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.
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.
1. De-parafinization
<ol>
 <li>Bake slides at 55 &deg;C for 20 min.</li>
 <li>In a chemical hood, immerse slides in xylene for 10 min. </li>
 <li>Immerse slides in fresh xylene for 10 min. </li>
 <li>Immerse slides in 100% ethanol for 5 min. </li>
 <li>Immerse slides in fresh 100% ethanol for 5 min. </li>
 <li>Immerse slides in 95% ethanol for 5 min. </li>
 <li>Immerse slides in 80% ethanol for 5 min. </li>
 <li>Immerse slides in deionized water for 5 min. </li>
</ol>
2. Antigen Retrieval
<ol>
 <li>Immerse slides in Tris-EDTA buffer (pH 9.0) and heat in a pressure cooker to 125 &deg;C for 30 sec, then 90 &deg;C for 10 sec. </li>
 <li>Let slides cool for 60 min. </li>
 <li>Immerse slides in TBST for 5 min; repeat twice. </li>
</ol>
3. Staining
<ol>
 <li>Wipe off solutions around the tissue on the slides. Mark a circle around the tissue with a hydrophobic pen.</li>
 <li>Place the slides in a humidity chamber and avoid the drying of the tissue on the slide throughout the entire staining process. Place one drop (approximately 200 &mu;l or more to cover the tissue area completely) of peroxidase block within the hydrophobic circle on the slide and incubate slides in peroxidase block for 5 min. </li>
 <li>Rinse slides with TBST, then place in TBST for 5 min. </li>
 <li>Place one drop of BLOCK ACE blocking buffer on each slide and incubate for 15 min. </li>
 <li>Rinse slides with TBST, then place in TBST for 5 min.</li>
 <li>Place one drop of Avidin solution on each slide and incubate for 15 min. </li>
 <li>Rinse slides with TBST, then place in TBST for 5 min. </li>
 <li>Place one drop of Biotin solution on each slide and incubate for 15 min.</li>
 <li>Rinse slides with TBST, then place in TBST for 5 min. </li>
 <li>Incubate slides with 1:1,000 dilution of mouse anti-B7-H1 monoclonal antibody (clone 5H1) or a mouse IgG1 &kappa; isotype control in 200 &mu;l TBST at 4 &deg;C overnight for 22 hr+/- 1 hr. The final antibody concentration in TBST is 2.57 &mu;g/ml. </li>
 <li>Rinse slides with TBST, then place in TBST for 5 min. Repeat twice. </li>
 <li>Incubate slides with a biotin conjugated rat anti-mouse IgG1 secondary antibody at a 1:500 concentration (in 200 &mu;l 2% goat serum solution) for 30 min. </li>
 <li>Rinse slides with TBST, then place in TBST for 5 min. Repeat twice.</li>
 <li>Place one drop of Streptavidin-Biotin Complex (must be prepared at least 30 min prior to use) on each slide and incubate for 15 min. </li>
 <li>Rinse slides with TBST, then place in TBST for 5 min. Repeat twice. </li>
 <li>Place one drop of Amplification Reagent (diluted 2:1 with TBST) on each slide and incubate for 4 min.</li>
 <li>Rinse slides with TBST, then place in TBST for 5 min. Repeat twice. </li>
 <li>Place one drop of Streptavidin-HRP on each slide and incubate for 15 min. </li>
 <li>Rinse slides with TBST, then place in TBST for 5 min. Repeat twice. </li>
 <li>Place one drop of 3,3-Diaminobenzidine (DAB) substrate and chromogen on each slide and develop the color for 2 min. </li>
 <li>Immediately after the color development, wash slides with ddH2O for 1-2 min. </li>
 <li>Incubate slides with one drop of hematoxylin for 90 sec. </li>
 <li>Wash with ddH2O for 1-2 min. </li>
 <li>Wash with soapy tap water for 1-2 min. </li>
 <li>Wash with ddH2O for 1-2 min. </li>
</ol>
4. Dehydration and Mounting
<ol>
 <li>Immerse the slides in 80% ethanol for 3 min.</li>
 <li>Immerse the slides in 95% ethanol for 3 min.</li>
 <li>Immerse the slides in 100% ethanol for 5 min. Repeat. </li>
 <li>Immerse the slides in xylene for 5 min. Repeat.</li>
 <li>Add one drop of Cytoseal 60 to the slide and place the coverslip.</li>
</ol>
Table 1 lists the reagents used in the above described procedures.

