x-ray microscopy. Osmium tetroxide provides an excellent stain for lipid and can delineate cell membrane. Uranyl acetate has high affinity for nucleotide and can stain nucleus. Immunolocalization of specific proteins and sub-cellular organelles was achieved by 3'3 diaminobenzidine (DAB) with nickel enhancement and nanogold-conjugated secondary antibody with silver enhancement. The x-rays emitted from synchrotron source was monochromatized by double crystal monochromator, the photon energy was fixed at 8 keV to optimize the focusing efficiency of the zone plates. The estimated resolution is about 60 nm. When compared with visible light and conventional confocal microscopy, the X-ray microscopy provides a superior resolution to both conventional optical microscopes.

  11. Silver staining of proteins in polyacrylamide gels

    PubMed Central

    Chevallet, Mireille; Luche, Sylvie; Rabilloud, Thierry

    2006-01-01

    Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) whilst using very simple and cheap equipment and chemicals. It is compatible with downstream processing such as mass spectrometry analysis after protein digestion. The sequential phases of silver staining are protein fixation, then sensitization, then silver impregnation and finally image development. Several variants of silver staining are described here, which can be completed in a time range from 2 hours to one day after the end of the electrophoretic separation. Once completed, the stain is stable for several weeks PMID:17487168

  12. Standardization of the Papanicolaou stain. I. A comparison of five nuclear stains.

    PubMed

    Schulte, E; Wittekind, D

    1990-06-01

    The staining characteristics of five nuclear stains used in a Papanicolaou staining procedure were investigated. Alcohol-fixed cervical smears were stained with a modified Papanicolaou procedure using hematoxylin, alcoholic thionin bromide, alcoholic Victoria blue B, gallocyanin or the thionin Feulgen reagent (thionin-SO2) as the nuclear stain. The same anionic counterstain was used for all slides, and the optical densities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyzer. Alcoholic thionin gave the most intense nuclear stain, with a very high reproducibility of the staining pattern. Hematoxylin showed the highest coefficient of variation of the staining intensity. Both hematoxylin and gallocyanin gave some nonspecific cytoplasmic staining. Thionin-SO2 allowed a quantitative assessment of DNA, but gave a low staining intensity. Staining with the metal complex dyes interfered with subsequent staining with the pararosaniline Feulgen reagent. Alcoholic thioinin is thus recommended as a nuclear stain for cervical cytology in the Papanicolaou procedure, both for image analysis and for visual microscopy. PMID:1695100

  13. A differential staining technique for vertebrate histology.

    PubMed

    Bhattacharyya, T K

    1979-03-01

    A staining method is described for studying micro-anatomy of different vertebrate tissues in the light microscope. A staining sequence of celestin blue--erythrosin--orange G--fast green with mordanting in phosphomolybdic acid yields a satisfactory differentiation and fine colour contrast in various tissues. The efficacy of the method was tested on different avian and mammalian tissues. PMID:86938

  14. Efficiency of staining hair with indocyanine green

    NASA Astrophysics Data System (ADS)

    Kulyabina, Tatyana V.; Kochubey, Vyacheslav I.

    2005-06-01

    The efficiency of staining hair with indocyanine green (ICG) solution depending on type of hair, natural color, staining time and other parameters was investigated. Bonding ICG with hair material occurs due to interaction between ICG molecules and keratinocyte albumin. The penetration of ICG dye into hair meets with difficulties owing to surface protective layer.

  15. 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.

  16. Tooth stain during 2 years' use of chlorhexidine- and fluoride-containing dentifrices.

    PubMed

    Dolles, O K; Eriksen, H M; Gjermo, P

    1979-08-01

    The purpose of the present study was to evaluate the effect of fluoride upon the stain-inducing capacity of chlorhexidine. Ninety-one children, 13 years of age, volunteered for the experiment. Three test dentifrices were used, containing 0.1% NaF, 0.1% NaF + 2% chlorhexidine, and 2% chlorhexidine respectively. The experiment was carried out as a double-blind test, and the degree of staining was recorded after 1 and 2 years. Four methods were used for stain scoring. The results after 1 year showed that chlorhexidine induced less stain when applied together with fluoride than when applied alone. After 2 years this difference disappeared, indicating that fluoride only retarded chlorhexidine-induced staining of teeth. PMID:295486

  17. Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators.

    PubMed

    Tuma, R S; Beaudet, M P; Jin, X; Jones, L J; Cheung, C Y; Yue, S; Singer, V L

    1999-03-15

    The highest sensitivity nucleic acid gel stains developed to date are optimally excited using short-wavelength ultraviolet or visible light. This is a disadvantage for laboratories equipped only with 306- or 312-nm UV transilluminators. We have developed a new unsymmetrical cyanine dye that overcomes this problem. This new dye, SYBR Gold nucleic acid gel stain, has two fluorescence excitation maxima when bound to DNA, one centered at approximately 300 nm and one at approximately 495 nm. We found that when used with 300-nm transillumination and Polaroid black-and-white photography, SYBR Gold stain is more sensitive than ethidium bromide, SYBR Green I stain, and SYBR Green II stain for detecting double-stranded DNA, single-stranded DNA, and RNA. SYBR Gold stain's superior sensitivity is due to the high fluorescence quantum yield of the dye-nucleic acid complexes ( approximately 0.7), the dye's large fluorescence enhancement upon binding to nucleic acids ( approximately 1000-fold), and its capacity to more fully penetrate gels than do the SYBR Green gel stains. We found that SYBR Gold stain is as sensitive as silver staining for detecting DNA-with a single-step staining procedure. Finally, we found that staining nucleic acids with SYBR Gold stain does not interfere with subsequent molecular biology protocols. PMID:10075818

  18. Porcine Intestinal Mast Cells. Evaluation of Different Fixatives for Histochemical Staining Techniques Considering Tissue Shrinkage

    PubMed Central

    Rieger, J.; Twardziok, S.; Huenigen, H.; Hirschberg, R.M.; Plendl, J.

    2013-01-01

    Staining of mast cells (MCs), including porcine ones, is critically dependent upon the fixation and staining technique. In the pig, mucosal and submucosal MCs do not stain or stain only faintly after formalin fixation. Some fixation methods are particularly recommended for MC staining, for example the fixation with Carnoy or lead salts. Zinc salt fixation (ZSF) has been reported to work excellently for the preservation of fixation-sensitive antigens. The aim of this study was to establish a reliable histological method for counting of MCs in the porcine intestinum. For this purpose, different tissue fixation and staining methods that also allow potential subsequent immunohistochemical investigations were evaluated in the porcine mucosa, as well as submucosa of small and large intestine. Tissues were fixed in Carnoy, lead acetate, lead nitrate, Zamboni and ZSF and stained subsequently with either polychromatic methylene blue, alcian blue or toluidine blue. For the first time our study reveals that ZSF, a heavy metal fixative, preserves metachromatic staining of porcine MCs. Zamboni fixation was not suitable for histochemical visualization of MCs in the pig intestine. All other tested fixatives were suitable. Alcian blue and toluidine blue co-stained intestinal goblet cells which made a prima facie identification of MCs difficult. The polychromatic methylene blue proved to be the optimal staining. In order to compare MC counting results of the different fixation methods, tissue shrinkage was taken into account. As even the same fixation caused shrinkagedifferences between tissue from small and large intestine, different factors for each single fixation and intestinal localization had to be calculated. Tissue shrinkage varied between 19% and 57%, the highest tissue shrinkage was found after fixation with ZSF in the large intestine, the lowest one in the small intestine after lead acetate fixation. Our study emphasizes that MC counting results from data using different

  19. Porcine intestinal mast cells. Evaluation of different fixatives for histochemical staining techniques considering tissue shrinkage.

    PubMed

    Rieger, J; Twardziok, S; Huenigen, H; Hirschberg, R M; Plendl, J

    2013-01-01

    Staining of mast cells (MCs), including porcine ones, is critically dependent upon the fixation and staining technique. In the pig, mucosal and submucosal MCs do not stain or stain only faintly after formalin fixation. Some fixation methods are particularly recommended for MC staining, for example the fixation with Carnoy or lead salts. Zinc salt fixation (ZSF) has been reported to work excellently for the preservation of fixation-sensitive antigens. The aim of this study was to establish a reliable histological method for counting of MCs in the porcine intestinum. For this purpose, different tissue fixation and staining methods that also allow potential subsequent immunohistochemical investigations were evaluated in the porcine mucosa, as well as submucosa of small and large intestine. Tissues were fixed in Carnoy, lead acetate, lead nitrate, Zamboni and ZSF and stained subsequently with either polychromatic methylene blue, alcian blue or toluidine blue. For the first time our study reveals that ZSF, a heavy metal fixative, preserves metachromatic staining of porcine MCs. Zamboni fixation was not suitable for histochemical visualization of MCs in the pig intestine. All other tested fixatives were suitable. Alcian blue and toluidine blue co-stained intestinal goblet cells which made a prima facie identification of MCs difficult. The polychromatic methylene blue proved to be the optimal staining. In order to compare MC counting results of the different fixation methods, tissue shrinkage was taken into account. As even the same fixation caused shrinkage-differences between tissue from small and large intestine, different factors for each single fixation and intestinal localization had to be calculated. Tissue shrinkage varied between 19% and 57%, the highest tissue shrinkage was found after fixation with ZSF in the large intestine, the lowest one in the small intestine after lead acetate fixation. Our study emphasizes that MC counting results from data using

  20. Compact, Automated Centrifugal Slide-Staining System

    NASA Technical Reports Server (NTRS)

    Feeback, Daniel L.; Clarke, Mark S. F.

    2004-01-01

    The Directional Acceleration Vector-Driven Displacement of Fluids (DAVD-DOF) system, under development at the time of reporting the information for this article, would be a relatively compact, automated, centrifugally actuated system for staining blood smears and other microbiological samples on glass microscope slides in either a microgravitational or a normal Earth gravitational environment. The DAVD-DOF concept is a successor to the centrifuge-operated slide stainer (COSS) concept, which was reported in Slide-Staining System for Microgravity or Gravity (MSC-22949), NASA Tech Briefs, Vol. 25, No. 1 (January, 2001), page 64. The COSS includes reservoirs and a staining chamber that contains a microscope slide to which a biological sample is affixed. The staining chamber is sequentially filled with and drained of staining and related liquids from the reservoirs by use of a weighted plunger to force liquid from one reservoir to another at a constant level of hypergravity maintained in a standard swing-bucket centrifuge. In the DAVD-DOF system, a staining chamber containing a sample would also be sequentially filled and emptied, but with important differences. Instead of a simple microscope slide, one would use a special microscope slide on which would be fabricated a network of very small reservoirs and narrow channels connected to a staining chamber (see figure). Unlike in the COSS, displacement of liquid would be effected by use of the weight of the liquid itself, rather than the weight of a plunger.

  1. Multicenter Assessment of Gram Stain Error Rates.

    PubMed

    Samuel, Linoj P; Balada-Llasat, Joan-Miquel; Harrington, Amanda; Cavagnolo, Robert

    2016-06-01

    Gram stains remain the cornerstone of diagnostic testing in the microbiology laboratory for the guidance of empirical treatment prior to availability of culture results. Incorrectly interpreted Gram stains may adversely impact patient care, and yet there are no comprehensive studies that have evaluated the reliability of the technique and there are no established standards for performance. In this study, clinical microbiology laboratories at four major tertiary medical care centers evaluated Gram stain error rates across all nonblood specimen types by using standardized criteria. The study focused on several factors that primarily contribute to errors in the process, including poor specimen quality, smear preparation, and interpretation of the smears. The number of specimens during the evaluation period ranged from 976 to 1,864 specimens per site, and there were a total of 6,115 specimens. Gram stain results were discrepant from culture for 5% of all specimens. Fifty-eight percent of discrepant results were specimens with no organisms reported on Gram stain but significant growth on culture, while 42% of discrepant results had reported organisms on Gram stain that were not recovered in culture. Upon review of available slides, 24% (63/263) of discrepant results were due to reader error, which varied significantly based on site (9% to 45%). The Gram stain error rate also varied between sites, ranging from 0.4% to 2.7%. The data demonstrate a significant variability between laboratories in Gram stain performance and affirm the need for ongoing quality assessment by laboratories. Standardized monitoring of Gram stains is an essential quality control tool for laboratories and is necessary for the establishment of a quality benchmark across laboratories. PMID:26888900

  2. De-staining and re-staining mucins in formalin fixed paraffin sections.

    PubMed

    Smith, A A; Glickfield, I

    2011-04-01

    Re-staining of formalin fixed paraffin sections sometimes is required and this requires prior de-staining. Some simple and effective protocols for de-staining are described. Mucihematoxylin and mucicarmine can be removed with acid alcohol. Zirconyl hematoxylin can be removed with periodic acid or Sinha's fixative. Alcian blue can be removed with 5% trifluoroacetic acid in dichloromethane. Colloidal iron can be bleached in 1% household bleach in alcohol. PAS can be removed with hydrogen peroxide or ammonium hydroxide. With few exceptions, de-stained sections can be re-stained with mucihematoxylin, PAS or Gabe's trichrome. PMID:20001228

  3. Comparison between ELISA and various stains techniques in laboratory diagnosis of cryptosporidiosis.

    PubMed

    Gabr, Nabil S; Abdellatif, Manal Z M; Abd El-Hafeez, Ekhlas; Abd Rabou, Reham A M

    2014-08-01

    Cryptosporidium spp. is an important parasitic protozoa causing diarrhea which is a severe life-threatening diarrhea especially in immunocompromised hosts. We aimed to evaluate the usefulness of detection of Cryptosporidium spp. copro-antigen from fecal specimens by using enzyme linked immunosorbent assay (ELISA) test and comparing its sensitivity and specificity with some staining methods. The results revealed that Modified Acid-Fast stain is considered better than Giemsa in detecting Cryptosporidium species oocysts in faecal smears as their sensitivity were 67.5% and 53.75% respectively. On contrary, ELISA technique is considered the best method used for detection of cryptosporidial infection as its sensitivity is 90%. PMID:25597165

  4. Antigenic sites in carcinoembryonic antigen.

    PubMed

    Hammarstrom, S; Shively, J E; Paxton, R J; Beatty, B G; Larsson, A; Ghosh, R; Bormer, O; Buchegger, F; Mach, J P; Burtin, P

    1989-09-01

    The epitope reactivities of 52 well-characterized monoclonal antibodies (Mabs) against carcinoembryonic antigen from 11 different research groups were studied using competitive solid-phase immunoassays. About 60% of all possible combinations of Mabs as inhibitors and as the primary binding antibody were investigated. The inhibition data were analyzed by a specially developed computer program "EPITOPES" which measures concordance and discordance in inhibition patterns between Mabs. The analysis showed that 43 of the 52 Mabs (83%) could be classified into one of five essentially noninteracting epitope groups (GOLD 1-5) containing between four and 15 Mabs each. The epitopes recognized by the Mabs belonging to groups 1 to 5 were peptide in nature. With one or two possible exceptions non-classifiable Mabs were either directed against carbohydrate epitopes (4 Mabs) or were inactive in the tests used. Within epitope groups GOLD 1, 4, and 5 two partially overlapping subgroups were distinguished. Mabs with a high degree of carcinoembryonic antigen specificity generally belonged to epitope groups GOLD 1 and 3. PMID:2474375

  5. MAGE-A antigens in lesions of the oral mucosa.

    PubMed

    Krauss, Eva; Rauthe, Stephan; Gattenlöhner, Stefan; Reuther, Tobias; Kochel, Michael; Kriegebaum, Ulrike; Kübler, Alexander C; Müller-Richter, Urs D A

    2011-06-01

    Oral squamous cell carcinoma develops continuously out of predamaged oral mucosa. For the physician and pathologist, difficulties arise in distinguishing precancerous from cancerous lesions. MAGE-A antigens are tumor antigens that are found solely in malignant transformed cells. These antigens might be useful in distinguishing precancerous from cancerous lesions. The aim of this study was to verify this assumption by comparing MAGE-A expression in benign, precancerous, and cancerous lesions of the oral mucosa. Retrospectively, biopsies of different oral lesions were randomly selected. The lesions that were included are 64 benign oral lesions (25 traumatic lesions (oral ulcers), 13 dental follicles, and 26 epulis), 26 oral lichen planus, 123 epithelial precursor lesions (32 epithelial hyperplasia found in leukoplakias, 24 epithelial dysplasia found in leukoplakias, 26 erythroplasia with oral epithelial dysplasia, and 41 carcinomas in situ in erythroleukoplakias). The lesions were immunohistochemically stained with the poly-MAGE-A antibody 57B, and the results were compared. Biopsies of oral lichen planus, oral ulcers, dental follicles, epulis, and leukoplakia without dysplasia showed no positive staining for MAGE-A antigens. Leukoplakia with dysplasia, dysplasia, and carcinomata in situ displayed positive staining in 33%, 65%, and 56% of the cases, respectively. MAGE-A antigens were not detectable via immunohistochemistry in benign lesions of the oral mucosa. The staining rate of dysplastic precancerous lesions or malignant lesions ranged from 33% to 65%. The MAGE-A antigens might facilitate better differentiation between precancerous and cancerous lesions of the oral mucosa. PMID:20174843

  6. Gram staining apparatus for space station applications

    NASA Technical Reports Server (NTRS)

    Molina, T. C.; Brown, H. D.; Irbe, R. M.; Pierson, D. L.

