अग्नाशय के Adenocarcinoma ऊतक B7-H1 immunohistochemical धुंधला (पीडी L1) आयल एम्बेडेड स्लाइड्स
Summary
B7-H1 (पी.डी. L1) और उसके PD-1 के लिए बाध्य एक प्रमुख ट्यूमर प्रेरित ट्यूमर microenvironment में immunosuppressive संकेत प्रदान करते हैं. एक immunohistochemical धुंधला अभिव्यक्ति और अग्नाशय ग्रंथिकर्कटता में B7-H1 का स्थानीयकरण विशेषताएँ तकनीक वर्णित है यहाँ.
Abstract
B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction 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 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 cancer 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.
Protocol
Representative Results
Discussion
B7-H1 cryosections पर immunohistochemical धुंधला हो जाना 3,4 के कैंसर के एक किस्म के लिए पूर्व अध्ययन की रिपोर्ट में किया गया है. हालांकि, नैदानिक ट्यूमर के नमूनों आमतौर पर आयल – एम्बेडेड ब्लॉकों के रूप में ही उपलब्ध हैं. 5H1 म?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
EB, KMB, सेमी और समान रूप से योगदान करते हैं. इस अध्ययन गैस्ट्रोइंटेस्टाइनल CA062924 P50 कैंसर, NIH CA148964 K23, Lustgarten फाउंडेशन, Viragh फाउंडेशन, ASCO युवा अन्वेषक पुरस्कार, NIH CA0090701-28 प्रशिक्षण अनुदान, सोल गोल्डमैन अग्नाशय के कैंसर केंद्र, और राष्ट्रीय अग्न्याशय फाउंडेशन 5T32 में NCI बीजाणु द्वारा समर्थित किया गया.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| CSA System | Dako | K1500 | Includes the following reagents mentioned in the protocol: peroxidase block, streptavidin-biotin complex, amplification reagent, streptavidin-HRP, and DAB substrate and chromogen |
| Avidin/Biotin Blocking Kit | Vector | SP-2001 | |
| Block ACE | Serotec | BUF029 | |
| Biotin anti-mouse IgG1 | BD | 553441 | |
| Mouse IgG1 K Isotype Control | eBioscience | 14-4714-85 | |
| TBS(Tris-buffered saline), 10X | Cellgro | 46-012-CM | 10xTBS contains 80 g/L NaCl and 24.2 g/L Tirs; Diluted to 1X with ddH2O for washes; |
| Tween 20 | Sigma-Aldrich | P1379 | 1 ml added to 1 L of 1X TBS to make TBST |
| Target Retrieval, 20x | Celerus Wave | 014-3000-000 | Diluted to 1x with ddH2O |
| 190 Proof Ethyl Alcohol | Pharmco-Aaper | 111000190 | |
| 200 Proof Ethyl Alcohol | Pharmo-Aaper | 111000200 | |
| Xylene | VWR | 95057-827 | |
| Hematoxylin | Richard-Allan Scientific | 72804 | |
| Cytoseal 60 | Richard-Allan Scientific | 8310-4 | |
| Coverslips | Corning | 2940-245 | |
| Humidity chamber | Sigma | H6644 | |
| Table 1. Reagents and equipment. |
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