<ol>
	<li>Flies, D. B., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+B7s:+playing+a+pivotal+role+in+tumor+immunity.">The new B7s: playing a pivotal role in tumor immunity.</a> J. Immunother. 30, 251-260 (2007).</li><li>Dong, H., Zhu, G., Tamada, K., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-H1,+a+third+member+of+the+B7+family,+co-stimulates+T-cell+proliferation+and+interleukin-10+secretion.">B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion.</a> Nat. Med. 5, 1365-1369 (1999).</li><li>Curiel, T. J., Wei, S., Dong, H., Alvarez, X., Cheng, P., Mottram, P., Krzysiek, R., Knutson, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+B7-H1+improves+myeloid+dendritic+cell-mediated+antitumor+immunity.">Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity.</a> Nat. Med. 9, 562-567 (2003).</li><li>Dong, H., Strome, S. E., Salomao, D. R., Tamura, H., Hirano, F., Flies, D. B., Roche, P. C., Lu, J., Zhu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-associated+B7-H1+promotes+T-cell+apoptosis:+a+potential+mechanism+of+immune+evasion.">Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion.</a> Nat. Med. 8, 793-800 (2002).</li><li>Dong, H., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-H1+pathway+and+its+role+in+the+evasion+of+tumor+immunity.">B7-H1 pathway and its role in the evasion of tumor immunity.</a> J. Mol. Med. (Berl). 81, 281-287 (2003).</li><li>Strome, S. E., Dong, H., Tamura, H., Voss, S. G., Flies, D. B., Tamada, K., Salomao, D., Cheville, J., Hirano, F., Lin, W., Kasperbauer, J. L., Ballman, K. V., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-H1+blockade+augments+adoptive+T-cell+immunotherapy+for+squamous+cell+carcinoma.">B7-H1 blockade augments adoptive T-cell immunotherapy for squamous cell carcinoma.</a> Cancer Res. 63, 6501-6505 (2003).</li><li>Hirano, F., Kaneko, K., Tamura, H., Dong, H., Wang, S., Ichikawa, M., Rietz, C., Flies, D. B., Lau, J. S., Zhu, G., Tamada, K., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+B7-H1+and+PD-1+by+monoclonal+antibodies+potentiates+cancer+therapeutic+immunity.">Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity.</a> Cancer Res. 65, 1089-1096 (2005).</li><li>Thompson, R. H., Gillett, M. D., Cheville, J. C., Lohse, C. M., Dong, H., Webster, W. S., Krejci, K. G., Lobo, J. R., Sengupta, S., Chen, L., Zincke, H., Blute, M. L., Strome, S. E., Leibovich, B. C., Kwon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Costimulatory+B7-H1+in+renal+cell+carcinoma+patients:+Indicator+of+tumor+aggressiveness+and+potential+therapeutic+target.">Costimulatory B7-H1 in renal cell carcinoma patients: Indicator of tumor aggressiveness and potential therapeutic target.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 17174-17179 (2004).</li><li>Geng, L., Huang, D., Liu, J., Qian, Y., Deng, J., Li, D., Hu, Z., Zhang, J., Jiang, G., Zheng, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-H1+up-regulated+expression+in+human+pancreatic+carcinoma+tissue+associates+with+tumor+progression.">B7-H1 up-regulated expression in human pancreatic carcinoma tissue associates with tumor progression.</a> J. Cancer Res. Clin. Oncol. 134, 1021-1027 (2008).</li><li>Loos, M., Giese, N. A., Kleeff, J., Giese, T., Gaida, M. M., Bergmann, F., Laschinger, M., WB, M., Friess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+significance+and+regulation+of+the+costimulatory+molecule+B7-H1+in+pancreatic+cancer.">Clinical significance and regulation of the costimulatory molecule B7-H1 in pancreatic cancer.</a> Cancer Lett. 268, 98-109 (2008).</li><li>Okudaira, K., Hokari, R., Tsuzuki, Y., Okada, Y., Komoto, S., Watanabe, C., Kurihara, C., Kawaguchi, A., Nagao, S., Azuma, M., Yagita, H., Miura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+B7-H1+or+B7-DC+induces+an+anti-tumor+effect+in+a+mouse+pancreatic+cancer+model.">Blockade of B7-H1 or B7-DC induces an anti-tumor effect in a mouse pancreatic cancer model.