Name: immunohistochemical staining of b7-h1 (pd-l1) on paraffin-embedded slides of pancreatic adenocarcinoma tissue
Uploaded: 2013-01-03T12:00:00
Description: Watch this Scientific Journal Video about Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue at JoVE.com

E.B., K.M.B, and H.X. contribute equally. This study was supported by the NCI SPORE in Gastrointestinal Cancers P50 CA062924, NIH K23 CA148964, Lustgarten Foundation, Viragh Foundation, ASCO Young Investigator Award, NIH 5T32 CA0090701-28 Training Grant, Sol Goldman Pancreatic Cancer Center, and National Pancreas Foundation.

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.
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<ol>
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Cancer Res. 13, 709s-715s (2007).</li><li>Karim, R., Jordanova, E. S., Piersma, S. J., Kenter, G. G., Chen, L., Boer, J. M., Melief, C. J., vander Burg, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-expressed+B7-H1+and+B7-DC+in+relation+to+PD-1++T-cell+infiltration+and+survival+of+patients+with+cervical+carcinoma.">Tumor-expressed B7-H1 and B7-DC in relation to PD-1+ T-cell infiltration and survival of patients with cervical carcinoma.</a> Clin. Cancer Res. 15, 6341-6347 (2009).</li><li>Inman, B. A., Sebo, T. J., Frigola, X., Dong, H., Bergstralh, E. J., Frank, I., Fradet, Y., Lacombe, L., Kwon, E. 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Immunohistochemical staining of B7-H1 on cryosections has been reported in prior studies for a variety of cancers3,4. However, clinical tumor samples usually are available only in the form of paraffin-embedded blocks. The 5H1 monoclonal antibody has more recently been successfully applied to the staining of paraffin embedded tumor tissues including renal cell carcinoma, cervical carcinoma, bladder cancer and melanoma13-18. B7-H1 staining on paraffin-embedded9,19 or cryostat20 s...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>CSA System</td>
			<td>Dako</td>
			<td>K1500</td>
			<td>Includes the following reagents mentioned in the protocol: peroxidase block, streptavidin-biotin complex, amplification reagent, streptavidin-HRP, and DAB substrate and chromogen</td>
		</tr>
		<tr>
			<td>Avidin/Biotin Blocking Kit</td>
			<td>Vector</td>
			<td>SP-2001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Block ACE</td>
			<td>Serotec</td>
			<td>BUF029</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biotin anti-mouse IgG1</td>
			<td>BD</td>
			<td>553441</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse IgG1 K Isotype Control</td>
			<td>eBioscience</td>
			<td>14-4714-85</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TBS(Tris-buffered saline), 10X</td>
			<td>Cellgro</td>
			<td>46-012-CM</td>
			<td>10xTBS contains 80 g/L NaCl and 24.2 g/L Tirs; 
			Diluted to 1X with ddH2O for washes;</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td>1 ml added to 1 L of 1X TBS to make TBST</td>
		</tr>
		<tr>
			<td>Target Retrieval, 20x</td>
			<td>Celerus Wave</td>
			<td>014-3000-000</td>
			<td>Diluted to 1x with ddH2O</td>
		</tr>
		<tr>
			<td>190 Proof Ethyl Alcohol</td>
			<td>Pharmco-Aaper</td>
			<td>111000190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>200 Proof Ethyl Alcohol</td>
			<td>Pharmo-Aaper</td>
			<td>111000200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR</td>
			<td>95057-827</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Richard-Allan Scientific</td>
			<td>72804</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cytoseal 60</td>
			<td>Richard-Allan Scientific</td>
			<td>8310-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Corning</td>
			<td>2940-245</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Humidity chamber</td>
			<td>Sigma</td>
			<td>H6644</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Table 1. Reagents and equipment.</td>
		</tr>
	</tbody>
</table>

immunohistochemical staining of b7-h1 (pd-l1) on paraffin-embedded slides of pancreatic adenocarcinoma tissue

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.

A successful immunohistochemical staining of B7-H1 will demonstrate the heterogeneous expression of B7-H1 (brown signals DAB color development) in pancreatic adenocarcinoma. Some pancreatic adenocarcinoma cells have predominantly membranous expression of B7-H1 (Figure 1A) whereas others have either predominantly cytoplasmic expression of B7-H1 or little expression of B7-H1 (Figures 2 and 3). Normal pancreatic duct epithelium expresses little B7-H1 (Figure 4). Staining ...

Watch this Scientific Journal Video about Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue at JoVE.com

Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor&#x2019;s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

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 ...

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