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Antigen Retrieval (antigen + retrieval)
Selected AbstractsThe production and characterization of monoclonal antibodies against Photobacterium damselae ssp. piscicida and initial observations using immunohistochemistryJOURNAL OF FISH DISEASES, Issue 2 2001T S Jung Five monoclonal antibodies (MAbs: F2B1, 1E4, 13B10, 4D4 and F3G12) were produced against lysed Photobacterium damselae ssp. piscicida (Ph. d. ssp. piscicida). The MAbs recognized three antigens of differing molecular weight on the Western blot of Ph. d. ssp. piscicida. They also cross-reacted with five different species of Vibrio. An enzyme linked immunosorbent assay (ELISA) with MAbs, F3G12 and 4D4 demonstrated differences between Ph. d. ssp. piscicida and three Ph. d. ssp. damselae type strains, indicating differences in the surface antigenicity between these two groups of bacteria. Antigen retrieval in conjunction with immunohistochemistry (IHC) using MAb 13B10, revealed colonies of bacteria in the kidney, spleen and liver of sea bass, Dicentrarchus labrax, infected with pasteurellosis. A number of positive colonies were observed around the mucosal layers of the intestinal tissue, especially within the lamina propria. In addition, a number of bacterial colonies were associated with red blood cells and blood vessels of the organs examined. [source] Comparison of antibodies to HBME-1 and calretinin for the detection of mesothelial cells in effusion cytology ,DIAGNOSTIC CYTOPATHOLOGY, Issue 3 2001Patricia A. Fetsch M.T. (A.S.C.P.) Abstract The distinction of mesothelial cells in cytologic samples is often a diagnostic challenge. This is particularly true in potentially malignant effusions in which reactive mesothelial cells may simulate adenocarcinoma (ACA) cells, and in the differentiation of ACA vs. mesothelioma. We sought to determine the superior antibody for the positive identification of mesothelial cells in these circumstances. Cell block sections of 25 reactive and 8 malignant mesothelioma effusions were immunostained with an avidin-biotin procedure, using antibodies to HBME-1 and calretinin. No pretreatment of samples was necessary for the HBME-1-stained slides; microwave antigen retrieval was performed on all slides stained for calretinin. A negative control was performed on each sample. The staining intensity of tumor cells was scored on a scale of 0,3+, with the proportion of immunoreactive cells categorized as <25%, 25,50%, 50,75%, and >75%. The predominant staining pattern for HBME-1 was surface, with rare samples also exhibiting cytoplasmic staining as well. The calretinin-staining pattern was cytoplasmic, with peripheral condensation/prominence and accompanying nuclear staining. All samples were immunoreactive with both antibodies. Fifty-five percent (18/33) of samples showed significantly stronger immunoreactivity with calretinin than with HBME-1; 45% (15/33) of samples showed equivalent staining with the two markers. None of the samples in this study showed stronger immunoreactivity with HBME-1 than with calretinin. Sixty-one percent (20/33) of samples stained with HBME-1 at a moderate (2+) intensity. Fifty-five percent (18/33) of samples stained with calretinin at a strong (3+) intensity. While only 12% of samples showed >75% immunoreactivity for HBME-1, 58% of samples showed >75% of cells immunoreactive for calretinin. Calretinin is the preferred marker in identifying mesothelial cells in cytologic samples, showing the highest sensitivity for mesothelial cells, as evidenced by a more intense staining reaction in a higher percentage of cells than with HBME-1. Diagn. Cytopathol. 2001;25:158,161. Published 2001 Wiley-Liss, Inc. [source] Quantification of immunohistochemistry,issues concerning methods, utility and semiquantitative assessment IHISTOPATHOLOGY, Issue 4 2006R A Walker Immunohistochemistry is no longer a technique used only for research but is employed increasingly for diagnosis and for the assessment of therapeutic biomarkers. The latter, in particular, often require a semiquantitative evaluation of the extent of their presence. There are many factors that can affect this that relate to the method: fixation of tissue, duration and type of antigen retrieval, antibody specificity, antibody dilution and detection systems. Other complexities relate to assessment. Different scoring systems are used for either the same or different antigens. Cut-off levels for assessing whether a tissue is ,positive' or ,negative' can vary for the same antigen. Whilst there are quality assurance schemes for the methodology that have improved standards of staining, there are no similar schemes that relate to interpretation, although errors here can create as many problems. There have been improvements in automated analysis but availability is limited and it is still predominantly a research tool. In order for quantification of immunohistochemistry to be a reliable and reputable tool, there must be easy to use, reproducible, standardized protocols for assessment which are international. Improvements in automated analysis with wider applicability could lead to standardization. [source] Generation of high-quality protein extracts from formalin-fixed, paraffin-embedded tissuesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2009Maria Filippa Addis Abstract A wealth of information on proteins involved in many aspects of disease is encased within formalin-fixed