EB，九巴，和HX的贡献相同。这项研究是由国家癌症研究所SPORE在胃肠道癌症P50 CA062924，NIH K23 CA148964，勒斯特加滕基金会，Viragh基金会，美国临床肿瘤学会青年研究者奖，美国国立卫生研究院5T32 CA0090701-28培训津贴，溶胶高盛胰腺癌中心，国立胰腺基金会的支持。

B7-H1/PD-L1，免疫调节的细胞表面蛋白的B7家族的成员，在负调控的细胞介导的​​免疫反应中起着重要的作用通过其与其受体的相互作用，程序性死亡-1（PD -1）1,2。已注意到在一个数量的人类癌症，包括黑色素瘤，胶质母细胞瘤，肺癌，乳腺癌，结肠癌，卵巢癌，肾细胞癌B7-H1由肿瘤细胞中的过表达，并已被证明是损害抗肿瘤T细胞免疫3-8。 近日，B7-H1表达的胰腺癌组织已被确定为一个潜在的预后标志物9,10。此外，封锁的B7-H1在胰腺癌的小鼠模型中已被证明产生的抗肿瘤反应11。这些数据表明，B7-H1的重要性，作为一个潜在的治疗靶。因此，在临床试验中被测试多种人体抗-B7-H1阻断抗体实体肿瘤，包括恶性黑色素瘤及癌症的肺，大肠，肾，胃和胰腺12。 为了最终能够确定患者将受益于B7-H1靶向疗法，重要的是调查的表达和定位的B7-H1和B7-H1阻断抗体治疗病人的反应之间的相关性。检查通过免疫组化的人类胰腺癌组织中B7-H1的表达，将能够更好地了解如何共同抑制信号分子有助于抑制肿瘤的微环境中的抗肿瘤免疫。抗B7-H1的单克隆抗体（克隆5H1）由Chen和他的同事开发已被证明在冷冻切片的多种类型的人类肿瘤组织4,8产生可靠的染色结果，但染色石蜡包埋的幻灯片已经是一个挑战，直到最近13-18。我们已经开发了B7-H1 staini的纳克协议石蜡包埋胰腺癌组织中的幻灯片。这里描述的B7-H1染色协议产生一致的膜和胞质染色B7-H1的一些背景知识。 1。去parafinization <ol><li>烘烤20分钟，在55℃下滑动。 </li><li>以一种化学遮光罩，浸泡在二甲苯中滑动10分钟。 </li><li>新鲜的二甲苯10分钟浸入滑动。 </li><li>浸没在100％乙醇中滑动5分钟。 </li><li>在新鲜的5分钟的100％乙醇中的浸入滑动。 </li><li>浸在95％乙醇中滑动5分钟。 </li><li>浸在80％乙醇中滑动5分钟。 </li><li>浸没在去离子水中滑动5分钟。 </li></ol> 2。抗原修复<ol><li>浸入滑动的Tris-EDTA缓冲液（pH9.0）中和在高压锅热至125℃，持续30秒，然后在90℃下持续10秒。 </li><li>让幻灯片冷却60分钟。 </li><li> TBST 5分钟的浸泡幻灯片; REPE的两倍。 </li></ol> 3。染色法<ol><li>擦去周围组织幻灯片上的解决方案。标记一圈的组织与疏水性笔。 </li><li>放置在湿度室中滑动，并避免在整个染色过程中的组织上的滑动的干燥。将一滴（约200μl或多个完全覆盖组织区域）的过氧化物酶块内的疏水圈上滑动并孵育过氧化物酶滑动块5分钟。 </li><li>用TBST冲洗幻灯片，然后在TBST 5分钟。 </li><li>将一滴的BLOCK ACE阻断缓冲每张幻灯片上孵育15分钟。 </li><li>用TBST冲洗幻灯片，然后在TBST 5分钟。 </li><li>将一滴每张幻灯片上的抗生物素蛋白溶液孵育15分钟。 </li><li>用TBST冲洗幻灯片，然后在TBST 5分钟。 </li><li>将一滴每张幻灯片上的生物素溶液孵育15分钟。 </li><li>冲洗SLIDES，然后用TBST TBST 5分钟。 </li><li>孵育1:1,000稀释的小鼠抗-B7-H1单克隆抗体（克隆5H1）或小鼠IgG1滑动κ型控制在200μlTBST中在4℃下22小时+ / - 1小时过夜。 TBST最后的抗体浓度为2.57微克/毫升。 </li><li>用TBST冲洗幻灯片，然后在TBST 5分钟。重复两次。 </li><li>孵育幻灯片与生物素共轭大鼠抗小鼠IgG1二次抗体在1:500浓度（在200μl2％山羊血清溶液）30分钟。 </li><li>用TBST冲洗幻灯片，然后在TBST 5分钟。重复两次。 </li><li>将一滴链霉亲和素生物素复合物（每张幻灯片上孵育15分钟，必须准备至少前30分钟使用）。 </li><li>用TBST冲洗幻灯片，然后在TBST 5分钟。重复两次。 </li><li>将一滴扩增试剂（2:1，TBST稀释），每张幻灯片上孵育4分钟。 </li><li>冲洗幻灯片与TBST，然后TBST 5分钟。重复两次。 </li><li>将一滴每张幻灯片上的链霉亲和素-HRP孵育15分钟。 </li><li>用TBST冲洗幻灯片，然后在TBST 5分钟。重复两次。 </li><li>将1滴3,3 - 二氨基联苯胺（DAB）的基板和色原体在每个幻灯片和发展的颜色，持续2分钟。 </li><li>后，立即发色，洗幻灯片DDH 2 O为1-2分钟。 </li><li>孵育90秒与一滴苏木滑动。 </li><li> DDH 2 O清洗1-2分钟。 </li><li>用肥皂水自来水冲洗1-2分钟。 </li><li> DDH 2 O清洗1-2分钟。 </li></ol> 4。脱水和安装<ol><li>沉浸在80％乙醇3分钟的幻灯片。 </li><li>浸泡在95％乙醇3分钟的幻灯片。 </li><li>沉浸在100％乙醇5分钟的幻灯片。重复以上步骤。 </li><li>沉浸在二甲苯中5分钟的幻灯片。重复以上步骤。 </li><li>加入一滴Cytoseal 60幻灯片和盖玻片。 </li></ol> 表1列出了在上述的程序中使用的试剂。