    1990-01-01

    A self-contained, portable Gram staining apparatus (GSA) has been developed for use in the microgravity environment on board the Space Station Freedom. Accuracy and reproducibility of this apparatus compared with the conventional Gram staining method were evaluated by using gram-negative and gram-positive controls and different species of bacteria grown in pure cultures. A subsequent study was designed to assess the performance of the GSA with actual specimens. A set of 60 human and environmental specimens was evaluated with the GSA and the conventional Gram staining procedure. Data obtained from these studies indicated that the GSA will provide the Gram staining capability needed for the microgravity environment of space.

  7. An alternative to India ink stain.

    PubMed

    Ibembe, Isaac Nicholas; Wiggin, Timothy Roger

    2015-07-01

    Accessing India ink in rural Uganda is difficult and costly. An alternative stain was sought to assist in microbiological diagnoses of cryptococcal infections in immunosuppressed patients with meningitis. Mascara proved to be an excellent and cheap alternative. PMID:25999353

  8. Gram staining apparatus for space station applications.

    PubMed Central

    Molina, T C; Brown, H D; Irbe, R M; Pierson, D L

    1990-01-01

    A self-contained, portable Gram staining apparatus (GSA) has been developed for use in the microgravity environment on board the Space Station Freedom. Accuracy and reproducibility of this apparatus compared with the conventional Gram staining method were evaluated by using gram-negative and gram-positive controls and different species of bacteria grown in pure cultures. A subsequent study was designed to assess the performance of the GSA with actual specimens. A set of 60 human and environmental specimens was evaluated with the GSA and the conventional Gram staining procedure. Data obtained from these studies indicated that the GSA will provide the Gram staining capability needed for the microgravity environment of space. Images PMID:1690529

  9. Comparison of Special Stains for Keratin with Routine Hematoxylin and Eosin Stain

    PubMed Central

    Rao, Roopa S; Patil, Shankargouda; Majumdar, Barnali; Oswal, Rakesh G

    2015-01-01

    Background: Keratins are the most abundant proteins and are characteristic findings in many epithelial pathologies, making it a diagnostically important marker, both histopathologically and immunohistochemically. Since, immunohistochemistry is an expensive diagnostic tool, special stains to detect the degree of keratinization could serve as a faster and economic option. The aim of the present study was to compare the efficacy of special stains for keratin with standard hematoxylin and eosin stain (H and E). Objectives include: (i) To subject the diagnosed cases of keratin disorders to the selected special stains: Ayoub-shklar method, Dane-Herman method, Alcian blue –periodic acid Schiff ’s (PAS), rapid papanicolaou (PAP) and Gram’s stain. (ii) To compare the staining specificity and staining intensity of special stains with respect to routine hematoxylin and eosin (H and E) stain. (iii) To compare the efficacy of special stains to routine H and E stain in identification of the type of keratin present in the selected cases. Materials and Methods: A total of 80 cases of known pathology for keratin were retrieved from the department archive, which included 10 each of normal gingiva, hyperkeratosis, squamous papilloma, verrucous hyperplasia, verrucous carcinoma, well-differentiated squamous cell carcinoma, orthokeratinized odontogenic cyst and keratocystic odontogenic tumors. Six sections of 4 µ each from the paraffin blocks were made, stained with H and E and the special stains and these were evaluated by 2 pathologists based on the modified scoring criteria from Rahma Al-Maaini and Philip Bryant 2008. Results: The results were tabulated using Chi square and kappa statistics. The statistical values for identification of the type of keratinization was insignificant showing that ortho and parakeratinized epithelia could be correctly identified by both H and E as well as all the special stains. Furthermore, all the special stains showed a positive result and

  10. Antibody Staining in C. Elegans Using "Freeze-Cracking"

    PubMed Central

    Duerr, Janet S.

    2013-01-01

    To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest. PMID:24145964

  11. New Grocott Stain without Using Chromic Acid

    PubMed Central

    Shiogama, Kazuya; Kitazawa, Kayo; Mizutani, Yasuyoshi; Onouchi, Takanori; Inada, Ken-ichi; Tsutsumi, Yutaka

    2015-01-01

    We established a new “ecological” Grocott stain for demonstrating fungi, based upon a 4R principle of refusal, reduction, reuse, and recycle of waste management. Conventional Grocott stain employs environmentally harsh 5% chromic acid for oxidization. Initially, we succeeded in reducing the concentration of chromic acid from 5% to 1% by incubating the solution at 60°C and using five-fold diluted chromic acid solution at which point it was reusable. Eventually, we reached the refusal level where 1% periodic acid oxidization was efficient enough, when combined with preheating of sections in the electric jar, microwave oven, or pressure pan. For convenience sake, we recommend pressure pan heating in tap water for 10 min. Stainability of fungi in candidiasis and aspergillosis was comparable with conventional Grocott stain, while Mucor hyphae showed enhanced staining. The modified sequence was further applicable to detecting a variety of mycotic pathogens in paraffin sections. Our environmentally-friendly Grocott stain also has the advantage of avoiding risk of human exposure to hexavalent chromium solution in the histopathology laboratory. The simple stain sequence is can be easily applied worldwide. PMID:25861133

  12. Induction of embryonic major histocompatibility complex antigen expression by gamma-IFN.

    PubMed

    Warner, C M; Almquist, C D; Toulimat, M H; Xu, Y

    1993-07-01

    Preimplantation mouse embryos were incubated in vitro with mouse recombinant gamma-interferon (IFN). The effect of the gamma-IFN on major histocompatibility complex (MHC) class I antigen expression was tested using an ELISA procedure. It was found that there is a doubling of Db antigens and a tripling of Qa-2 antigens on C57BL/6 mouse embryos cultured from the 8-cell stage for 24 h in the presence of 10(5) units/ml gamma-IFN. The effect of gamma-IFN on the rate of preimplantation embryonic development was tested by culturing 2-cell embryos for 48 h and 8-cell embryos for 24 h in the presence of varying concentrations of gamma-IFN up to 10(6) units/ml. Two methods were used to assess the cell number per embryo after the culture period: incorporation of [3H]thymidine into DNA, and direct counting of nuclei in fixed and stained embryos. Both methods showed that treatment with gamma-IFN increases the rate of development of preimplantation mouse embryos. Since rate of preimplantation embryonic development is genetically controlled by the Ped gene, it is suggested that gamma-IFN has a direct effect on the Ped gene phenotype of preimplantation mouse embryos. PMID:8229991

  13. Antigenic composition of single nano-sized extracellular blood vesicles.

    PubMed

    Arakelyan, Anush; Ivanova, Oxana; Vasilieva, Elena; Grivel, Jean-Charles; Margolis, Leonid

    2015-04-01

    Extracellular vesicles (EVs) are important in normal physiology and are altered in various pathologies. EVs produced by different cells are antigenically different. Since the majority of EVs are too small for routine flow cytometry, EV composition is studied predominantly in bulk, thus not addressing their antigenic heterogeneity. Here, we describe a nanoparticle-based technique for analyzing antigens on single nano-sized EVs. The technique consists of immuno-capturing of EVs with 15-nm magnetic nanoparticles, staining captured EVs with antibodies against their antigens, and separating them from unbound EVs and free antibodies in a magnetic field, followed by flow analysis. This technique allows us to characterize EVs populations according to their antigenic distribution, including minor EV fractions. We demonstrated that the individual blood EVs carry different sets of antigens, none being ubiquitous, and quantified their distribution. The physiological significance of antigenically different EVs and their correlation with different pathologies can now be directly addressed. From the clinical editor: This study reports a nanoparticle-based technique for analyzing antigens on single nano-sized extracellular vehicles (EV). The technique consists of immuno-capturing of EVs with 15-nm magnetic nanoparticles, followed by staining the captured EVs with antibodies and separating them via a magnetic field, followed by flow analysis. This technique enables studies of antigenic properties of individual EVs that conventionally can only be studied in bulk. PMID:25481806

  14. Expression patterns of the T antigen and the cryptic T antigen in rat fetuses: detection with the lectin amaranthin.

    PubMed

    Sata, T; Zuber, C; Rinderle, S J; Goldstein, I J; Roth, J

    1990-06-01

    The lectin amaranthin, purified from the seeds of Amaranthus caudatus, has been shown to react specifically with the Gal beta 1,3GalNAc-alpha and the NeuAc alpha 2,3Gal beta 1,3GalNAc-alpha sequence which represent the T antigen and the cryptic T antigen, respectively. We report here the development of labeling techniques that apply amaranthin to stain paraffin sections from rat fetuses. Amaranthin staining was inhibited by pre-incubation of lectin-gold complexes with 10 mM Gal beta 1,3GalNAc-alpha-O-benzyl (synthetic T antigen) or 10 mM Gal beta 1,3GalNAc-alpha-O-aminophenylethyl-human serum albumin (T antigen neoglycoprotein), asialoglycophorin, asialofetuin, and asialomucin. The beta-elimination reaction also abolished the lectin staining demonstrating specificity for O-glycosidically linked structures. A comparison with monoclonal anti-T antigen antibody immunostaining demonstrated that amaranthin detects the T antigen and its cryptic form in tissue sections. Application of the galactose oxidase-Schiff sequence abolished amaranthin (and anti-T antibody) binding to the T antigen but not to its cryptic form, and therefore permitted their differentiation in tissue sections. Histochemical evidence was obtained indicating that amaranthin is a more specific anti-T reagent than peanut lectin. Data are presented that show the differential expression of the T antigen and the cryptic T antigen in organs and cells of rat fetuses late in gestation. Therefore, amaranthin can be used for histochemical detection of the T antigen and the cryptic T antigen, and facilitates discrimination between them. PMID:2335739

  15. A Comparison of Acquired Port-wine Stain with Congenital Port-wine Stain Using an Image Analyzer

    PubMed Central

    Lee, Jung Ju; Lee, Jae Chul; Kim, Byung Soo; Lee, Weon Ju; Kim, Do Won; Jang, Yun Hwan; Bae, Han Ik

    2008-01-01

    Background Recent reports have proposed that there were no differences between acquired port-wine stain (APWS) and congenital port-wine stain (CPWS) except the onset of disease. Pulsed dye laser (PDL) therapy is regarded as the treatment of choice in PWS. Although in some articles, APWS might have shown a better response to PDL than CPWS, this is still controversial. It has been assumed however, that there might be some differences determining therapeutic responses between the two entities. Objective The purpose of this study is to find out some histopathologic differences between APWS and CPWS. Methods 14 patients with APWS and 17 patients with CPWS from our patient files were included in this study. Immunohistochemical staining by factor VIII-related antigen was carried out on the specimens of punch biopsy to better visualize the blood vessels. Histopathologic assessment of variables such as vessel area, percentage of vascular area and vessel depth was performed using a computer-assisted image analyzer program. Results The mean vessel area in APWS was 1014.7 ± 782.5µm2 and that of CPWS was 1341.5 ± 689.9µm2. The mean percentage of vascular area in APWS was 2.02 ± 1.38% and that of CPWS was 2.65 ± 1.56%. The mean vessel depth in APWS was 327.5 ± 120.7µm and 321.7 ± 93.1µm in CPWS. No histopathologic variable was statistically significant using the Mann-Whitney test (p>0.05). PMID:27303148

  16. Digital stain separation for histological images.

    PubMed

    Tadrous, P J

    2010-11-01

    It is often desirable to perform digital image analyses on sections prepared for human interpretation, e.g. nuclear chromatin texture analysis or three-dimensional reconstructions using sections requiring human delineation of structures of interest. Unfortunately such analyses are often more effective using stains with less complex contrast. Here an automated selective 'de-staining' method for digital images is presented. The method separates an image into its red, green and blue and hue, saturation and intensity components. A mask of stained tissue is prepared by automatic percentile thresholding. A single weighted inverted colour channel is then added to each of the three primary colour channels separately by an iterative algorithm that adjusts the weights to give minimum variance within the mask. The modified red, green and blue channels are then recombined. This method is automatic requiring no pre-definition of stain colours or special hardware. The method is demonstrated to 'de-stain' nuclei in haematoxylin and eosin (H&E) sections (and a separate haematoxylin image can be derived from this). An image of isolated brown reaction product is produced with immunoperoxidase preparations counterstained with haematoxylin. Furthermore trichrome (haematoxylin van Gieson, picrosirius red) and other common stains may be separated into their components with modifications of the same algorithm. Although other methods for colour separation do exist (e.g. spectral pathology and colour deconvolution) these require special apparatus or precise calibration and foreknowledge of pure dye colour spectra. The present method of digital stain separation is fully automatic with no such prerequisites. PMID:20946383

  17. Compositions for chromosome-specific staining

    DOEpatents

    Gray, J.W.; Pinkel, D.

    1998-05-26

    Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. The methods produce staining patterns that can be tailored for specific cytogenetic analyses. The probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods are provided to disable the hybridization capacity of shared, high copy repetitive sequences and/or remove such sequences to provide for useful contrast. Still further methods are provided to produce chromosome-specific staining reagents which are made specific to the targeted chromosomal material, which can be one or more whole chromosomes, one or more regions on one or more chromosomes, subsets of chromosomes and/or the entire genome. Probes and test kits are provided for use in tumor cytogenetics, in the detection of disease related loci, in analysis of structural abnormalities, such as translocations, and for biological dosimetry. Methods and prenatal test kits are provided to stain targeted chromosomal material of fetal cells, including fetal cells obtained from maternal blood. The invention provides for automated means to detect and analyze chromosomal abnormalities. 17 figs.

  18. Compositions for chromosome-specific staining

    DOEpatents

    Gray, Joe W.; Pinkel, Daniel

    1998-01-01

    Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods are provided to disable the hybridization capacity of shared, high copy repetitive sequences and/or remove such sequences to provide for useful contrast. Still further methods are provided to produce chromosome-specific staining reagents which are made specific to the targeted chromosomal material, which can be one or more whole chromosomes, one or more regions on one or more chromosomes, subsets of chromosomes and/or the entire genome. Probes and test kits are provided for use in tumor cytogenetics, in the detection of disease related loci, in analysis of structural abnormalities, such as translocations, and for biological dosimetry. Further, methods and prenatal test kits are provided to stain targeted chromosomal material of fetal cells, including fetal cells obtained from maternal blood. Still further, the invention provides for automated means to detect and analyse chromosomal abnormalities.

  19. Acridine orange staining reaction as an index of physiological activity in Escherichia coli

    NASA Technical Reports Server (NTRS)

    McFeters, G. A.; Singh, A.; Byun, S.; Callis, P. R.; Williams, S.

    1991-01-01

    The assumption that the acridine orange (AO) color reaction may be used as an index of physiological activity was investigated in laboratory grown Escherichia coli. Spectrofluorometric observations of purified nucleic acids, ribosomes and the microscopic color of bacteriophage-infected cells stained with AO confirmed the theory that single-stranded nucleic acids emit orange to red fluorescence while those that are double-stranded fluoresce green in vivo. Bacteria growing actively in a rich medium could be distinguished from cells in stationary phase by the AO reaction. Cells from log phase appeared red, whereas those in stationary phase were green. However, this differentiation was not seen when the bacteria were grown in a minimal medium or when a variation of the staining method was used. Also, shifting bacteria in stationary phase to starvation conditions rapidly changed their AO staining reaction. Boiling and exposure to lethal concentrations of azide and formalin resulted in stationary-phase cells that appeared red after staining but bacteria killed with chlorine remained green. These findings indicate that the AO staining reaction may be suggestive of physiological activity under defined conditions. However, variables in staining and fixation procedures as well as uncertainties associated with mixed bacterial populations in environmental samples may produce results that are not consistent with the classical interpretation of this reaction. The importance of validating the putative physiological implications of this staining reaction is stressed.

  20. Detection Of Concrete Deterioration By Staining

    DOEpatents

    Guthrie, Jr., George D.; Carey, J. William

    1999-09-21

    A method using concentrated aqueous solutions of sodium cobaltinitrite and a rhodamine dye is described which can be used to identify concrete that contains gels formed by the alkali-silica reaction (ASR), and to identify degraded concrete which results in a porous or semi-permeable paste due to carbonation or leaching. These solutions present little health or environmental risk, are readily applied, and rapidly discriminate between two chemically distinct gels; K-rich, Na--K--Ca--Si gels are identified by yellow staining, and alkali-poor, Ca--Si gels are identified by pink staining.

  1. Laser treatment of port-wine stains

    PubMed Central

    Brightman, Lori A; Geronemus, Roy G; Reddy, Kavitha K

    2015-01-01

    Port-wine stains are a type of capillary malformation affecting 0.3% to 0.5% of the population. Port-wine stains present at birth as pink to erythematous patches on the skin and/or mucosa. Without treatment, the patches typically darken with age and may eventually develop nodular thickening or associated pyogenic granuloma. Laser and light treatments provide improvement through selective destruction of vasculature. A variety of vascular-selective lasers may be employed, with the pulsed dye laser being the most common and well studied. Early treatment produces more optimal results. Advances in imaging and laser treatment technologies demonstrate potential to further improve clinical outcomes. PMID:25624768

  2. Automated single-slide staining device

    NASA Technical Reports Server (NTRS)

    Wilkins, J. R.; Mills, S. M. (Inventor)

    1977-01-01

    A simple apparatus and method is disclosed for making individual single Gram stains on bacteria inoculated slides to assist in classifying bacteria in the laboratory as Gram-positive or Gram-negative. The apparatus involves positioning a single inoculated slide in a stationary position and thereafter automatically and sequentially flooding the slide with increments of a primary stain, a mordant, a decolorizer, a counterstain and a wash solution in a sequential manner without the individual lab technician touching the slide and with minimum danger of contamination thereof from other slides.