</a> Int. J. Oncol. 35, 741-749 (2009).</li><li>Wang, S., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunobiology+of+cancer+therapies+targeting+CD137+and+B7-H1/PD-1+cosignal+pathways.">Immunobiology of cancer therapies targeting CD137 and B7-H1/PD-1 cosignal pathways.</a> Curr. Top Microbiol. Immunol. 344, 245-267 (2011).</li><li>Thompson, R. H., Webster, W. S., Cheville, J. C., Lohse, C. M., Dong, H., Leibovich, B. C., Kuntz, S. M., Sengupta, S., Kwon, E. D., Blute, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B7-H1+glycoprotein+blockade:+a+novel+strategy+to+enhance+immunotherapy+in+patients+with+renal+cell+carcinoma.">B7-H1 glycoprotein blockade: a novel strategy to enhance immunotherapy in patients with renal cell carcinoma.</a> Urology. 66, 10-14 (2005).</li><li>Thompson, R. H., Kuntz, S. M., Leibovich, B. C., Dong, H., Lohse, C. M., Webster, W. S., Sengupta, S., Frank, I., Parker, A. S., Zincke, H., Blute, M. L., Sebo, T. J., Cheville, J. C., Kwon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+B7-H1+is+associated+with+poor+prognosis+in+renal+cell+carcinoma+patients+with+long-term+follow-up.">Tumor B7-H1 is associated with poor prognosis in renal cell carcinoma patients with long-term follow-up.</a> Cancer Res. 66, 3381-3385 (2006).</li><li>Thompson, R. H., Dong, H., Kwon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+B7-H1+expression+in+clear+cell+carcinoma+of+the+kidney+for+prognostication+and+therapy.">Implications of B7-H1 expression in clear cell carcinoma of the kidney for prognostication and therapy.</a> Clin. 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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-L1+(B7-H1)+expression+by+urothelial+carcinoma+of+the+bladder+and+BCG-induced+granulomata:+associations+with+localized+stage+progression.">PD-L1 (B7-H1) expression by urothelial carcinoma of the bladder and BCG-induced granulomata: associations with localized stage progression.</a> Cancer. 109, 1499-1505 (2007).</li><li>Brahmer, J. R., Drake, C. G., Wollner, I., Powderly, J. D., Picus, J., Sharfman, W. H., Stankevich, E., Pons, A., Salay, T. M., McMiller, T. L., Gilson, M. M., Wang, C., Selby, M., Taube, J. M., Anders, R., Chen, L., Korman, A. J., Pardoll, D. M., Lowy, I., Topalian, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+I+study+of+single-agent+anti-programmed+death-1+(MDX-1106)+in+refractory+solid+tumors:+safety,+clinical+activity,+pharmacodynamics,+and+immunologic+correlates.">Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates.</a> J. Clin. Oncol. 28, 3167-3175 (2010).</li><li>Wang, L., Ma, Q., Chen, X., Guo, K., Li, J., Zhang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+significance+of+B7-H1+and+B7-1+expressions+in+pancreatic+carcinoma.">Clinical significance of B7-H1 and B7-1 expressions in pancreatic carcinoma.</a> World J. Surg. 34, 1059-1065 (2010).</li><li>Nomi, T., Sho, M., Akahori, T., Hamada, K., Kubo, A., Kanehiro, H., Nakamura, S., Enomoto, K., Yagita, H., Azuma, M., Nakajima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+significance+and+therapeutic+potential+of+the+programmed+death-1+ligand/programmed+death-1+pathway+in+human+pancreatic+cancer.">Clinical significance and therapeutic potential of the programmed death-1 ligand/programmed death-1 pathway in human pancreatic cancer.</a> Clin. Cancer Res. 13, 2151-2157 (2007).</li><li>Taube, J. M., Anders, R. A., Young, G. D., Xu, H., Sharma, R., McMiller, T. L., Chen, S., Klein, A. P., Pardoll, D. M., Topalian, S. L., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colocalization+of+inflammatory+response+with+B7-h1+expression+in+human+melanocytic+lesions+supports+an+adaptive+resistance+mechanism+of+immune+escape.">Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape.</a> Sci. Transl. Med. 4, 127ra37 (2012).</li></ol>