<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>

没有利益冲突的声明。

B7-H1对冷冻切片的免疫组织化学染色，已报道在以前的研究中，用于各种癌症的3,4。然而，临床肿瘤样本通常只适用于石蜡包埋块的形式。 5H1单克隆抗体已被成功地应用到染色的石蜡包埋的肿瘤组织，包括肾细胞癌，宫颈癌，膀胱癌，黑色素瘤13-18。 B7-H1染色石蜡包埋9,19或低温恒温器20内的与其他单克隆抗体的胰腺癌组织上也有报道。这里，我们已经示出成功染?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> CSA系统</td><td> Dako公司</td><td> K1500 </td><td>包括在协议中提及的下列试剂：过氧化物酶块，链霉抗生物素蛋白 - 生物素复合物，扩增试剂，链霉亲和素-HRP，和DAB底物和色原体</td></tr><tr><td>抗生物素蛋白/生物素阻断试剂盒</td><td>向量</td><td> SP-2001 </td><td></td></tr><tr><td>座ACE </td><td> Serotec公司</td><td> BUF029 </td><td></td></tr><tr><td>生物素的抗小鼠IgG1 </td><td> BD </td><td> 553441 </td><td></td></tr><tr><td> K表型对照小鼠IgG1 </td><td> eBioscience公司</td><td> 14-4714-85 </td><td></td></tr><tr><td> TBS（Tris缓冲盐水），10X </td><td> Cellgro </td><td> 46-012-CM </td><td> 10xTBS含有80克/ L的NaCl和24.2 g / L的TIRS; 为洗涤用ddH2O稀释至1X; </td></tr><tr><td>吐温20 </td><td> Sigma-Aldrich公司</td><td> P1379 </td><td> 1毫升添加到1 L的1X TBS TBST </td></tr><tr><td>目标检索，20X </td><td> Celerus波</td><td> 014-3000-000 </td><td> DDH 2 O稀释至1倍</td></tr><tr><td> 190标准酒精</td><td> Pharmco-Aaper </td><td> 111000190 </td><td></td></tr><tr><td> 200证明乙醇</td><td> Pharmo-Aaper </td><td> 111000200 </td><td></td></tr><tr><td>二甲苯</td><td> VWR </td><td> 95057-827 </td><td></td></tr><tr><td>苏木</td><td>理查德·阿伦科学的</td><td> 72804 </td><td></td></tr><tr><td> Cytoseal 60 </td><td>理查德·阿伦科学的</td><td> 8310-4 </td><td></td></tr><tr><td>盖玻片</td><td>康宁</td><td> 2940-245 </td><td></td></tr><tr><td>恒温恒湿试验箱</td><td>西格玛</td><td> H6644 </td><td></td></tr><tr><td></td><td></td><td></td><td> 表1试剂和设备。 </td></tr></tbody></table>