  3. Pleural and Pulmonary Staining at Inferior Phrenic Arteriography Mimicking a Tumor Staining of Hepatocellular Carcinoma

    SciTech Connect

    Lee, Deok Hee; Hwang, Jae Cheol; Lim, Soo Mee; Yoon, Hyun-Ki; Sung, Kyu-Bo; Song, Ho-Young

    2000-03-15

    Purpose: To describe the findings of pleural and pulmonary staining of the inferior phrenic artery, which can be confused with tumor staining during transarterial chemoembolization (TACE) of hepatoma.Methods: Fifteen patients who showed pleural and pulmonary staining without relationship to hepatic masses at inferior phrenic arteriography were enrolled. The staining was noted at initial TACE (n = 8), at successive TACE (n = 5), and after hepatic surgery (n = 2). The angiographic pattern, the presence of pleural change on computed tomography (CT), and clinical history were evaluated.Results: Draining pulmonary veins were seen in all cases. The lower margin of the staining corresponded to the lower margin of the pleura in 10 patients. CT showed pleural and/or pulmonary abnormalities in all cases. After embolization of the inferior phrenic artery, the accumulation of iodized oil in the lung was noted.Conclusion: Understanding the CT and angiographic findings of pleural and pulmonary staining during TACE may help differentiate benign staining from tumor staining.

  4. Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells

    PubMed Central

    Starkie, Dale. O; Compson, Joanne E.; Rapecki, Stephen; Lightwood, Daniel J.

    2016-01-01

    Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive). These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP) fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking antibody from mice

  5. Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells.

    PubMed

    Starkie, Dale O; Compson, Joanne E; Rapecki, Stephen; Lightwood, Daniel J

    2016-01-01

    Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive). These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP) fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking antibody from mice

  6. Antigenic characterisation of lyssaviruses in South Africa.

    PubMed

    Ngoepe, Ernest; Fehlner-Gardiner, Christine; Wandeler, Alex; Sabeta, Claude

    2014-01-01

    There are at least six Lyssavirus species that have been isolated in Africa, which include classical rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, Shimoni bat virus and Ikoma lyssavirus. In this retrospective study, an analysis of the antigenic reactivity patterns of lyssaviruses in South Africa against a panel of 15 anti-nucleoprotein monoclonal antibodies was undertaken. A total of 624 brain specimens, collected between 2005 and 2009, confirmed as containing lyssavirus antigen by direct fluorescent antibody test, were subjected to antigenic differentiation. The lyssaviruses were differentiated into two species, namely rabies virus (99.5%) and Mokola virus (0.5%). Furthermore, rabies virus was further delineated into two common rabies biotypes in South Africa: canid and mongoose. Initially, it was found that the canid rabies biotype had two reactivity patterns; differential staining was observed with just one monoclonal antibody. This difference was likely to have been an artefact related to sample quality, as passage in cell culture restored staining. Mongoose rabies viruses were more heterogeneous, with seven antigenic reactivity patterns detected. Although Mokola viruses were identified in this study, prevalence and reservoir host species are yet to be established. These data demonstrate the usefulness of monoclonal antibody typing panels in lyssavirus surveillance with reference to emergence of new species or spread of rabies biotypes to new geographic zones. PMID:25685866

  7. The Language of Stained-Glass Windows

    ERIC Educational Resources Information Center

    Brew, Charl Anne

    2010-01-01

    The splendor and beauty of stained glass punctuates any room. In this article, the author describes a cross-curriculum project which incorporated the French classes' research and written study of France in the Middle Ages. For the project the author suggested Sainte-Chapelle which is considered a reliquary and was built by Louis IX to house the…

  8. Method for copper staining of germanium crystals

    NASA Technical Reports Server (NTRS)

    Rivet, E. J.

    1969-01-01

    Proper conditions for copper staining of germanium crystals include a low solution temperature of 3 degrees C, illumination of the sample by infrared light, and careful positioning of the light source relative to the sample so as to minimize absorption of the infrared light.

  9. Asbestos identification by dispersion staining microscopy.

    PubMed

    Ganotes, J T; Tan, H T

    1980-01-01

    Asbestos can be detected and identified by an optical microscope procedure known as dispersion staining. This procedure can be carried out with most phase contrast equipped microscopes. The primary application is for material samples. Distinction between tremolite and anthophyllite asbestos requires examination between crossed polarizers. PMID:6153496

  10. Rapid contrast evaluation method based on affinity beads and backscattered electron imaging for the screening of electron stains.

    PubMed

    Kaku, Hiroki; Inoue, Kanako; Muranaka, Yoshinori; Park, Pyoyun; Ikeda, Kenichi

    2015-10-01

    Uranyl salts are toxic and radioactive; therefore, several studies have been conducted to screen for substitutes of electron stains. In this regard, the contrast evaluation process is time consuming and the results obtained are inconsistent. In this study, we developed a novel contrast evaluation method using affinity beads and a backscattered electron image (BSEI), obtained using scanning electron microscopy. The contrast ratios of BSEI in each electron stain treatment were correlated with those of transmission electron microscopic images. The affinity beads bound to cell components independently. Protein and DNA samples were enhanced by image contrast treated with electron stains; however, this was not observed for sugars. Protein-conjugated beads showed an additive effect of image contrast when double-stained with lead. However, additive effect of double staining was not observed in DNA-conjugated beads. The varying chemical properties of oligopeptides showed differences in image contrast when treated with each electron stain. This BSEI-based evaluation method not only enables screening for alternate electron stains, but also helps analyze the underlying mechanisms of electron staining of cellular structures. PMID:26199255

  11. HLA Class II Antigen Expression in Colorectal Carcinoma Tumors as a Favorable Prognostic Marker12

    PubMed Central

    Sconocchia, Giuseppe; Eppenberger-Castori, Serenella; Zlobec, Inti; Karamitopoulou, Eva; Arriga, Roberto; Coppola, Andrea; Caratelli, Sara; Spagnoli, Giulio Cesare; Lauro, Davide; Lugli, Alessandro; Han, Junyi; Iezzi, Giandomenica; Ferrone, Cristina; Ferlosio, Amedeo; Tornillo, Luigi; Droeser, Raoul; Rossi, Piero; Attanasio, Antonio; Ferrone, Soldano; Terracciano, Luigi

    2014-01-01

    The goal of this study was to determine the frequency of HLA class II antigen expression in colorectal carcinoma (CRC) tumors, its association with the clinical course of the disease, and the underlying mechanism(s). Two tissue microarrays constructed with 220 and 778 CRC tumors were stained with HLA-DR, DQ, and DP antigen-specific monoclonal antibody LGII-612.14, using the immunoperoxidase staining technique. The immunohistochemical staining results were correlated with the clinical course of the disease. The functional role of HLA class II antigens expressed on CRC cells was analyzed by investigating their in vitro interactions with immune cells. HLA class II antigens were expressed in about 25% of the 220 and 21% of the 778 tumors analyzed with an overall frequency of 23%. HLA class II antigens were detected in 19% of colorectal adenomas. Importantly, the percentage of stained cells and the staining intensity were significantly lower than those detected in CRC tumors. However, HLA class II antigen staining was weakly detected only in 5.4% of 37 normal mucosa tissues. HLA class II antigen expression was associated with a favorable clinical course of the disease. In vitro stimulation with interferon gamma (IFNγ) induced HLA class II antigen expression on two of the four CRC cell lines tested. HLA class II antigen expression on CRC cells triggered interleukin-1β (IL-1β) production by resting monocytes. HLA class II antigen expression in CRC tumors is a favorable prognostic marker. This association may reflect stimulation of IL-1β production by monocytes. PMID:24563618

  12. 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.

  13. A method for the staining of intraosseous nerve fibers using Sihler's staining technique.

    PubMed

    Shiozaki, K; Miida, K; Tanaka, R; Shimoda, S

    2013-08-01

    Understanding nerve fiber distribution in the jaw bone is important when performing invasive surgical treatments. Both microscopic and macroscopic anatomical techniques have been developed to study innervation. Conventional methods of removing and staining these structures, however, often alter structure and lack reproducibility of the resulting specimens. We sought to optimize Sihler's staining technique to stain intraosseous nerves in mandibles. Four cadaver specimens were used. The best staining of intraosseous nerve fibers was achieved by using the Plank-Rychlo solution. When the Styrene monomer was used, the resulting transparency was better than that obtained with glycerin under the same conditions. No significant differences were found between Sihler's staining procedure performed according to the conventional method and the procedure in which the second decalcification step was omitted. Our results demonstrate that applying Sihler's staining technique to bones makes them transparent and allows observation of nerves while preserving the external shape of the bone and maintaining the position of intraosseous nerve fibers. Our findings suggest our Sihler staining method for intraosseous nerve fibers can provide an intermediate resolution between macroscopic and microscopic techniques. PMID:23472877

  14. 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

  15. 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

  16. Improved Whole-Blood-Staining Device

    NASA Technical Reports Server (NTRS)

    Sams, Clarence F.; Crucian, Brian; Paul, Bonnie; Melton, Shannon; Guess, Terry

    2012-01-01

    Dramatic improvements have been made in NASA s Whole Blood Staining Device (WBSD) since it was last described in "Whole-Blood-Staining Device," NASA Tech Briefs, Vol. 23, No. 10 (October 1999), page 64. The new system has a longer shelf life, a simpler and more effective operational procedure, improved interface with instrumentation, and shorter processing time. More specifically, the improvements have targeted bag and locking clip materials, sampling ports, and air pocket prevention. The WBSD stains whole blood collected during spaceflight for subsequent flow cytometric analysis. In short, the main device stains white blood cells by use of monoclonal antibodies conjugated to various fluorochromes, followed by lysing and fixing of the cells by use of a commercial reagent that has been diluted according to NASA safety standards. This system is compact, robust, and does not require electric power, precise mixing, or precise incubation times. Figure 1 depicts the present improved version for staining applications, which is a poly(tetrafluoroethylene) bag with a Luer-lock port and plastic locking clips. An InterLink (or equivalent) intravenous- injection port screws into the Luer-lock port. The inflatable/collapsible nature of the bag facilitates loading and helps to minimize the amount of air trapped in the fully loaded bag. Some additional uses have been identified for the device beyond whole blood staining. The WBSD has been configured for functional assays that require culture of live cells by housing sterile culture media, mitogens, and fixatives prior to use [Figure 2(a)]. Simple injection of whole blood allows cell-stimulation culture to be performed in reduced gravity conditions, and product stabilization prior to storage, while protecting astronauts from liquid biohazardous materials. Also, the improved WBSD has reconstituted powdered injectable antibiotics by mixing them with diluent liquids [Figure 2(b)]. Although such mixing can readily be performed on

  17. DNA Stains as Surrogate Nucleobases in Fluorogenic Hybridization Probes.

    PubMed

    Hövelmann, Felix; Seitz, Oliver

    2016-04-19

    The increasing importance assigned to RNA dynamics in cells and tissues calls for probe molecules that enable fluorescence microscopy imaging in live cells. To achieve this goal, fluorescence dyes are conjugated with oligonucleotides so as to provide strong emission upon hybridization with the target molecule. The impressive 10(3)-fold fluorescence intensification observed when DNA stains such as thiazole orange (TO) interact with double-stranded DNA is intriguing and prompted the exploration of oligonucleotide conjugates. However, nonspecific interactions of DNA stains with polynucleotides tend to increase background, which would affect the contrast achievable in live-cell imaging. This Account describes the development of DNA-stain-labeled hybridization probes that provide high signal-to-background. We focus on our contributions in context with related advances from other laboratories. The emphasis will be on the requirements of RNA imaging in live cells. To reduce background, intercalator dyes such as TO were appended to peptide nucleic acid (PNA), which is less avidly recognized by DNA stains than DNA/RNA. Constraining the TO dye as a nucleobase surrogate in "forced intercalation (FIT) probes" improved the target specificity, presumably by helping to prevent unspecific interactions. The enforcement of TO intercalation between predetermined base pairs upon formation of the probe-target duplex provided for high brightness and enabled match/mismatch selectivity beyond stringency of hybridization. We show examples that highlight the use of PNA FIT probes in the imaging of mRNA, miRNA, and lncRNA in living cells. The "FIT approach" was recently extended to DNA probes. Signal brightness can become limiting when low-abundance targets ought to be visualized over cellular autofluorescence. We discuss strategies that further the brightness of signaling by FIT probes. Multilabeling with identical dyes does not solve the brightness issue. To avoid self-quenching, we

  18. Al adjuvants can be tracked in viable cells by lumogallion staining.

    PubMed

    Mile, Irene; Svensson, Andreas; Darabi, Anna; Mold, Matthew; Siesjö, Peter; Eriksson, Håkan

    2015-07-01

    The mechanism behind the adjuvant effect of aluminum salts is poorly understood notwithstanding that aluminum salts have been used for decades in clinical vaccines. In an aqueous environment and at a nearly neutral pH, the aluminum salts form particulate aggregates, and one plausible explanation of the lack of information regarding the mechanisms could be the absence of an efficient method of tracking phagocytosed aluminum adjuvants and thereby the intracellular location of the adjuvant. In this paper, we want to report upon the use of lumogallion staining enabling the detection of phagocytosed aluminum adjuvants inside viable cells. Including micromolar concentrations of lumogallion in the culture medium resulted in a strong fluorescence signal from cells that had phagocytosed the aluminum adjuvant. The fluorescence appeared as spots in the cytoplasm and by confocal microscopy and co-staining with probes presenting fluorescence in the far-red region of the spectrum, aluminum adjuvants could to a certain extent be identified as localized in acidic vesicles, i.e., lysosomes. Staining and detection of intracellular aluminum adjuvants was achieved not only by diffusion of lumogallion into the cytoplasm, thereby highlighting the presence of the adjuvant, but also by pre-staining the aluminum adjuvant prior to incubation with cells. Pre-staining of aluminum adjuvants resulted in bright fluorescent particulate aggregates that remained fluorescent for weeks and with only a minor reduction of fluorescence upon extensive washing or incubation with cells. Both aluminum oxyhydroxide and aluminum hydroxyphosphate, two of the most commonly used aluminum adjuvants in clinical vaccines, could be pre-stained with lumogallion and were easily tracked intracellularly after incubation with phagocytosing cells. Staining of viable cells using lumogallion will be a useful method in investigations of the mechanisms behind aluminum adjuvants' differentiation of antigen-presenting cells

  19. Dendritic cell and macrophage staining by monoclonal antibodies in tissue sections and epidermal sheets.

    PubMed Central

    Flotte, T. J.; Springer, T. A.; Thorbecke, G. J.

    1983-01-01

    Mouse tissue sections were stained by monoclonal antibodies to macrophage antigens (Mac-1 (M1/70), Mac-2 (M3/38), Mac-3 (M3/84) with the use of immunoperoxidase. Mac-1 was located diffusely in the cytoplasm of round cells in a high percentage of alveolar macrophages, resident peritoneal and bone marrow cells, in splenic red pulp, and in rare perivascular cells in the thymus. Mac-1 was absent in epithelial cells and Langerhans cells. Mac-2 was strongly positive in many dendritic cells in the thymic medulla, more than the cortex, in paracortex and medulla of lymph nodes, sparing the follicles, and in the marginal zone of spleen. There were a few positive cells in germinal centers. Mac-2 was located in a low percentage of bone marrow and a high percentage of resident peritoneal cells. When positive in sections Mac-3 always showed granular cytoplasmic staining. Bone marrow showed a high percentage of cytoplasmic staining (greater than 50%), as compared with low surface staining (less than 1%). It was found in hematopoietic cells, and in all endothelium, including postcapillary venules and lining of sinuses. It was probable that the resulting dendritic staining pattern for Mac-3 in paracortex of lymph node, white and red pulp, thymic cortex, and medulla included dendritic cells other than endothelial cells. Alveolar macrophages and Kupffer cells were positive for Mac-2 and Mac-3. Mac-3 also stained bile canaliculi. Clearly different staining patterns were found in epithelial cells for Mac-2 and Mac-3 in kidney tubules, intestinal mucosal lining, bronchi, choroid plexus, and epidermis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:6340516

  20. Flavonoid-specific staining of Arabidopsis thaliana.

    PubMed

    Sheahan, J J; Rechnitz, G A

    1992-12-01

    Crop yields may be threatened by increases in UV-B radiation resulting from depletion of the ozone layer. In higher plants, the presence of flavonols provides a protective mechanism, and we report a novel staining procedure for the visualization of such protectants in plant tissue. It is shown that the proposed technique provides sensitive and specific fluorescence of flavonoids in chlorophyll-bleached tissue of Arabidopsis thaliana. PMID:1282347

  1. Laser Treatment of Port Wine Stains

    NASA Astrophysics Data System (ADS)

    Majaron, Boris; Nelson, J. Stuart

    Port wine stain (PWS), also called nevus flammeus, is a congenital, cutaneous vascular malformation involving post-capillary venules which produce a light pink to red to dark-red-violet discoloration of human skin [1]. PWS occurs in an estimated 3 children per 1000 live births, affecting males and females and all racial groups equally [2]. There appears to be no hereditary predilection for PWS within families. There are no known risk factors or ways to prevent PWS.

  2. Histamine immunohistochemistry is superior to the conventional heparin-based routine staining methodology for investigations of human skin mast cells.

    PubMed

    Johansson, O; Virtanen, M; Hilliges, M; Yang, Q

    1994-05-01

    Conventional studies of mast cells are limited by methodological restrictions such as a selective fixative-dependent routine staining blockage. This is thought to depend on the biochemical differences of the mast cell granule contents suggesting a cellular heterogeneity. Investigations of human mast cells, using routine methods, also suffer from the problem of a low signal-to-noise ratio. In the present study, normal human skin was used to compare an immunohistochemical method for histamine with two recommended mast-cell fixatives and a new commercial fixative in combination with three routine stains. Mast cells were found throughout the dermis with all the routine stains used. However, immunohistochemistry gave profoundly better results. Small structures, such as thin cytoplasmatic extensions and single granules, were readily detectable. Double-staining (immunohistochemistry followed by routine staining) revealed differences in staining capacity. All immunoreactive cells were not stained by routine stains and sometimes the opposite was also seen. This supports earlier reported evidence of heterogeneity, not only between skin and intestinal mast cells but also among skin mast cells themselves. Furthermore, by focusing on histamine, instead of heparin, we probably overcame the problems of the selective fixative-dependent routine staining blockage. Finally, the immunofluorescence technique provides a high signal-to-noise ratio and is an excellent method for making high-quality microphotographs of human mast cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8045782

  3. Examination of electron stains as a substitute for uranyl acetate for the ultrathin sections of bacterial cells.

    PubMed

    Yamaguchi, Kaoru; Suzuki, Ken-ichiro; Tanaka, Kenji

    2010-01-01

    Electron staining reagents were examined to find a possible substitute for uranyl acetate (UA) in electron microscopy of bacterial ultrathin sections. Four kinds of stains, platinum blue (Pt-blue), oolong tea extract (OTE), potassium permanganate (KMnO(4)) and phosphotungstic acid (PTA), were examined in comparison with UA either with or without post-staining with lead citrate (Pb). Electron microscopy was performed on sections from Spurr-embedded cells of a Gram-positive bacterium, Bacillus cereus NBRC 13597, and a Gram-negative bacterium, Escherichia coli NBRC 3301. Both Pt-blue and OTE showed staining similar to each other and to that of double staining with UA and Pb in B. cereus, while in E. coli the cytoplasmic membrane appeared less dense when compared with UA and Pb. KMnO(4) stained excessively to some extent, but showed images of the best contrast in the cytoplasmic membrane comparable with UA and Pb among the four reagents. PTA could stain the peptidoglycan layer but gave images of low quality for both bacteria. This study demonstrated that none of the reagents examined showed staining results of the same quality or better than the conventional method with UA and Pb. However, stains of Pt-blue, OTE and KMnO(4) could possibly be an alternative candidate for the UA according to the structure in question. PMID:19767626

  4. Methods and compositions for chromosome-specific staining

    DOEpatents

    Gray, Joe W.; Pinkel, Daniel

    2003-07-22

    Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogenous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include methods for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes.

  5. Rotavirus antigen test

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003349.htm Rotavirus antigen test To use the sharing features on this page, please enable JavaScript. The rotavirus antigen test detects rotavirus in the feces. This ...

  6. Newer applications of the histological stain prepared from Pterocarpus santalinus.

    PubMed

    Sen Gupta, P C; Mukherjee, A K

    1981-03-01

    A histological stain prepared from the heartwood of Pterocarpus santalinus Linn. has been found to be an excellent nuclear stain for various cells of animal and plant origin. As an elastic tissue stain, the results are comparable to standard elastic tissue stains. The striations of voluntary muscle fibers are well shown. The Nissl granules and fibers of cranial nerves in the pons are visualized. When counterstained with light green, it differentially stains muscle and fibrous tissue. The stain can be used as counterstain with certain histochemical procedures with satisfactory results. The preparation and use of this versatile stain are described. PMID:6166099

  7. Evaluation of lanthanide salts as alternative stains to uranyl acetate.

    PubMed

    Hosogi, Naoki; Nishioka, Hideo; Nakakoshi, Masamichi

    2015-12-01

    Uranyl acetate (UAc) has been generally used not only as a superb staining reagent for ultrathin sections of plastic-embedded biological materials, but also as high-contrast negative stains for biological macromolecules such as particles of protein or virus. However, the use and purchase of radioactive UAc have been restricted. In this study, we determine the performance of ytterbium triacetate, lutetium triacetate, samarium triacetate and gadolinium triacetate as new staining reagents for biological electron microscopy. We observed chemically fixed spinach (Spinacia oleracea) leaves stained with these reagents. Ultrathin sections were stained with these reagents. Some of them were counterstained with lead citrate. The transmission electron microscopy contrast of spinach organelles was evaluated in sections exposed to the conventional stain and new stains. We show acetate salts of samarium, gadolinium, ytterbium and lutetium could be excellent substitutes for UAc for thin section staining and for negative staining. In addition, each reagent showed appreciable negative-staining effects. PMID:26374081

  8. An activation antigen on a subpopulation of B lymphocytes identified by the monoclonal antibody CMRF-17.

    PubMed

    Peach, S F; Davidson, S E; McKenzie, J L; Nimmo, J C; Hart, D N

    1989-07-01

    The identification of membrane molecules expressed on subpopulations of B lymphocytes is of potential significance because these molecules may be candidates for regulating the activation, proliferation and differentiation of B cells. A new monoclonal antibody, CMRF-17, which reacts with a subpopulation of tonsil B lymphocytes has been produced. The antibody did not react with T lymphocytes in tonsil or peripheral blood nor most peripheral blood B lymphocytes but did label erythrocytes and some platelets. In tonsil, the germinal centre cells, cells in the interfollicular region and endothelial cells were positive, but mantle zone B cells were negative. Double labelling experiments showed that CMRF-17 reacted with activated tonsillar lymphocytes. The antigen recognized by CMRF-17 was heat stable, resistant to treatment with proteolytic enzymes and neuraminidase and was shown to be a carbohydrate determinant on one or more glycolipids. These characteristics of the antigen recognized by CMRF-17 and its pattern of reactivity distinguish this antibody from other monoclonal antibodies recognizing B-cell activation markers. It was notable that of the B-lymphoid malignancies tested to date, including those of probable follicular origin, few stained with CMRF-17. PMID:2474491

  9. False-positive Gram-stained smears.

    PubMed

    Hoke, C H; Batt, J M; Mirrett, S; Cox, R L; Reller, L B

    1979-02-01

    The rate per 1,000 smears showing nonviable Gram-negative bacilli (false-positive smears) increased from a baseline of 10.8 to 38.5 following purchase of new culture-collection devices; the rate decreased to 8.0 following replacement of contaminated culture sets. False-positive reports led to changes in therapy for five patients. In addition to being sterile, commercial culture-collection devices should be certified by the manufacturer as being free of stainable microorganisms or as unsuitable for preparation of Gram-stained smears. PMID:83398

  10. Bleaching of fluorosis stains using sodium hypochlorite

    PubMed Central

    Penumatsa, Narendra Varma; Sharanesha, Rajashekhara Bhari

    2015-01-01

    Fluorosis staining is commonly considered an esthetic problem because of the psychological impact of unesthetic maxillary anterior teeth. Numerous treatment approaches have been proposed, ranging from bleaching to enamel reduction to restorative techniques. Bleaching of hypomineralized enamel lesions, using 5% sodium hypochlorite, has been useful clinically. The technique described, in this case, appears to have advantages over other methods for improving the appearance of fluorotic lesions. It is simple, low cost, noninvasive, so the enamel keeps its structure, relatively rapid, and safe; it requires no special materials, and it can be used with safety on young permanent teeth. PMID:26538964

  11. Histological Stains: A Literature Review and Case Study

    PubMed Central

    Alturkistani, Hani A; Tashkandi, Faris M; Mohammedsaleh, Zuhair M

    2016-01-01

    The history of histology indicates that there have been significant changes in the techniques used for histological staining through chemical, molecular biology assays and immunological techniques, collectively referred to as histochemistry. Early histologists used the readily available chemicals to prepare tissues for microscopic studies; these laboratory chemicals were potassium dichromate, alcohol and the mercuric chloride to harden cellular tissues. Staining techniques used were carmine, silver nitrate, Giemsa, Trichrome Stains, Gram Stain and Hematoxylin among others. The purpose of this research was to assess past and current literature reviews, as well as case studies, with the aim of informing ways in which histological stains have been improved in the modern age. Results from the literature review has indicated that there has been an improvement in histopathology and histotechnology in stains used. There has been a rising need for efficient, accurate and less complex staining procedures. Many stain procedures are still in use today, and many others have been replaced with new immunostaining, molecular, non-culture and other advanced staining techniques. Some staining methods have been abandoned because the chemicals required have been medically proven to be toxic. The case studies indicated that in modern histology a combination of different stain techniques are used to enhance the effectiveness of the staining process. Currently, improved histological stains, have been modified and combined with other stains to improve their effectiveness. PMID:26493433

  12. Changes in distribution of nuclear matrix antigens during the mitotic cell cycle.

    PubMed

    Chaly, N; Bladon, T; Setterfield, G; Little, J E; Kaplan, J G; Brown, D L

    1984-08-01

    We examined the distribution of nonlamin nuclear matrix antigens during the mitotic cell cycle in mouse 3T3 fibroblasts. Four monoclonal antibodies produced against isolated nuclear matrices were used to characterize antigens by the immunoblotting of isolated nuclear matrix preparations, and were used to localize the antigens by indirect immunofluorescence. For comparison, lamins and histones were localized using human autoimmune antibodies. At interphase, the monoclonal antibodies recognized non-nucleolar and nonheterochromatin nuclear components. Antibody P1 stained the nuclear periphery homogeneously, with some small invaginations toward the interior of the nucleus. Antibody I1 detected an antigen distributed as fine granules throughout the nuclear interior. Monoclonals PI1 and PI2 stained both the nuclear periphery and interior, with some characteristic differences. During mitosis, P1 and I1 were chromosome-associated, whereas PI1 and PI2 dispersed in the cytoplasm. Antibody P1 heavily stained the periphery of the chromosome mass, and we suggest that the antigen may play a role in maintaining interphase and mitotic chromosome order. With antibody I1, bright granules were distributed along the chromosomes and there was also some diffuse internal staining. The antigen to I1 may be involved in chromatin/chromosome higher-order organization throughout the cell cycle. Antibodies PI1 and PI2 were redistributed independently during prophase, and dispersed into the cytoplasm during prometaphase. Antibody PI2 also detected antigen associated with the spindle poles. PMID:6378926

  13. Stain removal from a pigmented silicone maxillofacial elastomer.

    PubMed

    Yu, R; Koran, A; Craig, R G; Raptis, C N

    1982-08-01

    The removal of environmental stains from a pigmented maxillofacial elastomer was carried out by solvent extraction under network swelling. Silastic 44210 was pigmented with 11 maxillofacial pigments prior to staining. Samples were stained with lipstick, methylene blue, and disclosing solution. These stains were then removed by solvent extraction with 1,1,1-trichloroethane. Color parameter measurements both before and after staining and after solvent extraction demonstrated the effectiveness of removing these stains by solvent extraction while causing little or no change in the color of the pigmented samples. PMID:6955345

  14. Treatment of port-wine stains: analysis

    SciTech Connect

    van Gemert, M.J.; Welch, A.J.

    1987-08-01

    Port-wine stains (PWS) are bluish red skin stains that are caused by enlarged, ectatic blood vessels in the dermis. Laser treatment of PWS is analyzed from computation of the spatial distribution of heat production by direct absorption of the laser light and subsequent heat conduction. The absorption and scattering caused by oxyhemoglobin, epidermis, and dermis as a function of wavelength are utilized in this analysis. Ideal treatment is defined as coagulating the ectatic blood vessels without irreversible damage to the epidermis and dermis. The analysis shows that a millisecond pulsed, yellow dye laser at 577 nm (one of the large absorption bands in blood) is the laser of choice to treat PWS, offering as close to the ''ideal treatment'' as possible. The blue-green argon laser, which is currently the most frequently used laser for this purpose, is strongly recommended with irradiation times in milliseconds. Other lasers that are in clinical use, such as the red ruby and near-infrared Nd-YAG lasers, can provide selective treatment only when the epidermis is cooled concurrently. The CO/sub 2/ laser, on the other hand, can coagulate the blood vessels only through heat conduction from the hot epidermis; hence, it has neither the treatment selectivity nor any other physical option to force this selectivity.

  15. Co-staining for Keratins 8/18 plus Ubiquitin Improves Detection of Hepatocyte Injury in Nonalcoholic Fatty Liver Disease

    PubMed Central

    Guy, Cynthia D; Suzuki, Ayako; Burchette, James L; Brunt, Elizabeth M; Abdelmalek, Manal F; Cardona, Diana; McCall, Shannon J; Ünalp, Aynur; Belt, Patricia; Ferrell, Linda D; Diehl, Anna Mae

    2011-01-01

    Nonalcoholic fatty liver disease (NAFLD) is a global health dilemma. The gold standard for diagnosis is liver biopsy. Ballooned hepatocytes (BH) are histologic manifestations of hepatocellular injury and are characteristic features of steatohepatitis (SH), the more severe form of NAFLD. Definitive histologic identification of BH on routine stains, however, can be difficult. Immunohistochemical (IHC) evidence for loss of the normal hepatocytic keratins 8/18 (K8/18) can serve as an objective marker of BH. We sought to explore the utility of a K8/18 plus ubiquitin (Ub) double IHC stain for the histologic evaluation of adult NAFLD. Double IHC staining for K8/18 and Ub was analyzed using 40 adult human NAFLD core liver biopsies. Ballooned hepatocytes lack K8/18 staining (KBH) as previously shown by others, but normal size hepatocytes with keratin loss (KH) are approximately five times greater in number than KBH. KBH, KH, and Ub deposits show a zonal distribution, are positively associated with each other, and are frequently found adjacent to or intermixed with fibrous matrix. All three lesions correlate with fibrosis stage and the H&E diagnosis of SH (all p values < 0.05). Compared to H&E staining, IHC staining improves the receiver operating characteristics curve for advanced fibrosis (0.77 vs. 0.83, 0.89, and 0.89 for KBH, KH, and Ub, respectively) because IHC is more sensitive and specific for fibrogenic hepatocellular injury than H&E staining. K8/18+Ub double IHC stain improves detection of hepatocyte injury in NAFLD. Thus, it may help differentiate NASH from NAFL. PMID:22036053

  16. Ki-67 Membranous Staining: Biologically Relevant or an Artifact of Multiplexed Immunofluorescent Staining.

    PubMed

    Wang, Dan; Pang, Zhengyu; Clarke, Gina M; Nofech-Mozes, Sharon; Liu, Kela; Cheung, Alison M Y; Filkins, Robert J; Yaffe, Martin J

    2016-07-01

    In the process of developing a multiplex of 8 common breast cancer biomarkers (Her2/neu, estrogen receptor, progesterone receptor, Ki-67, aldehyde dehydrogenase-1, NaK-ATPase, cytokeratin 8/18, and myosin smooth muscle) on a single formalin-fixed paraffin-embedded slide using a sequential staining, imaging, and dye bleaching technology developed by General Electric Company, membranous Ki-67 staining was observed and colocalized with Her2/neu staining. Using immunohistochemistry as gold standards, we discovered that membranous Ki-67 was an artifact caused by the binding of cyanine 5-conjugated rabbit polyclonal Ki-67 antibody to a secondary cyanine 3-conjugated donkey anti-rabbit antibody which was previously applied and bound to rabbit Her2/neu antibody in our multiplexing experiment. After blocking with rabbit serum, a successful protocol for 8 biomarker multiplexing without cross-reactivity of antibodies from the same species was developed. PMID:26258752

  17. Production, fixation, and staining of cells on slides for maximum photometric sensitivity

    NASA Astrophysics Data System (ADS)

    Leif, Robert C.; Harlow, Patrick M.; Vallarino, Lidia M.

    1994-07-01

    The need to detect increasingly low levels of antigens or polynucleotides in cells requires improvements in both the preparation and the staining of samples. The combination of centrifugal cytology with the use of glyoxal as cross-linking fixative produces monolayers of cells having minimum background fluorescence. Detection can be further improved by the use of a recently developed type of luminescent tag containing a lanthanide(III) ion as the light- emitting center. These novel tags are macrocyclic complexes functionalized with an isothiocyanate group to allow covalent coupling to a biosubstrate. The Eu(III) complex possesses a set of properties -- water solubility, inertness to metal release over a wide pH range, ligand-sensitized narrow-band luminescence, large Stoke's shift, and long excited-state lifetime -- that provides ease of staining as well as maximum signal with minimum interference from background autofluorescence. Luminescence efficiency studies indicate significant solvent effects.

  18. Nuclear dot antigens may specify transcriptional domains in the nucleus.

    PubMed

    Xie, K; Lambie, E J; Snyder, M

    1993-10-01

    A bank of 892 human autoimmune serum samples was screened by indirect immunofluorescence on human tissue culture HT-29 cells. Seven serum samples that stain 4 to 10 bright dots in cell lines of several different mammals, including humans, monkeys, rats, and pigs, were identified. Immunofluorescence experiments indicate that these antigens, called nuclear dot (ND) antigens, are distinct from splicing complexes, kinetochores, and other known nuclear structures. An ND antigen recognized by these sera was cloned by immunoscreening a human lambda gt11 expression library. Analysis of seven cDNA clones for the ND antigen indicates that several mRNAs exist, perhaps derived through alternative splicing mechanisms. One major form of the message has an open reading frame of 1,440 bp capable of encoding a 53,000-M(r) protein. Treatment of cells with detergent, salt, or RNase A fails to remove the ND antigen from the nucleus. However, incubation with DNase I obliterates ND staining, indicating that the ND protein directly or indirectly associates with nuclear DNA. Fusion of the ND protein to a LexA DNA binding domain activates transcription in Saccharomyces cerevisiae. A 75-amino-acid domain that activates transcription in both yeast and primate cells has been identified. We suggest that ND antigens may participate in the activation of transcription of specific regions of the genome. PMID:8413218

  19. Staining Protocols for Human Pancreatic Islets

    PubMed Central

    Campbell-Thompson, Martha L.; Heiple, Tiffany; Montgomery, Emily; Zhang, Li; Schneider, Lynda

    2012-01-01

    Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region. The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&E and IHC stains. The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database

  20. In Vitro Targeted Photodynamic Therapy with a Pyropheophorbide-a Conjugated Inhibitor of Prostate Specific Membrane Antigen

    PubMed Central

    Liu, Tiancheng; Wu, Lisa Y.; Choi, Joseph K.; Berkman, Clifford E.

    2009-01-01

    BACKROUND The lack of specific delivery of photosensitizers (PSs), represents a significant limitation of photodynamic therapy (PDT) of cancer. The biomarker prostate-specific membrane antigen (PSMA) has attracted considerable attention as a target for imaging and therapeutic applications for prostate cancer. Although recent efforts have been made to conjugate inhibitors of PSMA with imaging agents, there have been no reports on photosensitizer-conjugated PSMA inhibitors for targeted PDT of prostate cancer. The present study focuses on the use of a PSMA inhibitor-conjugate of pyropheophorbide-a (Ppa-conjugate 2) for targeted PDT to achieve apoptosis in PSMA+ LNCaP cells. METHODS Confocal laser scanning microscopy with a combination of nuclear staining and immunofluorescence methods were employed to monitor the specific imaging and PDT-mediated apoptotic effects on PSMA-positive LNCaP and PSMA-negative (PC-3) cells. RESULTS Our results demonstrated that PDT-mediated effects by Ppa-conjugate 2 were specific to LNCaP cells, but not PC-3 cells. Cell permeability was detected as early as 2 h by HOE33342/PI double-staining, becoming more intense by 4 h. Evidence for the apoptotic caspase cascade being activated was based on the appearance of PARP p85 fragment. TUNEL assay detected DNA fragmentation 16 h post-PDT, confirming apoptotic events. CONCLUSIONS Cell permeability by HOE33342/PI double-staining as well as PARP p85 fragment and TUNEL assays confirm cellular apoptosis in PSMA+ cells when treated with PS-inhibitor conjugate 2 and subsequently irradiated. It is expected that the PSMA targeting small-molecule of this conjugate can serve as a delivery vehicle for PDT and other therapeutic applications for prostate cancer. PMID:19142895

  1. Port wine stain on a child's face (image)

    MedlinePlus

    Port wine stains are always present at birth. In an infant, they are flat, pink, vascular lesions. Common locations ... may be present anywhere on the body. Port wine stains may appear in association with other syndromes.

  2. Increased CD4+/CD8+ Double-Positive T Cells in Chronic Chagasic Patients

    PubMed Central

    Giraldo, Nicolas A.; Bolaños, Natalia I.; Cuellar, Adriana; Guzman, Fanny; Uribe, Ana Maria; Bedoya, Astrid; Olaya, Natalia; Cucunubá, Zulma M.; Roa, Nubia; Rosas, Fernando; Velasco, Víctor; Puerta, Concepción J.; González, John M.

    2011-01-01

    Background CD4+/CD8+ double positive (DP) T cells have been described in healthy individuals as well as in patients with autoimmune and chronic infectious diseases. In chronic viral infections, this cell subset has effector memory phenotype and displays antigen specificity. No previous studies of double positive T cells in parasite infections have been carried out. Methodology/Principal Findings Seventeen chronic chagasic patients (7 asymptomatic and 10 symptomatic) and 24 non-infected donors, including 12 healthy and 12 with non-chagasic cardiomyopathy donors were analyzed. Peripheral blood was stained for CD3, CD4, CD8, HLA-DR and CD38, and lymphocytes for intracellular perforin. Antigen specificity was assessed using HLA*A2 tetramers loaded with T. cruzi K1 or influenza virus epitopes. Surface expression of CD107 and intracellular IFN-γ production were determined in K1-specific DP T cells from 11 chagasic donors. Heart tissue from a chronic chagasic patient was stained for both CD8 and CD4 by immunochemistry. Chagasic patients showed higher frequencies of DP T cells (2.1%±0.9) compared with healthy (1.1%±0.5) and non-chagasic cardiomyopathy (1.2%±0.4) donors. DP T cells from Chagasic patients also expressed more HLA-DR, CD38 and perforin and had higher frequencies of T. cruzi K1-specific cells. IFN-γ production in K1-specific cells was higher in asymptomatic patients after polyclonal stimulation, while these cells tended to degranulate more in symptomatic donors. Immunochemistry revealed that double positive T cells infiltrate the cardiac tissue of a chagasic donor. Conclusions Chagasic patients have higher percentages of circulating double positive T cells expressing activation markers, potential effector molecules and greater class I antigenic specificity against T. cruzi. Although K1 tetramer positive DP T cell produced little IFN-γ, they displayed degranulation activity that was increased in symptomatic patients. Moreover, K1-specific DP T cells can

  3. Identification of a purified complement-fixing antigen as the Epstein-Barr-virus determined nuclear antigen (EBNA) by its binding to metaphase chromosomes.

    PubMed

    Ohno, S; Luka, J; Lindahl, T; Klein, G

    1977-04-01

    A soluble complement-fixing antigen carried by Epstein-Barr virus (EBV)-transformed human cells has been previously extracted from cell nuclei and purified by DNA-cellulose chromatography [Luka, J., Siegert, W. & Klein, G. (1977) J. Virol., in press]. On addition of this antigen to methanol/acetic acid-fixed metaphase chrmosomes, followed by exposure to human sera containing antibodies against the EBV-determined nuclear antigen (EBNA), brilliant positive staining was obtained by anti-complement immunofluorescence. There was no staining after exposure to EBV-negative sera. Moreover, a nuclear protein fraction, prepared from an EBV-negative cell line in an analogous fashion, failed to induce the staining reaction. These data identify the soluble purified antigen as the EBV-determined nuclear antigen. The purified antigen has a molecular weight of 174,000 +/- 15,000, as determined by sucrose gradient centrifugation and gel filtration experiments. In neutral buffers containing 0.5-1.0 M NaCl, the antigen dissociates into a form of approximately one-half the original molecular weight with retained complement-fixing activity. This "monomer" has a molecular weight of 98,000 +/- 8,000. PMID:67603

  4. Ultrafast tissue staining with chemical tags

    PubMed Central

    Kohl, Johannes; Ng, Julian; Cachero, Sebastian; Ciabatti, Ernesto; Dolan, Michael-John; Sutcliffe, Ben; Tozer, Adam; Ruehle, Sabine; Krueger, Daniel; Frechter, Shahar; Branco, Tiago; Tripodi, Marco; Jefferis, Gregory S. X. E.

    2014-01-01

    Genetically encoded fluorescent proteins and immunostaining are widely used to detect cellular and subcellular structures in fixed biological samples. However, for thick or whole-mount tissue, each approach suffers from limitations, including limited spectral flexibility and lower signal or slow speed, poor penetration, and high background labeling, respectively. We have overcome these limitations by using transgenically expressed chemical tags for rapid, even, high-signal and low-background labeling of thick biological tissues. We first construct a platform of widely applicable transgenic Drosophila reporter lines, demonstrating that chemical labeling can accelerate staining of whole-mount fly brains by a factor of 100. Using viral vectors to deliver chemical tags into the mouse brain, we then demonstrate that this labeling strategy works well in mice. Thus this tag-based approach drastically improves the speed and specificity of labeling genetically marked cells in intact and/or thick biological samples. PMID:25157152

  5. Investigation of a modified gallocyanin chrome alum staining technique in cytology compared to thionine and haematoxylin as nuclear stains.

    PubMed

    Schulte, E

    1988-01-01

    The present paper describes the staining characteristics of a modified Gallocyanin-chrome alum stain as compared to the original gallocyanin stain. Thionine, haematoxylin and the Feulgen reaction were used as controls. Tissue imprints of rabbit liver and spleen and smears of human venous blood were stained and controlled microscopically. Nuclear extinction was measured with the image analysis system IBAS 2000. Both GCA variants were examined by spectrophotometry and thin layer chromatography. The most striking difference between the GCA variants is the short staining time required for the modified stain (4 min) as compared to the original method (24 h). Both stains are stoichiometric for nucleic acids; the staining pattern, hue and intensity of nuclear colour and spectrophotometric and chromatographic data were absolutely consistent for both GCA-stains. These results and preliminary data from the analysis of the structure of the dye molecules seem to indicate that the molecular structure of the modified GCA is not changed by treatment with concentrated sulphuric acid. Differences in the staining kinetics might be due to differences in solubility. As nuclear chromatin texture after GCA staining is well appropriate for computerized image analysis the modified GCA-stain can be recommended as a simple and reproducible nuclear stain for automated feature extraction in cytology. PMID:2471227

  6. Cigarette staining and cleaning of a maxillofacial silicone

    SciTech Connect

    Yu, R.; Koran, A.; Raptis, C.N.; Craig, R.G.

    1983-07-01

    In this study, a maxillofacial silicone elastomer was stained with cigarette smoke. The stain was then removed by solvent extraction using 1,1,1-trichloroethane. The cigarette smoke produced large color changes in the elastomer as measured from spectrophotometric reflectance curves. The solvent was totally effective in removing the cigarette stain without changing the color of the silicone base.

  7. A simple cost-effective modification improves the quality of immunocytochemical staining in cervical scrape samples characterized by presence of excess mucus

    PubMed Central

    Pawar, Sagar; Mahantshetty, Umesh; Deodhar, Kedar; Teni, Tanuja

    2014-01-01

    Immunocytochemistry (ICC) is a very important tool in a diverse range of biomedical research as well as in diagnostic cytopathology. Smears prepared from cervical scrapes contain a large amount of overlying mucus that interferes with the standard immunocytochemical staining protocol. A modified ICC protocol is described, which involves pretreatment of these smears with 1 mg/ml solution of Ambroxol hydrochloride in methanol for 1 hour. Source of Ambroxol hydrochloride was a 30 mg Mucolite™ tablet, at a cost of 1.70 rupees (∼3·5 US cents) per tablet. This mucolytic solution effectively clears the mucus, facilitating the accessibility of the antibody to the antigenic determinants. This pretreatment resulted in the increased percentage of positively stained cells as well as staining intensity, leading to improved overall ICC staining and score. This is a novel modification that can be cost-effectively applied in ICC staining protocols for cytology samples characterized by the presence of excess mucus. PMID:25620823

  8. 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

  9. Red food coloring stain: new, safer procedures for staining nematodes in roots and egg masses on root surfaces.

    PubMed

    Thies, Judy A; Merrill, Sharon B; Corley, E Luther

    2002-06-01

    Acid fuchsin and phloxine B are commonly used to stain plant-parasitic nematodes in roots and egg masses on root surfaces, respectively. Both stains can be harmful to both the user and the environment and require costly waste disposal procedures. We developed safer methods to replace both stains using McCormick Schilling red food color. Eggs, juveniles, and adults of Meloidogyne incognita stained in roots with red food color were equally as visible as those stained with acid fuchsin. Egg masses stained with red food color appeared as bright-red spheres on the root surfaces and were highly visible even without magnification. Replacement of acid fuchsin and phloxine B with red food color for staining nematodes is safer for the user and the environment, and eliminates costly waste disposal of used stain solutions. PMID:19265929

  10. Efficacy of rapid, economical, acetic acid, Papanicolaou stain in cervical smears as an alternative to conventional Papanicolaou stain

    PubMed Central

    Izhar, Shabnam; Kaur, Rupinder; Masih, Kanwal

    2014-01-01

    Background: Papanicolaou (Pap) stain has been used over the years for cervical cytology screening. However; it utilizes a considerable amount of alcohol which is expensive and difficult to procure. In one of the modifications, ethyl alcohol is replaced by 1% acetic acid and is termed as rapid, economical, acetic acid Papanicolaou (REAP) stain. It is cost effective, easily available and provides a suitable and rapid staining alternative. Aim: This study was undertaken to assess the efficacy of REAP stain as an alternative method to conventional Pap stain. Materials and Methods: This study was done over a period of 18 months in a tertiary care hospital. Two sets of cervical smears were prepared of which one was stained with conventional Pap stain, and other was stained with REAP stain. The smears were examined for cytomorphological parameters and were evaluated using a modification of parameters given by Ng et al. Results: A total of 737 smears were examined in duplicate. Most of the conventional Pap smears showed excellent preservation (91.6%) with very few showing optimal (7.6%) and sub-optimal staining (0.8%). In contrast to this excellent preservation was seen in just 33.6% of the REAP stained smears with majority showing optimal and sub-optimal preservation (46.5% and 20% respectively). The P value was statistically significant (<0.0001) depicting inferior staining quality of REAP stain. Conclusion: Rapid, economical, acetic acid Papanicolaou stain undoubtly is a simple, fast and cost effective stain which can be adopted mainly in resource limited settings, but cannot be utilized for research purpose in a tertiary care setup due to poor preservation of the staining quality. PMID:25538385

  11. Centrifuge-operated specimen staining method and apparatus

    NASA Technical Reports Server (NTRS)

    Clarke, Mark S. F. (Inventor); Feeback, Daniel L. (Inventor)

    1999-01-01

    A method of staining preselected, mounted specimens of either biological or nonbiological material enclosed within a staining chamber where the liquid staining reagents are applied and removed from the staining chamber using hypergravity as the propelling force. In the preferred embodiment, a spacecraft-operated centrifuge and method of diagnosing biological specimens while in orbit, characterized by hermetically sealing a shell assembly. The assembly contains slide stain apparatus with computer control therefor, the operative effect of which is to overcome microgravity, for example on board an International Space Station.

  12. Automated single-slide staining device. [in clinical bacteriology

    NASA Technical Reports Server (NTRS)

    Wilkins, J. R.; Mills, S. M.

    1975-01-01

    An automatic single-slide Gram staining device is described. A timer-actuated solenoid controls the dispensing of gentian violet, Gram iodine solution, decolorizer, and 1% aqueous safranin in proper sequence and for the time required for optimum staining. The amount of stain or reagent delivered is controlled by means of stopcocks below each solenoid. Used stains and reagents can be flushed automatically or manually. Smears Gram stained automatically are equal in quality to those prepared manually. The time to complete one Gram cycle is 4.80 min.

  13. An antigenic study of human plasma cells in normal tissue and in myeloma: identification of a novel plasma cell associated antigen.

    PubMed Central

    Nathan, P D; Walker, L; Hardie, D; Richardson, P; Khan, M; Johnson, G D; Ling, N R

    1986-01-01

    A mouse monoclonal antibody named BU11 which detects an antigen strongly expressed on human plasma cells is described. The antibody stains plasma cells in tonsil sections, fresh and cultured plasmacytoid cells from the bone marrow of patients with multiple myeloma and cells of the plasmacytoid cell line RPMI 8226 used as the immunogen. In vitro studies of pokeweed mitogen (PWM) stimulated peripheral blood B cells and Epstein-Barr virus (EBV) stimulated tonsil B cells show that the antigen is present mainly on cells coexpressing the OKT10 antigen and containing cytoplasmic immunoglobulin (cIg). The BU11 antigen is expressed weakly on some normal B cells and is not present on T cells, monocytes or granulocytes. The antigen is of molecular weight 58kD under reducing conditions and is biochemically distinct from previously described plasma cell antigens. Images Fig. 4 PMID:3024883

  14. Studies on the Thomsen-Friedenreich antigen in human colon with the lectin Amaranthin. Normal and neoplastic epithelium express only cryptic T antigen.

    PubMed

    Sata, T; Roth, J; Zuber, C; Stamm, B; Rinderle, S J; Goldstein, I J; Heitz, P U

    1992-02-01

    The lectin Amaranthin has been shown to be highly specific for the galactose beta 1,3 N-acetylgalactosamine-alpha and sialic acid alpha 2,3 galactose beta 1,3 N-acetylgalactosamine-alpha sequence which represents the Thomsen-Friedenreich (T) antigen and its cryptic form, respectively. Previously, we demonstrated the usefulness of gold-labeled Amaranthin for the histochemical detection of the T antigen and its cryptic form. Application of the galactose oxidase (GO)-Schiff sequence abolished lectin binding to the T antigen but not its cryptic form, and therefore permitted their differentiation. In the present study we have analyzed by light and electron microscopy the distribution and subcellular localization of Amaranthin binding sites in normal, dysplastic and neoplastic colonic epithelium. Furthermore, a monoclonal antibody raised against synthetic galactose bera 1,3 N-acetylgalactosamine-alpha-bovine serum albumin was applied as a reagent for the T antigen. In normal colonic mucosa, two different Amaranthin staining patterns existed: (a) reactivity restricted to the lower portion of the crypts which was principally observed in the left colon, and (b) reactivity along the entire length of the crypts and in the surface epithelium with goblet cell staining in the upper portion of the crypts which was principally observed in the right colon. This Amaranthin staining was resistant to GO-Schiff treatment. No immunostaining with the monoclonal anti-T antigen was observed. Investigation of transitional mucosa, adenocarcinomas of different degrees of differentiation and mucinous carcinomas as well as adenomas with different degrees of dysplasia all revealed positive Amaranthin staining. The lectin staining was resistant to GO-Schiff treatment, and immunolabeling with the monoclonal antibody against the T antigen was absent. These results indicate that only the cryptic form of the T antigen is expressed in normal, dysplastic and neoplastic human colonic epithelium. PMID

  15. Case of Mycobacterium tuberculosis meningitis: Gram staining as a useful initial diagnostic clue for tuberculous meningitis.