No conflicts of interest declared.

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 ...

immunohistochemical-staining-b7-h1-pd-l1-on-paraffin-embedded-slides

Research

JoVE Journal

Medicine

23.0K Views.  The Johns Hopkins University School of Medicine. B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor’s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.

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

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Application of End-to-end Anastomosis in Robotic Central Pancreatectomy

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of pancreatic body. With technological development, the robotic surgical system has shown its advantage in minimally invasive surgery and been increasingly applied in central pancreatectomy. However, reconstruction of the continuity of pancreas with end-to-end anastomosis after robotic central pancreatectomy has not been applied. In this study, we report surgical techniques for robotic central pancreatectomy with end-to-end anastomosis. The pancreas is reconstructed by duct-to-duct anastomosis of the pancreatic duct with a pancreatic stent inserted in the two stumps of pancreatic duct, and by end-to-end anastomosis of the pancreatic parenchyma. Compared with traditional central pancreatectomy with pancreaticoenteric anastomosis, this approach decreases the operative injury to the patient, and also conserves the integrity and continuity of the digestive duct and pancreatic duct. The robotic surgical system integrated with multiple instruments with flexible and precise movement is particularly suitable for the dissection and reconstruction of the pancreatic duct. We found that robotic central pancreatectomy with end-to-end anastomosis is safe and feasible, and we need more experience to evaluate its best indications and long-term outcomes.

The robotic central pancreatectomy with end-to-end anastomosis is feasible and safe for tumor in the pancreatic neck and proximal portion of pancreatic body. The operative techniques of this operation are presented.

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...

Robotic Enucleation of an Intra-Pancreatic Insulinoma in the Pancreatic Head

Pancreatic parenchyma sparing surgery for insulinomas avoids the risk of endocrine and exocrine insufficiency, and potential high-risk anastomoses associated with pancreatic resection. Robotic surgery may be used as an alternative for open pancreatic enucleation without compromising dexterity and 3D-vision.
We present the case of a 42-year old woman who presented with sweating, tremor and episodes of hypoglycemia. A fasting test confirmed endogenic insulin overproduction. After inconclusive CT- and MRI imaging, endoscopic ultrasonography showed a hypoechoic lesion, which was fully within the pancreatic head. Although consent was obtained for pancreatoduodenectomy, robotic enucleation seemed feasible. After mobilization, intraoperative ultrasonography was used to identify the lesion and its relation with the pancreatic duct. Dissection was performed using a traction suture, hot shears and bipolar diathermia. A sealant patch was applied for hemostasis and a drain placed. The patient developed a grade B pancreatic fistula for which endoscopic sphincterotomy was performed; the surgical drain could be removed in the outpatient clinic after 20 days. Prospective studies should confirm the short- and long-term benefits of robotic enucleation of insulinomas.

Here, we present a robotic approach to enucleate an insulinoma in the pancreatic head.

Pancreatic parenchyma sparing surgery for insulinomas avoids the risk of endocrine and exocrine insufficiency, and potential high-risk anastomoses ...

Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The use of experimental animal models of iron overload or deficiency has been instrumental to advance knowledge of the mechanisms involved in the systemic and cellular regulation of iron homeostasis. The measurement of total iron levels in animal tissues is commonly performed with atomic absorption spectroscopy or with a colorimetric assay based on the reaction of non-heme iron with a bathophenanthroline reagent. For many years, the colorimetric assay has been used for the measurement of the non-heme iron content in a wide range of animal tissues. Unlike atomic absorption spectroscopy, it excludes the contribution of heme iron derived from hemoglobin contained in red blood cells. Moreover, it does not require sophisticated analytical skills or highly expensive equipment, and can thus be easily implemented in most laboratories. Finally, the colorimetric assay can be either cuvette-based or adapted to a microplate format, allowing higher sample throughput. The present work provides a well-established protocol that is suited for the detection of alterations in tissue iron levels in a variety of experimental animal models of iron overload or iron deficiency.

Here, a protocol for the measurement of the non-heme iron content in animal tissues is provided, using a simple, well-established colorimetric assay that can be easily implemented in most laboratories.

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The ...

Tissue Embedding and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

Assessing Retinal Thickness and Ganglion Cell Layer Counts in Rodent Eyeballs

Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many more. Preclinical experiments enable researchers to examine the efficacy of novel drugs, develop new methods of treatment, or seek new pathomechanisms involved in the disease's onset or progression. A histological examination provides a lot of information necessary to assess the effects of the conducted experiments and can reveal degeneration, tissue remodeling, infiltration, and many other pathologies. In clinical research, there is rarely any chance of obtaining eye tissue suitable for a histological examination, which is why researchers should take advantage of the opportunity offered by the examination of eyeballs from rodents. This manuscript presents a protocol for the histological preparation of rodent eyeballs' sections. The procedure is presented for the eyeballs of mice and rats and has the following steps: (i) harvesting the eyeball, (ii) preserving the eyeball for further analysis, (iii) processing the tissue in paraffin, (iv) preparing slides, (v) staining with hematoxylin and eosin, (vi) assessing the tissue under a light microscope. With the proposed method, the retina can be easily visualized and assessed in detail.

This manuscript presents a protocol for the histological preparation of slides from rodent eyeballs. With the proposed method, the inner retina can be easily visualized and assessed under a light microscope. The procedure is presented for the eyeballs of mice and rats.

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many ...