Dehydration and Mounting Immerse the slides in 80% ethanol for 3 min. Immerse the slides in 95% ethanol for 3 min. Immerse the slides in 100% ethanol for 5 min. B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor’s microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol explained here produces consistent membranous and cytoplasmic staining of B7-H1 with little background. strong class=”kwd-title” Keywords: Malignancy Biology, Issue 71, Medicine, Immunology, Biochemistry, Molecular Biology, Cellular Biology, Chemistry, Oncology, immunohistochemistry, B7-H1 (PD-L1), pancreatic adenocarcinoma, pancreatic malignancy, pancreas, tumor, T-cell immunity, malignancy video preload=”none” poster=”/pmc/articles/PMC3582653/bin/jove-71-4059-thumb.jpg” width=”480″ height=”360″ source type=”video/x-flv” src=”/pmc/articles/PMC3582653/bin/jove-71-4059-pmcvs_normal.flv” /source source type=”video/mp4″ src=”/pmc/articles/PMC3582653/bin/jove-71-4059-pmcvs_normal.mp4″ Oaz1 /source source type=”video/webm” src=”/pmc/articles/PMC3582653/bin/jove-71-4059-pmcvs_normal.webm” /source /video Download video file.(60M, mov) Protocol B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the unfavorable regulation of cell-mediated immune responses through its conversation with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell Rebaudioside C immunity3-8. Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic malignancy has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12. In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor’s microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background. 1. De-parafinization Bake slides at 55 C for 20 min. In a chemical hood, immerse slides in xylene for 10 min. Immerse slides in new xylene for 10 min. Immerse slides in 100% ethanol for 5 min. Immerse slides in new 100% ethanol for 5 min. Immerse slides in 95% ethanol for 5 min. Immerse slides in 80% ethanol for 5 min. Immerse slides in deionized water for 5 min. 2. Antigen Retrieval Immerse slides in Tris-EDTA buffer (pH 9.0) and warmth in a pressure cooker to 125 C for Rebaudioside C 30 sec, then 90 C for 10 sec. Let slides cool for 60 min. Immerse slides in TBST for 5 min; repeat twice. 3. Staining Wipe off solutions round the tissue around the slides. Mark a circle round the tissue with a hydrophobic pen. Place the slides in a humidity chamber and avoid the drying of the tissue around the slide throughout the entire staining process. Place one drop (approximately 200 l or more to protect the tissue area completely) of peroxidase block within the hydrophobic circle on the slide and incubate slides in peroxidase block for 5 min. Rinse slides with TBST, then place in TBST for Rebaudioside C 5 min. Place one drop of BLOCK ACE blocking buffer on each slide and incubate for 15 min. Rinse slides with TBST, then place in TBST for 5 min. Place one drop of Avidin answer on each slide and incubate for 15 min. Rinse slides with TBST, then place in TBST for 5 min. Place one drop of Biotin answer on each slide and incubate for 15 min. Rinse slides with TBST, then place in.