    PubMed

    Kawakami, Sayoko; Kawamura, Yasuyosi; Nishiyama, Kyouhei; Hatanaka, Hiroki; Fujisaki, Ryuichi; Ono, Yasuo; Miyazawa, Yukihisa; Nishiya, Hajime

    2012-12-01

    A 32-year-old man was admitted to our hospital because of fever, headache, and loss of consciousness. Four days before admission, he had had difficulty speaking. On the day of admission, his colleague had found him to be unconscious and lying on his back. He was admitted to our hospital. The temperature at the eardrum was 35.2°C. Neurologic evaluation was negative. Computed tomography (CT) scan of the brain showed slight ventricular enlargement bilaterally. An X-ray film of the chest showed no abnormality. On the second hospital day, neck stiffness was noted. The cerebrospinal fluid (CSF) contained 870 white cells/μl, most of which were neutrophils; the glucose level in the CSF was 10 mg/dl, and the protein level was 140 mg/dl. Stained smears of the CSF, including Gram staining and India-ink preparations, disclosed no microorganisms. Capsular antigen tests for several bacteria were negative. Antimicrobial agents were started. However, by changing the microscope focus slightly while viewing Gram stains of the CSF, we could see brightened and Gram-positive bacilli that had been phagocytosed by neutrophils. This finding suggested the presence of Mycobacterium tuberculosis. Ziehl-Neelsen staining of the CSF and gastric juice revealed anti-acid bacilli. Polymerase chain reaction for M. tuberculosis in the gastric juice was positive. This case showed that Gram staining could be useful as an initial adjunct for the diagnosis of tuberculous meningitis, particularly when the CSF shows predominantly neutrocytic pleocytosis, but no other evidence of bacterial meningitis. PMID:22476652

  16. A six-week clinical efficacy study of four commercially available dentifrices for the removal of extrinsic tooth stain.

    PubMed

    Yankell, S L; Emling, R C; Petrone, M E; Rustogi, K; Volpe, A R; DeVizio, W; Chaknis, P; Proskin, H M

    1999-01-01

    The objective of this double-blind clinical study was to compare the efficacy for extrinsic tooth stain removal of four commercially available dentifrices: Colgate Tartar Control with Baking Soda & Peroxide Fluoride Toothpaste; Aquafresh Advanced Whitening Toothpaste with Fluoride; Rembrandt Tartar Control Low Abrasion Fluoride Whitening Toothpaste; and Crest Regular Fluoride Toothpaste. Following a baseline examination for extrinsic tooth stain on the anterior six mandibular and maxillary teeth, qualifying adult male and female subjects from the Philadelphia, Pennsylvania area were randomized into four treatment groups which were balanced for age, gender, tobacco habits, and level of extrinsic tooth stain. Subjects were instructed to brush their teeth twice daily (morning and evening) for one minute with their assigned dentifrice using a soft-bristled toothbrush. Examinations for extrinsic tooth stain were repeated after six weeks' use of the study dentifrices. One hundred eighty (180) subjects complied with the protocol, and completed the entire study. At the six-week examination, subjects assigned to the Colgate Tartar Control with Baking Soda & Peroxide Fluoride Toothpaste treatment group exhibited statistically lower levels of extrinsic tooth stain area and extrinsic tooth stain intensity than did those subjects assigned to the Crest Regular Fluoride treatment group. Subjects assigned to the Aquafresh Advanced Whitening treatment group exhibited significantly lower levels of extrinsic tooth stain area than did those assigned the Crest Regular Fluoride group. No other significant differences among the four study dentifrices were noted. Thus, the results of this double-blind clinical study support the conclusion that Colgate Tartar Control with Baking Soda & Peroxide Fluoride Toothpaste provides significantly greater control of extrinsic tooth stain than does Crest Regular Fluoride, a sodium fluoride/silica dentifrice. PMID:10825858

  17. Transcutaneous antigen delivery system

    PubMed Central

    Lee, Mi-Young; Shin, Meong-Cheol; Yang, Victor C.

    2013-01-01

    Transcutaneous immunization refers to the topical application of antigens onto the epidermis. Transcutaneous immunization targeting the Langerhans cells of the skin has received much attention due to its safe, needle-free, and noninvasive antigen delivery. The skin has important immunological functions with unique roles for antigen-presenting cells such as epidermal Langerhans cells and dermal dendritic cells. In recent years, novel vaccine delivery strategies have continually been developed; however, transcutaneous immunization has not yet been fully exploited due to the penetration barrier represented by the stratum corneum, which inhibits the transport of antigens and adjuvants. Herein we review recent achievements in transcutaneous immunization, focusing on the various strategies for the enhancement of antigen delivery and vaccination efficacy. [BMB Reports 2013; 46(1): 17-24] PMID:23351379

  18. Safranine fluorescent staining of wood cell walls.

    PubMed

    Bond, J; Donaldson, L; Hill, S; Hitchcock, K

    2008-06-01

    Safranine is an azo dye commonly used for plant microscopy, especially as a stain for lignified tissues such as xylem. Safranine fluorescently labels the wood cell wall, producing green/yellow fluorescence in the secondary cell wall and red/orange fluorescence in the middle lamella (ML) region. We examined the fluorescence behavior of safranine under blue light excitation using a variety of wood- and fiber-based samples of known composition to interpret the observed color differentiation of different cell wall types. We also examined the basis for the differences in fluorescence emission using spectral confocal microscopy to examine lignin-rich and cellulose-rich cell walls including reaction wood and decayed wood compared to normal wood. Our results indicate that lignin-rich cell walls, such as the ML of tracheids, the secondary wall of compression wood tracheids, and wood decayed by brown rot, tend to fluoresce red or orange, while cellulose-rich cell walls such as resin canals, wood decayed by white rot, cotton fibers and the G-layer of tension wood fibers, tend to fluoresce green/yellow. This variation in fluorescence emission seems to be due to factors including an emission shift toward red wavelengths combined with dye quenching at shorter wavelengths in regions with high lignin content. Safranine fluorescence provides a useful way to differentiate lignin-rich and cellulose-rich cell walls without counterstaining as required for bright field microscopy. PMID:18802812

  19. Immunogold silver staining for light microscopy.

    PubMed

    Lackie, P M

    1996-07-01

    The immunogold silver staining method (IGSS) is widely used as a sensitive and specific immunohistochemical visualisation technique. IGSS involves the specific deposition of metallic silver at the site of immunogold labelling and provides a means of visualisation at low magnification by light or electron microscopy. Silver developers for IGSS rapidly deposit metallic silver only at the site of heavy metals, including gold and silver, because of their catalytic activity. The developing solution contains the silver ions and reducing agent necessary for this reaction. Using different silver salts as ion donors and by selecting an appropriate temperature and pH, visible amounts of silver can be deposited in a few minutes at the site of colloidal gold labelling while little non-specific background deposition occurs. Inclusion of protective colloids in the solution can also be used to control the reaction. Although studies of the chemical basis of silver deposition around unlabelled colloidal gold date back to 1939, immunogold enhancement by silver was established in 1983. The IGSS method evolved from the combination of disparate photographic, histochemical and immunogold techniques which have been effectively combined and optimised over the last 10 years to provide a visualisation system which is well suited to many immunohistochemical studies. PMID:8858363

  20. Differential staining of interspecific chromosomes in somatic cell hybrids by alkaline Giemsa stain.

    PubMed

    Friend, K K; Chen, S; Ruddle, F H

    1976-03-01

    Staining of chromosome preparations of Chinese hamster-human hybrid cells and mouse-chimpanzee hybrids with alkaline Giemsa has yielded color differentiation of the interspecific chromosomes. Bicolor chromosomes, indicating apparent translocations also are observed for each of these hybrids. The specific color differences observed provide a rapid means of recognizing and aiding in the identification of the interspecific chromosomes and apparent translocations in these somatic cell hybrids. PMID:1028166

  1. Multi-class stain separation using independent component analysis

    NASA Astrophysics Data System (ADS)

    Trahearn, Nicholas; Snead, David; Cree, Ian; Rajpoot, Nasir

    2015-03-01

    Stain separation is the process whereby a full colour histology section image is transformed into a series of single channel images, each corresponding to a given stain's expression. Many algorithms in the field of digital pathology are concerned with the expression of a single stain, thus stain separation is a key preprocessing step in these situations. We present a new versatile method of stain separation. The method uses Independent Component Analysis (ICA) to determine a set of statistically independent vectors, corresponding to the individual stain expressions. In comparison to other popular approaches, such as PCA and NNMF, we found that ICA gives a superior projection of the data with respect to each stain. In addition, we introduce a correction step to improve the initial results provided by the ICA coefficients. Many existing approaches only consider separation of two stains, with primary emphasis on Haematoxylin and Eosin. We show that our method is capable of making a good separation when there are more than two stains present. We also demonstrate our method's ability to achieve good separation on a variety of different stain types.

  2. IgG Subclass Staining in Routine Renal Biopsy Material.

    PubMed

    Hemminger, Jessica; Nadasdy, Gyongyi; Satoskar, Anjali; Brodsky, Sergey V; Nadasdy, Tibor

    2016-05-01

    Immunofluorescence staining plays a vital role in nephropathology, but the panel of antibodies used has not changed for decades. Further classification of immunoglobulin (Ig)G-containing immune-type deposits with IgG subclass staining (IgG1, IgG2, IgG3, and IgG4) has been shown to be of diagnostic utility in glomerular diseases, but their value in the evaluation of renal biopsies has not been addressed systematically in large renal biopsy material. Between January 2007 and June 2014, using direct immunofluorescence, we stained every renal biopsy for the IgG subclasses if there was moderate to prominent glomerular IgG staining and/or IgG-predominant or IgG-codominant glomerular staining. The total number of biopsies stained was 1084, which included 367 cases of membranous glomerulonephritis, 307 cases of lupus nephritis, 74 cases of fibrillary glomerulonephritis, 53 cases of proliferative glomerulonephritis with monoclonal IgG deposits, and 25 cases of antiglomerular basement membrane disease, among others. We found that monoclonality of IgG deposits cannot always be reliably determined on the basis of kappa and lambda light chain staining alone, particularly if concomitant (frequently nonspecific) IgM staining is present. In IgG heavy and heavy and light chain deposition disease (3 cases), subclass staining is very helpful, and in proliferative glomerulonephritis with monoclonal IgG deposits subclass staining is necessary. IgG subclass staining is useful in differentiating primary from secondary membranous glomerulonephritis. In proliferative glomerulonephritis with polyclonal IgG deposition, IgG1 dominance/codominance with concomitant IgG3 and IgG2 but weak or absent IgG4 staining favors an underlying autoimmune disease. IgG subclass staining is a very useful diagnostic method in a selected cohort of renal biopsies, particularly in biopsies with glomerulonephritis with monoclonal IgG deposits. PMID:26848798

  3. B-cell antigens within normal and activated human T cells

    PubMed Central

    Sandilands, G P; Perry, M; Wootton, M; Hair, J; More, I A R

    1999-01-01

    In this study we compared cell surface staining for human peripheral blood lymphocyte (PBL) CD antigens by flow cytometry, with staining obtained following permeabilization of PBL using the Cytoperm method (Serotec). Six CD antigens (CD20, CD21, CD22, CD32, CD35 and major histocompatibility complex class II antigen) normally found on the surface of B cells, were also found to be expressed within T cells. We also showed, by immunoelectron microscopy, that these inappropriately expressed (‘occult’) CD antigens are located within cytoplasmic vesicles or within the rough endoplasmic reticulum. Following in vitro activation of T cells a distinct increase in expression of all of these cytoplasmic antigens was observed but staining at the cell surface was, by comparison, weak. We therefore propose that up-regulation of various B-cell CD antigens occurs within the cytoplasm of T cells following activation and that these antigens may be synthesized and released into the fluid-phase as soluble immunoregulatory molecules. PMID:10233724

  4. B-cell antigens within normal and activated human T cells.

    PubMed

    Sandilands, G P; Perry, M; Wootton, M; Hair, J; More, I A

    1999-03-01

    In this study we compared cell surface staining for human peripheral blood lymphocyte (PBL) CD antigens by flow cytometry, with staining obtained following permeabilization of PBL using the Cytoperm method (Serotec). Six CD antigens (CD20, CD21, CD22, CD32, CD35 and major histocompatibility complex class II antigen) normally found on the surface of B cells, were also found to be expressed within T cells. We also showed, by immunoelectron microscopy, that these inappropriately expressed ('occult') CD antigens are located within cytoplasmic vesicles or within the rough endoplasmic reticulum. Following in vitro activation of T cells a distinct increase in expression of all of these cytoplasmic antigens was observed but staining at the cell surface was, by comparison, weak. We therefore propose that up-regulation of various B-cell CD antigens occurs within the cytoplasm of T cells following activation and that these antigens may be synthesized and released into the fluid-phase as soluble immunoregulatory molecules. PMID:10233724

  5. Efficacy of baking soda-containing chewing gum in removing natural tooth stain.

    PubMed

    Mankodi, S M; Conforti, N; Berkowitz, H

    2001-07-01

    A 14-week, double-blind, randomized clinical trial was conducted with 126 healthy volunteers to compare the efficacy of twice-daily use of 3 baking soda-containing chewing gums in removing natural tooth stain when used in conjunction with a program of regular oral hygiene. All 3 chewing gums significantly reduced extrinsic stain (P < .0001) and improved the whitened appearance of teeth (P < .0001) at both the 2-week interim and the final 4-week evaluations. ARM & HAMMER DENTAL CARE The Baking Soda Gum (AHDC) reduced dental stain by 70.8%, compared to reductions of 71.9% and 65.3%, after use of 2 experimental gum formulations. Whitened appearance improved by 1.73 shade tabs using AHDC gum, and up to 2.49 shade tabs with the experimental formulations. These results suggest that the use of baking soda-containing gum after meals, in conjunction with good oral hygiene, can improve both extrinsic dental staining and the whitened appearance of teeth. PMID:11913307

  6. Harmonization of the intracellular cytokine staining assay.

    PubMed

    Welters, Marij J P; Gouttefangeas, Cécile; Ramwadhdoebe, Tamara H; Letsch, Anne; Ottensmeier, Christian H; Britten, Cedrik M; van der Burg, Sjoerd H

    2012-07-01

    Active immunotherapy for cancer is an accepted treatment modality aiming to reinforce the T-cell response to cancer. T-cell reactivity is measured by various assays and used to guide the clinical development of immunotherapeutics. However, data obtained across different institutions may vary substantially making comparative conclusions difficult. The Cancer Immunotherapy Immunoguiding Program organizes proficiency panels to identify key parameters influencing the outcome of commonly used T-cell assays followed by harmonization. Our successes with IFNγ-ELISPOT and peptide HLA multimer analysis have led to the current study on intracellular cytokine staining (ICS). We report the results of three successive panels evaluating this assay. At the beginning, 3 out of 9 participants (33 %) were able to detect >6 out of 8 known virus-specific T-cell responses in peripheral blood of healthy individuals. This increased to 50 % of the laboratories in the second phase. The reported percentages of cytokine-producing T cells by the different laboratories were highly variable with coefficients of variation well over 60 %. Variability could partially be explained by protocol-related differences in background cytokine production leading to sub-optimal signal-to-noise ratios. The large number of protocol variables prohibited identification of prime guidelines to harmonize the assays. In addition, the gating strategy used to identify reactive T cells had a major impact on assay outcome. Subsequent harmonization of the gating strategy considerably reduced the variability within the group of participants. In conclusion, we propose that first basic guidelines should be applied for gating in ICS experiments before harmonizing assay protocol variables. PMID:22714399

  7. Visible luminescence from silicon wafers subjected to stain etches

    NASA Technical Reports Server (NTRS)

    Fathauer, R. W.; George, T.; Ksendzov, A.; Vasquez, R. P.

    1992-01-01

    Etching of Si in a variety of solutions is known to cause staining. These stain layers consist of porous material similar to that produced by anodic etching of Si in HF solutions. In this work, photoluminescence peaked in the red from stain-etched Si wafers of different dopant types, concentrations, and orientations produced in solutions of HF:HNO3:H2O was observed. Luminescence is also observed in stain films produced in solutions of NaNO2 in HF, but not in stain films produced in solutions of CrO3 in HF. The luminescence spectra are similar to those reported recently for porous Si films produced by anodic etching in HF solutions. However, stain films are much easier to produce, requiring no special equipment.

