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Various Antibodies (various + antibody)
Selected AbstractsValidated protocol for FoxP3 reveals increased expression in type 1 diabetes patients,CYTOMETRY, Issue 2 2009Jean Grant Abstract Background FoxP3 has become a key identifier of regulatory T cells. Investigators have used a variety of antibodies and methods for detecting FoxP3 by flow cytometry. To standardize FoxP3 antibody staining for use in clinical trial samples, we tested various antibodies from different vendors, cell preparation protocols and fix/perm reagents, and cell isolation procedures. Using this optimized staining protocol, we evaluated clinical specimens from patients with multiple sclerosis (MS) or type 1 diabetes. Methods FoxP3 antibodies from eBioscience (236A/E7 and PCH101) and BioLegend (206D) were evaluated along with their respective methods and fix/perm reagents for preparation and staining of FoxP3 for flow cytometry. Fresh washed blood and frozen or fresh PBMC were evaluated. Upon optimization of the protocol, clinical samples (frozen PBMC) from patients with MS or type 1 diabetes and healthy control donors were evaluated with the BioLegend antibody. Results Clone 206D from BioLegend yielded optimal staining and the fix/perm reagents from both eBioscience and BioLegend were comparable. Data were also comparable between cells separated by Ficoll (fresh or frozen) and washed blood samples, allowing this protocol to be applicable to different types of samples. We validated this protocol using clinical samples and saw a significant increase in FoxP3 expression in the patients with type 1 diabetes but not in the MS. Conclusions The results from this study will allow the assessment of FoxP3 by flow cytometry on samples from clinical sites that are analyzed in real time on fresh blood or frozen PBMC. © 2008 Clinical Cytometry Society [source] The effects of antibody clone and pretreatment method on the results of HER2 immunostaining in cytologic samples of metastatic breast cancer: A query and a review of the literature,,§DIAGNOSTIC CYTOPATHOLOGY, Issue 6 2007Patricia A. Fetsch M.T. (ASCP) Abstract The standardization and use of heat-induced epitope retrieval (HIER) is particularly important with immunohistochemical markers that direct the course of cancer treatment, such as Herceptin therapy. Increasingly, many laboratories are performing immunohistochemical analysis using various antibodies and methodologies for HER2/neu. We attempted to determine the effects of antibody clone and pretreatment methods on the interpretation of HER-2/neu staining in cytologic samples. Cell block sections from 54 cases of metastatic breast cancer (24 FNAs, 30 effusions) were analyzed for HER2 expression using antibodies to CB-11, TAB250, and A0485. Antibodies were analyzed with and without HIER. One pathologist using the FDA-approved scoring system for the HercepTest reviewed all slides in a blinded fashion. Five of fifty-four cases (9%) using CB-11 showed a significant increase in HER2 immunoreactivity using HIER (i.e. from 0/1+ to 2,3+). However, in twenty-nine of fifty-four cases (54%), the cytoplasmic background was significantly higher after HIER. With the A0485 antibody, two of fifty four cases (4%) showed a significant increase in immunoreactivity using HIER, while seventeen of fifty-four cases (31%) exhibited only more pronounced cytoplasmic staining. HIER pretreatment did not increase HER2 staining in any TAB250 stained sample, rather four of fifty-four cases (7%) showed a significant decrease in staining with HIER. We conclude that HIER may enhance membrane staining with the CB-11 and A0485 antibodies, but also increases cytoplasmic background. Loss of antigenicity is seen when HIER is used with TAB250. Diagn. Cytopathol. 2007;35:319,328. © 2007 Wiley-Liss, Inc. [source] Proteomic analysis of RCL2 paraffin-embedded tissuesJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 5b 2008V. Belief Abstract Histopathological diagnosis in most of the world's hospitals is based upon formalin-fixed and paraffin-embedded (FFPE) tissues. Although this standard fixation and embedding procedure keeps the tissue in excellent form for morphological and immunohistological analysis, FFPE is inappropriate for nucleic acids and protein studies. We investigated the potential value of RCL2, a new non-toxic fixative, for sparing proteins preserved in paraffin-embedded tissues. Normal colonic mucosa tissue was fixed in RCL2 prior to paraffin embedding (RCL2P), and then processed for quality and quantity of protein conservation, as compared to frozen and FFPE tissues using complementary proteomic analysis approaches. Using 4 different protein extraction protocols, RCL2P tissue consistently showed the highest protein yield. Similar protein patterns were observed with RCL2P and frozen tissues using mono and bi-dimensional electrophoresis. Moreover, membrane, cytoplasmic and nuclear proteins, as well as phosphorylated proteins, were successfully detected using western-blot. Furthermore, protein patterns observed by mass spectrometry analysis after laser-captured microdissection were found to be identical for frozen and RCL2-fixed tissues. At last, immunohistochemistry using various antibodies showed comparable results between both tissue