paraffin-embedded (FFPE) tissue repositories stored in hospitals worldwide. Recently, access to this "hidden treasure" is being actively pursued by the application of two main extraction strategies: digestion of the entangled protein matrix with generation of tryptic peptides, or decrosslinking and extraction of full-length proteins. Here, we describe an optimised method for extraction of full-length proteins from FFPE tissues. This method builds on the classical "antigen retrieval" technique used for immunohistochemistry, and allows generation of protein extracts with elevated and reproducible yields. In model animal tissues, average yields of 16.3,,g and 86.8,,g of proteins were obtained per 80,mm2 tissue slice of formalin-fixed paraffin-embedded skeletal muscle and liver, respectively. Protein extracts generated with this method can be used for the reproducible investigation of the proteome with a wide array of techniques. The results obtained by SDS-PAGE, western immunoblotting, protein arrays, ELISA, and, most importantly, nanoHPLC-nanoESI-Q-TOF MS of FFPE proteins resolved by SDS-PAGE, are presented and discussed. An evaluation of the extent of modifications introduced on proteins by formalin fixation and crosslink reversal, and their impact on quality of MS results, is also reported. [source] Vascular endothelial growth factor, FLT-1, and FLK-1 analysis in a pancreatic cancer tissue microarray,CANCER, Issue 8 2006Gina G. Chung M.D. Abstract BACKGROUND Measures of vascular endothelial growth factor (VEGF) expression in pancreatic cancer typically have been qualitative or semiquantitative. The objective of this study was to use a series of algorithms called AQUA that quantitatively assesses protein expression on tissue microarrays (TMAs) to compare in situ expression of VEGF and its primary receptors, VEGF receptor 1 (FLT-1) and VEGF receptor 1 (FLK-1), on a pancreatic cancer TMA. METHODS TMAs were constructed by arraying 1.5-mm cores from 76 samples of pancreatic adenocarcinoma (1996-2002) that were obtained from the archives of the Yale Department of Pathology. The staining for AQUA was similar to standard immunohistochemistry and involved antigen retrieval and the application of primary antibodies, but with epifluorescence detection. Slides were counterstained with 4,,6-diamidino-2-phenylindole for nuclear visualization and cytokeratin for membrane visualization. The primary antibodies used were VEGF, FLT-1, FLK-1, and cytokeratin. RESULTS Disease stage was highly prognostic for outcome, as expected. Total amounts of VEGF and its receptors were assessed within the tumor mask and were divided into quartiles. Kaplan,Meier survival curves showed that VEGF and FLK-1 were not associated clearly with outcome. However, the expression of FLT-1 was correlated significantly, and the patients who had tumors with the lowest expression FLT-1 levels had the worst survival (P = .0038). In multivariate analysis, FLT-1 expression was an independent prognostic factor for overall survival (P = .0044). CONCLUSIONS VEGF and its 2 principal receptors were expressed to varying degrees in tumors of the pancreas. A significant association was found between low expression of FLT-1 and both poor prognosis and advanced stage, suggesting that tumor expression of this VEGF receptor is a marker of less aggressive disease. Cancer 2006. © 2006 American Cancer Society. [source] Evaluation of HER-2/neu expression in prostatic adenocarcinomaCANCER, Issue 8 2002A request for a standardized, organ specific methodology Abstract BACKGROUND Some evidence suggests a role for HER-2/neu overexpression in prostate carcinoma progression. Reported rates of HER-2/neu overexpression in patients with prostate carcinoma vary greatly. METHODS The authors studied radical prostatectomy specimens from 38 patients who had biochemical failure after undergoing radical prostatectomy for prostate carcinoma. Immunohistochemistry for HER-2/neu overexpression using the HercepTest kit (Dako Corporation, Carpenteria, CA) was employed. Two different antigen-retrieval techniques were used: 1) the standard U.S. Food and Drug Administration (FDA)-approved HercepTest assay and 2) a modified HercepTest, which employed an alkaline citrate buffer, pH 9.0, for antigen retrieval and a 1-hour primary antibody incubation time. The level of HER-2/neu expression was evaluated on a scale from 0 (no staining) to 3+ according to the published guidelines. Fluorescent in situ hybridization for gene amplification was performed on all specimens. RESULTS With the standard technique, only one specimen had 2+ staining, and no specimens had 3+ staining. With the modified technique, 10 specimens (26%) had 2+ staining, and 9 specimens (24%) had 3+ staining. There was a significant association between the level of HER-2/neu expression shown with the modified technique and tumor stage (P = 0.03) as well as Gleason grade (P = 0.01). None of the specimens had HER-2/neu gene amplification. CONCLUSIONS The authors report a simple modification of the HercepTest that resulted in an increased rate of HER-2/neu expression, which was correlated with poor-risk pathologic findings. The findings suggest that adenocarcinoma of the prostate should be evaluated for HER-2/neu expression with a prostate specific immunohistochemical procedure that differs from the FDA-approved standard procedure. Cancer 2002;95:1650,5. © 2002 American Cancer Society. DOI 10.1002/cncr.10839 [source] | ||||||||||