  8. Manual hematoxylin and eosin staining of mouse tissue sections.

    PubMed

    Cardiff, Robert D; Miller, Claramae H; Munn, Robert J

    2014-06-01

    The hematoxylin and eosin (H&E) stain is the standard used for microscopic examination of tissues that have been fixed, processed, embedded, and sectioned. It can be performed manually or by automation. For economic reasons, the manual technique is generally the method of choice for facilities with a low sample volume. This protocol describes manual H&E staining of fixed, processed, paraffin-embedded, and sectioned mouse tissues. In H&E-stained tissues, the nucleic acids stain dark blue and the proteins stain red to pink or orange. For accurate phenotyping and delineation of tissue detail, the protocol must be adhered to rigorously. This includes frequent reagent changes as well as the use of "in-date" reagents. Appropriate color in a good H&E stain allows for identification of many tissue subtleties that are necessary for accurate diagnosis. PMID:24890205

  9. Isolation and antigenicity of a 45-kilodalton Paracoccidioides brasiliensis immunodominant antigen.

    PubMed Central

    Ferreira-da-Cruz, M F; Galvão-Castro, B; Daniel-Ribeiro, C

    1992-01-01

    In the present study, we analyzed human antibody responses to Paracoccidioides brasiliensis cellular antigens by the immunoblot technique to identify specific cellular components and to investigate the existence of antigen profile differences among serological responses of paracoccidioidomycosis (PCM) patients. Among the 64 PCM serum samples analyzed, a relatively homogeneous immunoglobulin G response to P. brasiliensis antigens was observed. The polypeptide with a mass of 45 kDa was the most clinically important, since antibody to this antigen was detectable in 90.6% of PCM patients studied and the six individuals who did not produce antibody were either at the end of treatment or in the posttherapy period and had shown clinical recovery. These facts suggested that the presence of this antibody may be an indicator of active disease. The 45-kDa antigen was also the most specific antigen of the PCM humoral immune response, since it reacted with only 2 of 79 (2.5%) heterologous serum samples tested: 1 histoplasmosis case and 1 tuberculosis case. This polypeptide was isolated from gels by electroelution and, when tested by an immunoradiometric assay and immunoblotting, maintained its reactivity with PCM sera and also with anti-P. brasiliensis polyclonal antibodies raised in rabbits at the same sensitivity levels as those obtained in immunoblotting with a crude antigen. Since in our assays the 45-kDa polypeptide was the major P. brasiliensis antigen and seemed to be specific for PCM, its use in alternative diagnostic methods is promising, especially in patients suspected of having the juvenile clinical form of PCM often associated with negative double-immunodiffusion results. Images PMID:1612736

  10. Pericyte Antigens in Perivascular Soft Tissue Tumors

    PubMed Central

    Shen, Jia; Shrestha, Swati; Yen, Yu-Hsin; Asatrian, Greg; Mravic, Marco; Soo, Chia; Ting, Kang; Dry, Sarah M.; Peault, Bruno; James, Aaron W.

    2015-01-01

    Introduction Perivascular soft tissue tumors are relatively uncommon neoplasms of unclear line of differentiation, although most are presumed to originate from pericytes or modified perivascular cells. Among these, glomus tumor, myopericytoma, and angioleiomyoma share a spectrum of histologic findings and a perivascular growth pattern. In contrast, solitary fibrous tumor (previously termed hemangiopericytoma) was once hypothesized to have pericytic differentiation. Methods Here, we systematically examine pericyte immunohistochemical markers among glomus tumor (including malignant glomus tumor), myopericytoma, angioleiomyoma, and solitary fibrous tumor. Immunohistochemical staining and semiquantification was performed using well-defined pericyte antigens, including αSMA, CD146, and PDGFRβ. Results Glomus tumor and myopericytoma demonstrate diffuse staining for all pericyte markers, including immunohistochemical reactivity for αSMA, CD146, and PDGFRβ. Malignant glomus tumors all showed some degree of pericyte marker immunoreactivity, although it was significantly reduced. Angioleiomyoma shared a similar αSMA + CD146 + PDGFRβ+ immunophenotype; however, this was predominantly seen in the areas of perivascular tumor growth. Solitary fibrous tumors showed patchy PDGFRβ immunoreactivity only. Discussion In summary, pericyte marker expression is a ubiquitous finding in glomus tumor, myopericytoma, and angioleiomyoma. Malignant glomus tumor shows a comparative reduction in pericyte marker expression, which may represent partial loss of pericytic differentiation. Pericyte markers are essentially not seen in solitary fibrous tumor. The combination of αSMA, CD146, and PDGFRβ immunohistochemical stainings may be of utility for the evaluation of pericytic differentiation in soft tissue tumors. PMID:26085647

  11. Evaluation of Diagnos Malaria Stix test (antigen detection assay) for diagnosis of malaria.

    PubMed

    Khan, Haris M; Shujatullah, Fatima; Shahid, M; Raza, Adil; Malik, Ritu

    2010-06-01

    Malaria is one of the most common parasitic infection in India. The diagnosis largely depends on peripheral blood smear examination. Newer diagnostic methods like various antigen detection assays are now in use for prompt diagnosis and treatment. This study was done to determine the effectiveness of Diagnos Malaria Stix (antigen detection) assay in diagnosis of malaria. This involves detection of PfHRP-2 antigen and P.V. specific pLDH antigen. 162 patients with signs and symptoms of malaria included in the study. Leishman stained blood smear examination was done for all patients. Commercially available Diagnos Malaria Stix assay was used. Diagnos Malaria Stix showed sensitivity, specificity positive and negative predictive values of 100% each while Sensitivity, specificity, positive and negative predictive values of Leishman stained blood smear examination were 45.45%, 100%, 100% and 92% respectively. PMID:22471175

  12. Deletion of antigens of the Lewis a/b blood group family in human prostatic carcinoma.

    PubMed Central

    Young, W. W.; Mills, S. E.; Lippert, M. C.; Ahmed, P.; Lau, S. K.

    1988-01-01

    The expression of antigens of the blood group Lewis a/b family were studied in a series of 42 prostatectomy specimens from patients with adenocarcinoma clinically confined to the prostate; 19 of these were later reclassified as pathologic Stage C. Staining of normal or hyperplastic versus neoplastic epithelium was assessed in routinely processed, paraffin-embedded tissue using murine monoclonal antibodies and an avidin-biotin immunoperoxidase technique. Antigens screened and the antibodies used to recognize them were Lewis a (CF4C4), Lewis b and Type 1 H (NS10), monosialosyl Lewis a I (19.9), and disialosyl Lewis a and monosialosyl Lewis a II (FH7). FH7 strongly stained the benign epithelium of all 39 Lewis positive cases, suggesting that the sialyltransferase responsible for synthesis of FH7-reactive determinants is highly active in benign prostatic tissue. When compared to the reactivity of benign epithelium in Lewis positive cases, the staining of the carcinomas was markedly reduced in 18 cases (46%) and absent in 16 cases (41%). This reduction or loss of staining of the malignant epithelium was observed for all antibodies that stained the corresponding benign epithelium of each case. In only five of the cases (13%) was the intensity of staining in the carcinoma equal to that of the surrounding benign epithelium. No cases in this latter group had recurrence of disease, whereas in the other staining groups 25-33% of the cases had recurrences; median follow-up for the entire group was 78 months. No correlation was apparent between Gleason score and the staining pattern with these antigens. In summary, antigens of the Lewis a/b family are deleted in a high percentage of cases of prostatic adenocarcinoma. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:2454582

  13. Hepatitis B surface antigen positive skin lesions. Two case reports with an immunoperoxidase study.

    PubMed

    Rosen, L B; Rywlin, A M; Resnick, L

    1985-12-01

    This study represents the first two case reports of skin lesions positive for hepatitis B surface antigen (HBsAg) with the immunoperoxidase technique. A 25-year-old man and a 64-year-old woman with serologic evidence of acute B viral hepatitis and concurrent skin lesions are presented. Immunoperoxidase study of the skin lesions for HBsAg revealed strong positive staining of squamous epidermal cells, eccrine sweat glands, and endothelial cells in the superficial papillary dermis. Immunoperoxidase staining for hepatitis B core antigen (HBcAg) was negative in both cases. Electron microscopy failed to reveal viral particles. PMID:3911798

  14. Disaccharides Protect Antigens from Drying-Induced Damage in Routinely Processed Tissue Sections.

    PubMed

    Boi, Giovanna; Scalia, Carla Rossana; Gendusa, Rossella; Ronchi, Susanna; Cattoretti, Giorgio

    2016-01-01

    Drying of the tissue section, partial or total, during immunostaining negatively affects both the staining of tissue antigens and the ability to remove previously deposited antibody layers, particularly during sequential rounds of de-staining and re-staining for multiple antigens. The cause is a progressive loss of the protein-associated water up to the removal of the non-freezable water, a step which abolishes the immunoavailability of the epitope. In order to describe and prevent these adverse effects, we tested, among other substances, sugars, which are known to protect unicellular organisms from freezing and dehydration, and stabilize drugs and reagents in solid state form in medical devices. Disaccharides (lactose, sucrose) prevented the air drying-induced antigen masking and protected tissue-bound antigens and antibodies from air drying-induced damage. Complete removal of the bound antibody layers by chemical stripping was permitted if lactose was present during air drying. Lactose, sucrose and other disaccharides prevent air drying artifacts, allow homogeneous, consistent staining and the reuse of formalin-fixed, paraffin-embedded tissue sections for repeated immunostaining rounds by guaranteeing constant staining quality in suboptimal hydration conditions. PMID:26487185

  15. Comparative study of subculture, Gram staining and acridine orange staining for early detection of positive blood cultures.

    PubMed Central

    Mascart, G; Bertrand, F; Mascart, P

    1983-01-01

    In view of the importance of a rapid aetiological diagnosis in septicaemia, we compared the results of subculture, Gram staining and acridine orange staining in the detection of positive blood cultures. The study was based on 1013 blood cultures of which 138 were positive by culture. The three techniques were applied 12 h after the specimen was taken in 210 instances, at 24 h in 540 instances and after 48 h in 525. We were able to demonstrate the value of direct examination. Staining with acridine orange yields more positive results than Gram staining and is also simpler. PMID:6188764

  16. Comparison of tetrachromic VOF stain to other histochemical staining techniques for characterizing stromal soft and hard tissue components.

    PubMed

    Belaldavar, C; Hallikerimath, S; Angadi, P V; Kale, A D

    2014-11-01

    The components of hard tissues including dentin, enamel, cementum, bone and other calcified deposits, and mature and immature collagen pose problems for identification in routine hematoxylin and eosin (H & E) stained sections. Use of combinations of stains can demonstrate the components of hard tissues and soft tissues distinctly. We assessed the efficacy of the Verde Luz-orange G-acid fuchsin (VOF) stain for differentiating hard and soft connective tissues and compared results with other histochemical staining techniques. Eighty tissue sections comprising developing tooth (30), ossifying fibroma (30) and miscellaneous pathologies (20) expected to contain varying types of calcified tissues were stained with H & E, VOF, and Masson's trichrome (MT). In developing tooth, VOF demonstrated better differentiation of hard tissues, while it was comparable to MT for ossifying fibroma and miscellaneous pathologies. The intensity of staining was greater with VOF than with the other stains studied. VOF stains hard tissue components distinctly and gives good contrast with the surrounding connective tissue. VOF is comparable to MT, but has added advantages including single step staining, rapid and easy procedures, and it distinguishes the maturity of the tissues. PMID:24830362

  17. 7 CFR 3201.87 - Wood and concrete stains.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 15 2014-01-01 2014-01-01 false Wood and concrete stains. 3201.87 Section 3201.87... Designated Items § 3201.87 Wood and concrete stains. (a) Definition. Products that are designed to be applied as a finish for concrete and wood surfaces and that contain dyes or pigments to change the...

  18. 7 CFR 3201.87 - Wood and concrete stains.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 15 2013-01-01 2013-01-01 false Wood and concrete stains. 3201.87 Section 3201.87... Designated Items § 3201.87 Wood and concrete stains. (a) Definition. Products that are designed to be applied as a finish for concrete and wood surfaces and that contain dyes or pigments to change the...

  19. The effect of selected staining techniques on bull sperm morphometry.

    PubMed

    Banaszewska, Dorota; Andraszek, Katarzyna; Czubaszek, Magdalena; Biesiada-Drzazga, Barbara

    2015-08-01

    Sperm morphometry has some value as an indicator of reproductive capacity in males. In laboratory practice a variety of slide-staining methods are used during morphological evaluation of semen to predict male fertility. The aim of this study was to determine the effect of staining of semen using four different techniques on the morphometry of the bull sperm cell. The material for the study consisted of semen collected from test bulls of the Black-and-White variety of Holstein-Friesians. The results obtained in the study indicate differences in the dimensions of bull sperm heads when different slide staining techniques were used. The most similar results for sperm head dimensions were obtained in the case of SpermBlue(®) and eosin+gentian violet complex, although statistically significant differences were found between all the staining techniques. Extreme values were noted for the other staining techniques - lowest for the Papanicolaou and highest for silver nitrate, which may indicate more interference in the cell by the reagents used in the staining process. However, silver nitrate staining was best at identifying the structures of the sperm cell. Hence it is difficult to determine which of the staining methods most faithfully reveals the dimensions and shape of the bull sperm. PMID:26149220

  20. 21 CFR 864.1850 - Dye and chemical solution stains.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Dye and chemical solution stains. 864.1850 Section 864.1850 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and...

  1. 21 CFR 864.1850 - Dye and chemical solution stains.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Dye and chemical solution stains. 864.1850 Section 864.1850 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and...

  2. 21 CFR 864.1850 - Dye and chemical solution stains.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Dye and chemical solution stains. 864.1850 Section 864.1850 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and...

  3. 21 CFR 864.1850 - Dye and chemical solution stains.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Dye and chemical solution stains. 864.1850 Section 864.1850 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and...

  4. 21 CFR 864.1850 - Dye and chemical solution stains.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Dye and chemical solution stains. 864.1850 Section 864.1850 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1850 Dye and...

  5. Identification and characterization of specific hydatid antigen fraction(s).

    PubMed

    Maher, K M; Kaddah, M A; Hassanein, H I; Shaker, Z A; Khalafallah, A M

    1992-08-01

    A specific hydatid antigen was prepared in this study from Echinococcus granulosus cyst in livers and lungs of camels. Elimination of host "camel" protein from crude hydatid fluid was achieved by two methods: Salting out using ammonium sulfate precipitation method and immunoaffinity purification using coupled anticamel antibody to cyanogenbromide activated sepharose 4B gel. Testing the prepared hydatid antigen against anticamel serum, using immunodiffusion method, indicated that the affinity purified hydatid antigen was almost completely purified from camel protein. Characterization of the affinity purified hydatid antigen, using immunoelectrophoresis, showed positive arc 5 precipitation when tested against known positive antihydatid sera. Further characterization with gradient gel electrophoresis, showed with silver stain that the dominant and most consistently demonstrable proteins occurred as a complex in the 52/62 KDa region. Strong reaction with the 52/62 KDa complex was consistently observed when the affinity purified hydatid antigen was probed with known positive reference antihydatid sera. The identified hydatid antigen fraction(s) with 52/62 KDa complex can provide promising non-invasive parameter for diagnosis of Hydatidosis. PMID:1500792

  6. In vivo photoacoustic imaging of model of port wine stains.

    PubMed

    Yuan, Kaihua; Yuan, Yi; Gu, Ying; Gao, Jianhua; Xing, Da

    2012-01-01

    Port wine stains are categorized as a benign capillary vascular malformation, which is hard to cure. In this paper, a photoacoustic microscopy system, which integrated a two-dimensional scanning galvanometer, an objective lens and a focused ultrasound transducer, was designed for noninvasive imaging of blood vessels of port wine stains model in vivo. Cock comb was chosen as the port wine stains model in the experiment. The blood vessels in x-y plane and x-z plane were imaged clearly. Experimental results demonstrate that photoacoustic microscopy can image the blood vessels of port wine stains model in vivo with high contrast and high resolution. It has the potential for clinical applications in detecting the blood vessels in port wine stains skin. PMID:22635179

  7. Effects of fixation, dehydration and staining on dimensions of myxosporidan.

    PubMed

    Parker, J D; Warner, M C

    1970-10-01

    The effects of fixation, dehydration and staining on the morphological dimensions of myxo- and microsporidan spores were tested. Seven fixatives, two dehydrants and five stains were tested. Ten % formalin produced the least shrinkage and provided the best cytological detail of fixed material in both types of spores. All fixatives caused shrinkage of myxosporidan spore length and polar capsule length. Spore capsule width and polar capsule width were unaffected by 10% formalin. Ethyl alcohol caused no significant change in spore width. Microsporidan spore length shrunk with all fixatives, but spore width was generally unaffected. Dehydration, with either isopropyl alcohol or acetone, produced additional, significant shrinkage. The influence of stains on spore size was negligible. Heidenhains iron hematoxylin followed by eosin, and Mallory's analine-blue collagen stain, effectively stained myxo- and microsporidan spores. PMID:16512155

  8. Staining and histomorphometry of microcracks in the human femoral head.

    PubMed

    Villanueva, A R; Longo, J A; Weiner, G

    1994-03-01

    We developed staining techniques that permit identification and histomorphometric analysis of microcracks in the human femoral head 1) from thick, ground bone sections (100 microns) by prestaining with the Villanueva mineralized bone stain (MIBS), and 2) from plastic embedded, undecalcified thin bone sections (5-15 microns) by staining in gallocyanin chrome alum-Villanueva blood stain methods. Both methods represent a significant improvement in the stainability of the microcracks, cellular and tissue elements, and the simultaneous assessment of osteoid seams and tetracycline markers by histomorphometry. Shrinkage and other artifacts were minimized, which helped to clarify some of the uncertainties arising from artifacts resulting from some bone staining methods. Histomorphometric analyses of microcracks were conducted on thick, ground sections of subchondral and trabecular bone. Microcracks were more prevalent in the subchondral bone and osteochondral junction than in the more distant trabeculae. We have consistently localized microcrack areas in bone tissues prepared in these ways. PMID:7515700

  9. Presentation of hepatocellular antigens

    PubMed Central

    Grakoui, Arash; Crispe, Ian Nicholas

    2016-01-01

    The liver is an organ in which antigen-specific T-cell responses manifest a bias toward immune tolerance. This is clearly seen in the rejection of allogeneic liver transplants, and multiple other phenomena suggest that this effect is more general. These include tolerance toward antigens introduced via the portal vein, immune failure to several hepatotropic viruses, the lack of natural liver-stage immunity to malaria parasites, and the frequent metastasis of cancers to the liver. Here we review the mechanisms by which T cells engage with hepatocellular antigens, the context in which such encounters occur, and the mechanisms that act to suppress a full T-cell response. While many mechanisms play a role, we will argue that two important processes are the constraints on the cross-presentation of hepatocellular antigens, and the induction of negative feedback inhibition driven by interferons. The constant exposure of the liver to microbial products from the intestine may drive innate immunity, rendering the local environment unfavorable for specific T-cell responses through this mechanism. Nevertheless, tolerance toward hepatocellular antigens is not monolithic and under specific circumstances allows both effective immunity and immunopathology. PMID:26924525

  10. Automated detection of cells from immunohistochemically-stained tissues: application to Ki-67 nuclei staining

    NASA Astrophysics Data System (ADS)

    Cinar Akakin, Hatice; Kong, Hui; Elkins, Camille; Hemminger, Jessica; Miller, Barrie; Ming, Jin; Plocharczyk, Elizabeth; Roth, Rachel; Weinberg, Mitchell; Ziegler, Rebecca; Lozanski, Gerard; Gurcan, Metin N.