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

B7-H1/PD-L1，免疫调节的细胞表面蛋白的B7家族的成员，在负调控的细胞介导的​​免疫反应中起着重要的作用通过其与其受体的相互作用，程序性死亡-1（PD -1）1,2。已注意到在一个数量的人类癌症，包括黑色素瘤，胶质母细胞瘤，肺癌，乳腺癌，结肠癌，卵巢癌，肾细胞癌B7-H1由肿瘤细胞中的过表达，并已被证明是损害抗肿瘤T细胞免疫3-8。 近日，B7-H1表达的胰腺癌组织已被确定为一个潜在的预后标志物9,10。此外，封锁的B7-H1在胰腺癌的小鼠模型中已被证明产生的抗肿瘤反应11。这些数据表明，B7-H1的重要性，作为一个潜在的治疗靶。因此，抗-B7-H1阻断抗体在临床试验中被测试MULtiple人类固体肿瘤，包括恶性黑色素瘤及癌症的肺，大肠，肾，胃和胰腺12。 为了最终能够确定患者将受益于B7-H1靶向疗法，重要的是调查的表达和定位的B7-H1和B7-H1阻断抗体治疗病人的反应之间的相关性。检查通过免疫组化的人类胰腺癌组织中B7-H1的表达，将能够更好地了解如何共同抑制信号分子有助于抑制肿瘤的微环境中的抗肿瘤免疫。抗B7-H1的单克隆抗体（克隆5H1）由Chen和他的同事开发已被证明在冷冻切片的多种类型的人类肿瘤组织4,8产生可靠的染色结果，但染色石蜡包埋的幻灯片已经是一个挑战，直到最近13-18。我们必须开发私奔的B7-H1染色协议，石蜡包埋胰腺癌组织中的幻灯片。这里描述的B7-H1染色协议产生一致的膜和胞质染色B7-H1的一些背景知识。

一个成功的免疫组化染色B7-H1在胰腺癌会表现出异质性表达B7-H1（茶色的信号DAB显色）。有些胰腺癌细胞有主要是膜表达B7-H1（ 图1A），而另一些具有要么主要细胞质表达B7-H1或很少表达B7-H1（ 图2和图3）。一般的胰管上皮表达一点B7-H1（ 图4）。抗B7-H1的抗体与小鼠IgG1κ型对照抗体染色，而不是示出小的背景染色（ 图1B）。 <img ...

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）和PD-1的结合提供了重要的肿瘤引起的免疫抑制肿瘤的微环境中的信号。免疫组织化学技术的特点B7-H1在胰腺癌组织中的表达和定位的描述。

B7-H1（PD-L1）免疫组化染色石蜡的嵌入式幻灯片的胰腺癌组织

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

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

23.0K 次观看.  The Johns Hopkins University School of Medicine. B7-H1（PD-L1）和PD-1的结合提供了重要的肿瘤引起的免疫抑制肿瘤的微环境中的信号。免疫组织化学技术的特点B7-H1在胰腺癌组织中的表达和定位的描述。

视频: 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.

人类胰岛染色协议

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

科研

医学

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.

人类前列腺上皮细胞形成的使用鼠类泌尿生殖窦间质细胞和人类干细胞的组织重组

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

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

生物荧光原位模型胰腺癌进展

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

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.

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

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.

端到端吻合技术在机器人中心胰腺中的应用

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

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

胰岛门静脉移植小鼠模型的研究

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

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.

石蜡内嵌组织中人与鼠中性粒细胞外陷阱的免疫荧光标记

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.

胰腺内胰岛素瘤在胰腺头的机器人Enuen

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.

使用基于菲咯啉的比色法测量组织非血红素铁含量

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

Network Pharmacology Prediction and Experimental Validation of Trichosanthes-Fritillaria thunbergii Action Mechanism Against Lung Adenocarcinoma

We aimed to study the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma (LUAD) based on network pharmacology and experimental verification. The effective components and potential targets of Trichosanthis and Fritillaria thunbergii were collected by high-throughput experiment and reference-guided (HERB) database of traditional Chinese medicine and a similarity ensemble approach (SEA) database, and the LUAD-related targets were queried by the GeneCards and Online Mendelian Inheritance in Man (OMIM) databases. A drug-component-disease-target network was constructed by Cytoscape software. Protein-protein interaction (PPI) network, gene ontology (GO) function, and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses were conducted to obtain core targets and key pathways. An aqueous extract of Trichosanthes-Fritillaria thunbergii and A549 cells were used for the subsequent experimental validation. Through the HERB database and literature search, 31 effective compounds and 157 potential target genes of Trichosanthes-Fritillaria thunbergii were screened, of which 144 were regulatory targets of Trichosanthes-Fritillaria thunbergii in the treatment of lung adenocarcinoma. The GO functional enrichment analysis showed that the mechanism of action of Trichosanthes-Fritillaria thunbergii against lung adenocarcinoma is mainly protein phosphorylation. The KEGG pathway enrichment analysis suggested that the treatment of lung adenocarcinoma by Trichosanthes-Fritillaria thunbergii mainly involves the PI3K/AKT signaling pathway. The experimental validation showed that an aqueous extract of Trichosanthes-Fritillaria thunbergii could inhibit the proliferation of A549 cells and the phosphorylation of AKT. Through network pharmacology and experimental validation, it was verified that the PI3K/AKT signaling pathway plays a vital role in the action of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma.

Network Pharmacology Prediction and Experimental Validation of Trichosanthes-Fritillaria thunbergii Action Mechanism Against Lung Adenocarcinoma

This study reveals the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma based on network pharmacology and experimental verification. The study also demonstrates that the PI3K/AKT signaling pathway plays a vital role in the action of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma.