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
1The Sidney Kimmel Comprehensive Cancer Center,The Johns Hopkins University School of Medicine, 2Department of Oncology,The Johns Hopkins University School of Medicine, 3Department of Dermatology,The Johns Hopkins University School of Medicine, 4Department of Surgery,Johns Hopkins University School of Medicine, 5The Sol Goldman Pancreatic Cancer Center,The Johns Hopkins University School of Medicine, 6Yale Cancer Center,Yale School of Medicine, 7The Skip Viragh Center for Pancreatic Cancer,The Johns Hopkins University School of Medicine, 8Department of Pathology,The Johns Hopkins University School of Medicine
Summary
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。
Abstract
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。
Protocol
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?…
Representative Results
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 …
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
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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Tags
Immunohistochemical StainingB7-H1PD-L1Paraffin-embedded SlidesImmune-regulatory Cell-surface ProteinsNegative RegulationCell-mediated Immune ResponsesReceptorProgrammed Death-1OverexpressionTumor CellsHuman CancersPrognostic MarkerAnti-tumor T-cell ImmunityTherapeutic TargetBlockade AntibodiesClinical TrialsSolid TumorsExpression And LocalizationPatient ResponseTreatment
Citazione di questo articolo
Bigelow, E., Bever, K. M., Xu, H., Yager, A., Wu, A., Taube, J., Chen, L., Jaffee, E. M., Anders, R. A., Zheng, L. Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue. J. Vis. Exp. (71), e4059, doi:10.3791/4059 (2013).