storage methods. We concluded that RCL2 has great potential for performing both morphological and molecular analyses on the same archival paraffin-embedded tissue sample, and can be a new method for investigating protein biomarkers. [source] G1 cyclins in oral epithelial dysplasiaJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 2 2001R. J. Oliver Abstract: The G1 cyclins, D1, D3 and E, were investigated in 38 lesions of oral epithelial dysplasia from the floor of the mouth or the lateral border of the tongue. Their immunohistochemical expression was observed and compared with that of Ki-67 and with the degree of dysplasia assessed by the semi-objective technique of Smith & Pindborg. Antibody labelled cells were quantified and expressed as a percentage (LI%) of the total nucleated cell population and per mm basement membrane length (LI/mm). The labelling indices of all of the antibodies were high and quantitatively similar. There were no significant correlations with the degree of dysplasia assessed by the atypia scores. There was a correlation between labelling indices for the various antibodies expressed as LI/mm but little correlation between the indices expressed as LI%. The distribution of the D cyclins was similar to that of Ki-67 with relatively few of the basal cells demonstrating immunoreactivity. The reasons for this are discussed in the paper. Some cross-reactivity was observed with the cyclin antibodies. We conclude that the antibodies against the cyclins used in the present study are not a useful adjunct in the study of the cell kinetics of oral epithelial dysplasia. [source] Insights into the molecular basis of rhegmatogenous retinal detachmentACTA OPHTHALMOLOGICA, Issue 2009PN BISHOP Purpose Factors that determine the likelihood of developing posterior vitreous detachment and subsequent rhegmatogenous retinal detachment (RRD) include (i) the degree of vitreous liquefaction (ii) the strength of post-basal vitreoretinal adhesion and (iii) the topology of the posterior border of the vitreous base. The purpose of these studies was to investigate each of these using a combination of ultrastructural and molecular techniques. Methods Ultrastructural studies of the human vitreous and vitreoretinal interface were performed in combination with various antibodies and cationic dyes. Biochemical studies were performed on extracted vitreous components. Results The resultant data suggest that: (i) vitreous liquefaction is caused by the aggregation of vitreous collagen fibrils and this is due to a loss of type IX collagen proteoglycan from the fibril surfaces; (ii) interactions between heparan sulphate proteoglycans in the inner limiting lamina and components on the surface of cortical vitreous collagen fibrils contribute to postbasal vitreoretinal adhesion; (iii) the posterior border of the vitreous base migrates posteriorly with aging due to the synthesis of new vitreous collagen by the peripheral retina. Conclusion The molecular basis of RRD is starting to be unravelled. Furthering our understanding of the underlying molecular processes may lead to the development of novel therapeutic strategies to treat RRD and other vitreoretinal disorders. [source] D-TAT transporter as an ocular peptide delivery systemCLINICAL & EXPERIMENTAL OPHTHALMOLOGY, Issue 6 2005Daniel F Schorderet MD Abstract Background:, Future treatment for genetic diseases may involve the replacement of malfunctioning genes through virus-mediated gene therapy. However, this approach is plagued with many problems, both ethical and scientific. Therefore, alternative treatments based on new molecules may represent a safer option. Molecular treatment of many eye diseases will need to bring active molecules into the photoreceptors. Recently, the trans -activator protein (TAT) human immunodeficiency virus type 1 (HIV-1) transcriptional factor has proven to be effective in transporting molecules across cellular membranes. The half-life of these molecules does not exceed 48 hours. The potential use of the retro-inverso form of the TAT (D-TAT) peptide, the protein transducing domain of the HIV-1 transcriptional factor, as a molecular transporter was investigated. Methods:, FITC-labelled D-TAT (D-TAT FITC) was applied to the 661W murine photoreceptor cell line in culture. The labelled peptide was also injected into the vitreous body or the subretinal space of adult mice. Cells and cryosections of eyes were analysed under fluorescence microscopy at various time points after peptide treatment. Coimmunostaining with various antibodies was performed in order to characterize the transduces cells. Results:, D-TAT was effective in transducing photoreceptor cells in culture. Transduction of D-TAT FITC was also effective when injected into the vitreous or subretinal space and was observed for a longer period of time than L-TAT FITC. Conclusions:, The retro-inverso form of the TAT sequence is effective in transducing cells from various compartments of the eye. After 14 days, the D-TAT FITC was clearly visible in the retina whereas L-TAT FITC had almost disappeared. The D-TAT peptide represents an interesting molecular transporter that, when coupled to a specific effector, may have potential therapeutic future, especially when a long-lasting action is needed. [source] | ||||||||||