    2012-03-01

    An automated cell nuclei detection algorithm is described to be used for the quantification of immunohistochemicallystained tissues. Detection and segmentation of positively stained cells and their separation from the background and negatively-stained cells is crucial for fast, accurate, consistent and objective analysis of pathology images. One of the major challenges is the identification, hence accurate counting of individual cells, when these cells form clusters. To identify individual cell nuclei within clusters, we propose a new cell nuclei detection method based on the well-known watershed segmentation, which can lead to under- or over-segmentation for this problem. Our algorithm handles oversegmentation by combining H-minima transformed watershed algorithm with a novel region merging technique. To handle under-segmentation problem, we develop a Laplacian-of-Gaussian (LoG) filtering based blob detection algorithm, which estimates the range of the scales from the image adaptively. An SVM classifier was trained in order to separate non-touching single cells and touching cell clusters with five features representing connected region properties such as eccentricity, area, perimeter, convex area and perimeter-to-area ratio. Classified touching cell clusters are segmented with the H-minima based watershed algorithm. The resulting over-segmented regions are improved with the merging algorithm. The remaining under-segmented cell clusters are convolved with LoG filters to detect the cells within them. Cell-by-cell nucleus detection performance is evaluated by comparing computer detections with cell locations manually marked by eight pathology residents. The sensitivity is 89% when the cells are marked as positive at least by one resident and it increases to 99% when the evaluated cells are marked by all eight residents. In comparison, the average reader sensitivity varies between 70% +/- 18% and 95% +/- 11%.

  11. Novel Process for Laser Stain Removal from Archaeological Oil Paintings

    NASA Astrophysics Data System (ADS)

    El-Nadi, Lotfia; El-Feky, Osama; Abdellatif, Galila; Darwish, Sawsan

    2013-03-01

    Some samples of oil paintings (5 × 5 cm) were prepared on wooden panel with four types of fungi commonly encountered on oil paintings were selected for this study. Each of the fungi is associated with different colored stains. Fungus Alternaria tenuis is associated by a dense black stain, Chetomium globosum by a brownish gray stain, Aspergillus flavus by a yellowish stain, and Fusaruim oxysporum by a pinkish stain. Fungi growing on oil paintings affect the surface characteristics by forming a variety of colored patches typically composed of many complex chemical substances that are produced during metabolic processes. These colored stains may be encrusted in spores, present in mycelium or secreted to a substance such as oil paintings surfaces. While the fungal stains can sometimes be extracted with appropriate solvents, there are some stains that resist solvent extraction entirely. Developing new solvent system that might attack the paint structure, and is time consuming and requires a great deal of trial and error. Mechanical stain removal is also problematic in that it often produces abrasion of the surface, markedly deteriorating the artwork, and is extra ordinarily fine and tedious. For these reasons, we decided to examine an alternative physical technique as a new approach to deal with stain removal. Since the stains are due to the existence of fungi, we thought it a good idea to remove them by singlet oxygen. We applied the photo dynamic process through which the fungi stains were covered with organic dye derivatives in solution under controlled illumination in the lab. The samples were then irradiated by low power Laser light from a He-Ne laser, the dye will be photodecomposed and produce singlet oxygen. We report in this work the results obtained as a function of: - The concentration and types of the organic dye in solution, - The presence of certain amounts of liquids added to the solution, - The scanning speed of the laser beam on the sample surface

  12. Confusion over live/dead stainings for the detection of vital microorganisms in oral biofilms - which stain is suitable?

    PubMed Central

    2014-01-01

    Background There is confusion over the definition of the term “viability state(s)” of microorganisms. “Viability staining” or “vital staining techniques” are used to distinguish live from dead bacteria. These stainings, first established on planctonic bacteria, may have serious shortcomings when applied to multispecies biofilms. Results of staining techniques should be compared with appropriate microbiological data. Discussion Many terms describe “vitality states” of microorganisms, however, several of them are misleading. Authors define “viable” as “capable to grow”. Accordingly, staining methods are substitutes, since no staining can prove viability. The reliability of a commercial “viability” staining assay (Molecular Probes) is discussed based on the corresponding product information sheet: (I) Staining principle; (II) Concentrations of bacteria; (III) Calculation of live/dead proportions in vitro. Results of the “viability” kit are dependent on the stains’ concentration and on their relation to the number of bacteria in the test. Generally this staining system is not suitable for multispecies biofilms, thus incorrect statements have been published by users of this technique. To compare the results of the staining with bacterial parameters appropriate techniques should be selected. The assessment of Colony Forming Units is insufficient, rather the calculation of Plating Efficiency is necessary. Vital fluorescence staining with Fluorescein Diacetate and Ethidium Bromide seems to be the best proven and suitable method in biofilm research. Regarding the mutagenicity of staining components users should be aware that not only Ethidium Bromide might be harmful, but also a variety of other substances of which the toxicity and mutagenicity is not reported. Summary – The nomenclature regarding “viability” and “vitality” should be used carefully. – The manual of the commercial “viability” kit itself points out that

  13. Pathways of Antigen Processing

    PubMed Central

    Blum, Janice S.; Wearsch, Pamela A.; Cresswell, Peter

    2014-01-01

    T cell recognition of antigen presenting cells depends on their expression of a spectrum of peptides bound to Major Histocompatibility Complex class I (MHC-I) and class II (MHC-II) molecules. Conversion of antigens from pathogens or transformed cells into MHC-I and MHC-II-bound peptides is critical for mounting protective T cell responses, and similar processing of self proteins is necessary to establish and maintain tolerance. Cells use a variety of mechanisms to acquire protein antigens, from translation in the cytosol to variations on the theme of endocytosis, and to degrade them once acquired. In this review we highlight the aspects of MHC-I and MHC-II biosynthesis and assembly that have evolved to intersect these pathways and sample the peptides that are produced. PMID:23298205

  14. Western Blot of Stained Proteins from Dried Polyacrylamide Gels

    NASA Technical Reports Server (NTRS)

    Gruber, Claudia; Stan-Lotter, Helga

    1996-01-01

    Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.

  15. Lipid antigens in immunity

    PubMed Central

    Dowds, C. Marie; Kornell, Sabin-Christin

    2014-01-01

    Lipids are not only a central part of human metabolism but also play diverse and critical roles in the immune system. As such, they can act as ligands of lipid-activated nuclear receptors, control inflammatory signaling through bioactive lipids such as prostaglandins, leukotrienes, lipoxins, resolvins, and protectins, and modulate immunity as intracellular phospholipid- or sphingolipid-derived signaling mediators. In addition, lipids can serve as antigens and regulate immunity through the activation of lipid-reactive T cells, which is the topic of this review. We will provide an overview of the mechanisms of lipid antigen presentation, the biology of lipid-reactive T cells, and their contribution to immunity. PMID:23999493

  16. Quantitative assessment of Tn antigen in breast tissue micro-arrays using CdSe aqueous quantum dots.

    PubMed

    Au, Giang H T; Mejias, Linette; Swami, Vanlila K; Brooks, Ari D; Shih, Wan Y; Shih, Wei-Heng

    2014-03-01

    In this study, we examined the use of CdSe aqueous quantum dots (AQDs) each conjugated to three streptavidin as a fluorescent label to image Tn antigen expression in various breast tissues via a sandwich staining procedure where the primary monoclonal anti-Tn antibody was bound to the Tn antigen on the tissue, a biotin-labeled secondary antibody was bound to the primary anti-Tn antibody, and finally the streptavidin-conjugated AQDs were bound to the biotin on the secondary antibody. We evaluated the AQD staining of Tn antigen on tissue microarrays consisting of 395 cores from 115 cases including three tumor cores and one normal-tissue core from each breast cancer case and three tumor cores from each benign case. The results indicated AQD-Tn staining was positive in more than 90% of the cells in the cancer cores but not the cells in the normal-tissue cores and the benign tumor cores. As a result, AQD-Tn staining exhibited 95% sensitivity and 90% specificity in differentiating breast cancer against normal breast tissues and benign breast conditions. These results were better than the 90% sensitivity and 80% specificity exhibited by the corresponding horse radish peroxidase (HRP) staining using the same antibodies on the same tissues and those of previous studies that used different fluorescent labels to image Tn antigen. In addition to sensitivity and specificity, the current AQD-Tn staining with a definitive threshold was quantitative. PMID:24411673

  17. Unique Appearance of Proliferating Antigen-Presenting Cells Expressing DC-SIGN (CD209) in the Decidua of Early Human Pregnancy

    PubMed Central

    Kämmerer, Ulrike; Eggert, Andreas O.; Kapp, Michaela; McLellan, Alexander D.; Geijtenbeek, Teunis B. H.; Dietl, Johannes; van Kooyk, Yvette; Kämpgen, Eckhart

    2003-01-01

    Intact human pregnancy can be regarded as an immunological paradox in that the maternal immune system accepts the allogeneic embryo without general immunosuppression. Because dendritic cell (DC) subsets could be involved in peripheral tolerance, the uterine mucosa (decidua) was investigated for DC populations. Here we describe the detailed immunohistochemical and functional characterization of HLA-DR-positive antigen-presenting cells (APCs) in early pregnancy decidua. In contrast to classical macrophages and CD83+ DCs, which were found in comparable numbers in decidua and nonpregnant endometrium, only decidua harbored a significant population of HLA-DR+/DC-SIGN+ APCs further phenotyped as CD14+/CD4+/CD68+/−/CD83−/CD25−. These cells exhibited a remarkable proliferation rate (9.2 to 9.8% of all CD209+ cells) by double staining with Ki67 and proliferating cell nuclear antigen. Unique within the DC-family, the majority of DC-SIGN+ decidual APCs were observed in situ to have intimate contact with CD56+/CD16−/ICAM-3+ decidual natural killer cells, another pregnancy-restricted cell population. In vitro, freshly isolated CD14+/DC-SIGN+ decidual cells efficiently took up antigen, but could not stimulate naive allogeneic T cells at all. Treatment with an inflammatory cytokine cocktail resulted in down-regulation of antigen uptake capacity and evolving capacity to effectively stimulate resting T cells. Fluorescence-activated cell sorting analysis confirmed the maturation of CD14+/DC-SIGN+ decidual cells into CD25+/CD83+ mature DCs. In summary, this is the first identification of a uterine immature DC population expressing DC-SIGN, that appears only in pregnancy-associated tissue, has a high proliferation rate, and a conspicuous association with a natural killer subset. PMID:12598322

  18. Efficacy of Extrinsic Stain Removal by Novel Dentifrice Containing Papain and Bromelain Extracts

    PubMed Central

    Chakravarthy, PK; Acharya, S

    2012-01-01

    To evaluate the stain removal efficacy of a novel dentifrice containing papain and bromelain extracts (Glodent) in comparison with a control dentifrice (Colgate regular). This was a randomized, positive-controlled, double-blinded, clinical study. Subjects were randomly divided into one of the two study groups. Pre-treatment and post treatment photographs of the 4 anterior teeth were recorded under standardized conditions and analyzed for lightness or luminosity values using Adobe Photoshop. The difference between the mean pre-treatment luminosity of test and control groups was not statistically significant. In both test and control groups, the post-treatment luminosity was significantly higher than pre- treatment luminosity (P < 0.001 and P = 0.003 respectively). The mean post-treatment luminosity for test group was found to be significantly higher than control group. The mean percentage removal of stains for test group was significantly higher than control group. The test dentifrice showed significant stain removal when compared to control which could be attributed to the role of proteolytic enzymes in the test dentifrice. PMID:23493413

  19. IMMUNOHISTOCHEMICAL DOUBLE-STAINING FOR AH RECEPTOR AND ARNT IN HUMAN EMBRYONIC PALATAL SHELVES

    EPA Science Inventory

    The aryl hydrocarbon receptor (AhR) and the AhR nuclear translocation protein (ARNT) are helix-loop-helix (HLH) proteins involved in transcriptional regulation. olycyclic aromatic halogenated chemicals, of which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent, bind ...

  20. Antigen detection systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissue using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular methodology is chosen ...

  1. Antigen smuggling in tuberculosis.

    PubMed

    Hudrisier, Denis; Neyrolles, Olivier

    2014-06-11

    The importance of CD4 T lymphocytes in immunity to M. tuberculosis is well established; however, how dendritic cells activate T cells in vivo remains obscure. In this issue of Cell Host & Microbe, Srivastava and Ernst (2014) report a mechanism of antigen transfer for efficient activation of antimycobacterial T cells. PMID:24922567

  2. Antigen detection systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissues or other specimens, using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular m...

  3. Steinway piano and stained glass clerestory window in lounge area, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Steinway piano and stained glass clerestory window in lounge area, upper deck. Hot water radiators can be seen at base of wall. These run throughout the houseboat. - Houseboat LA DUCHESSE, The Antique Boat Museum, Clayton, Jefferson County, NY

  4. 18. INTERIOR DETAIL VIEW OF STAINED GLASS WINDOW LOCATED AT ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    18. INTERIOR DETAIL VIEW OF STAINED GLASS WINDOW LOCATED AT SOUTH SIDE OF ALTAR, NOTE INSCRIPTION DEDICATED IN THE MEMORY OF FATHER DAMIEN - St. Francis Catholic Church, Moloka'i Island, Kalaupapa, Kalawao County, HI

  5. VIEW OF THREE SOUTH FACING STAINED GLASS WINDOWS. THESE WINDOWS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    VIEW OF THREE SOUTH FACING STAINED GLASS WINDOWS. THESE WINDOWS ARE LOCATED ADJACENT TO THE ALTER. - U.S. Naval Base, Pearl Harbor, Chapel, Corner of Oakley & Nimitz Street, Pearl City, Honolulu County, HI

  6. Interior, detail closeup shot of window with stained glass inserts ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Interior, detail closeup shot of window with stained glass inserts in top southeast room taken from ther west - J. Weingartner & Son Cigar Factory, 414 East Walnut Street, North Wales, Montgomery County, PA

  7. 6. Vick Farm, interior perspective of stained glass window, added ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    6. Vick Farm, interior perspective of stained glass window, added as part of deck addition on west side. - Vick Farm, North side Idlewild Road, 0.2 mile northwest of Idlewild & Maplewood Drive, Burlington, Boone County, KY

  8. VIEW OF THREE NORTH FACING STAINED GLASS WINDOWS. THESE WINDOWS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    VIEW OF THREE NORTH FACING STAINED GLASS WINDOWS. THESE WINDOWS ARE LOCATED ADJACENT TO THE ALTAR. - U.S. Naval Base, Pearl Harbor, Chapel, Corner of Oakley & Nimitz Street, Pearl City, Honolulu County, HI

  9. 18. INTERIOR OF KITCHEN NO. 1 SHOWING STAINED CABINETRY ON ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    18. INTERIOR OF KITCHEN NO. 1 SHOWING STAINED CABINETRY ON OPPOSITE WALL FROM PAINTED CABINETS. VIEW TO NORTHEAST. - Bishop Creek Hydroelectric System, Plant 6, Cashbaugh-Kilpatrick House, Bishop Creek, Bishop, Inyo County, CA

  10. VIEW OF THREE NORTH FACING STAINED GLASS WINDOWS. THESE WINDOWS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    VIEW OF THREE NORTH FACING STAINED GLASS WINDOWS. THESE WINDOWS ARE LOCATED JUST BELOW THE CHOIR LOFT. - U.S. Naval Base, Pearl Harbor, Chapel, Corner of Oakley & Nimitz Street, Pearl City, Honolulu County, HI