人类胰岛染色协议
Summary
本视频演示了人体胰腺胰岛细胞用苏木精伊红（H＆E）和免疫组化法（IHC）的鉴定程序。从头部，身体，和尾区域胰腺部分是染色都Ĥ＆é和免疫组化确定胰岛内分泌（胰岛素，胰高血糖素，和胰多肽）组成，细胞复制（Ki67的）和（H＆E，CD3）炎性浸润。钩突地区的本地化使用胰多肽免疫组化。
Abstract
胰岛面积和数字，在成人胰腺内分泌细胞组成的估计各不相同，从几十万到几百万和β的质量范围从500至1500毫克1-3。这个著名的异质性，开发一个标准的加工和染色过程，使胰腺地区进行了明确规定和胰岛细胞的特点，用严格的组织病理学和免疫组化检查。
本系列第1部分中描述的标准化程序处理人体器官捐赠者的胰腺恢复。胰腺被加工成横截面的3个主要地区（头，身，尾）。横截面从胰头进一步划分，如根据大小，按字母顺序和编号表示小节。这种标准化允许的头部区域的一个完整的交叉截面分析包括钩突的区域，其中包含小岛组成主要的胰多肽细胞的尾部区域。
目前的报告，包括本系列的第2部分，介绍了用于强调胰岛内分泌细胞，复制，和T细胞的浸润胰腺石蜡切片连续切片和病理特征的程序。病理学胰腺节的目的是表征两个外分泌腺，胆管，内分泌组件。在的外分泌腺车厢被评为存在胰腺炎（主动或慢性），萎缩，纤维化，脂肪，以及管道系统，特别是在关系到存在的胰腺导管内肿瘤4。评估胰岛形态，大小，密度，内分泌细胞，炎症，纤维化，淀粉样蛋白，并使用H＆E和免疫组化污渍复制或凋亡细胞的存在。
第2部分中描述的最后一个组件是提供彩色幻灯片作为数字化整体幻灯片图像。数字化幻灯片的情况下，在网上创建一个虚拟的生物资料库的病理数据库胰腺地区举办。访问这个网上收集目前提供超过200名医生和1型糖尿病的研究中科学家参与。联机数据库提供了一个快速和完整的数据共享和调查选择石蜡或冰冻连续切片块的手段。
Protocol
1。为不染节的切片成立水浴和标准的石蜡切片切片。正电的幻灯片和前标签使用数量的情况下，胰腺区域（样品）和幻灯片编号。广场左侧的磁带标签的切片机夹头的石蜡块。 按照正常切片程序，节成块，直到组织均匀遇到。产生一个连续切片（4μm厚3-6（见案例提交表格， 附录1）根据初期所需的污渍）剪彩。色带单独的章节，以便拿起，和幻灯片的地方用铅笔编号?…
Discussion
免疫组化标准化程序是图像分析的关键，特别是当使用大量的幻灯片之间随着时间的推移以计算机为基础的算法。在本报告所述的免疫组化染色过程将使一批在8小时工作制Autostainer自动给定的捐赠者的样品的分析和修改是从以 前的报告5。每个彩色幻灯片的全数字化的幻灯片图像可通过基于web的在线病理系统nPOD附属调查。研究人员可以审查与幻灯片一起捐助者的人口统计资料，实验?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢捐助者的家庭和组织的专家协助参与这项研究和刘慧卿蒙哥马利罗伯特Pietras，安富，Mitali阿加瓦尔，罗山阿加瓦尔器官采购。这项工作是由青少年糖尿病研究基金会（MC-T.）对糖尿病患者的胰腺器官捐赠者的网络支持。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| 30% Hydrogen Peroxide (H2O2) | FisherScientific | H325-500 | Endogenous peroxidase blocking |
| ABC-Alkaline Phosphatase (AP) | Vector | AK-5000 | AP conjugate |
| Antibody Diluent | Invitrogen | 003218 | Primary antibody |
| Avidin blocker | Vector | SP-2001 | Block endogenous biotin |
| Biotinylated Goat anti-guinea pig | Vector | BA-7000 | Secondary antibody |
| Bluing Reagent | FisherScientific | 7301 | Leica autostainer |
| Citrate buffer, pH 6.0 | BioGenex | HK086-9K | Antigen retrieval |
| Clarifier 1 | FisherScientific | 7401 | Leica autostainer |
| Cytoseal XYL | FisherScientific | 8312-4 | Coverslip mountant |
| DAB | Vector | SK-4100 | HRP chromogen |
| DEEB | DAKO | S2003 | Endogenous AP blocking reagent |
| Eosin Y Alcoholic | FisherScientific | 71204 | Leica autostainer |
| Ethanols- 100%, 95%, 90%, 70% | FisherScientific | Various | Deparaffinization and coverslipping |
| Hematoxylin | DAKO | S3301 | Dako autostainer |
| Hematoxylin 7211 | FisherScientific | 7211 | Leica autostainer |
| IgG (all host species for primary antibodies) | Various | Various | Negative controls for primaries |
| Liquid Permanent Red (LPR) | DAKO | K0640 | AP chromogen |
| Mach2 AP | Biocare Medical | MALP521L | Goat anti-Mouse AP conjugate |
| Mach2 HRP | Biocare Medical | MHRP520L | Goat anti-Mouse HRP conjugate |
| Mach2 HRP | Biocare Medical | RHRP520L | Goat anti-Mouse HRP conjugate |
| Methanol | FisherScientific | Various | H2O2 diluent |
| Normal goat serum | Vector | S-1000 | IHC blocking reagent |
| Sniper | Biocare Medical | BS966M | IHC blocking reagent |
| TBST 20X | ThermoScientific | TA-999-TT | IHC buffer |
| Triology 20x | Cell Marque | 920P-06 | Antigen Retrieval |
| Xylene | FisherScientific | Various | Deparaffinization and coverslipping |
Table 1. Specific reagents.
| Primary Antibody | Host species | Vendor | Cat. # | Comments and Antigen Retrieval |
| Paraffin | ||||
| Amylase | Mouse | Santa Cruz | SC-46657 | Citrate |
| Amylin | Mouse | Serotec | MCA11267 | Citrate |
| Carbonic anhydrase 19.9 | Mouse | Abcam | ab15146 | |
| Caspase-3, cleaved | Rabbit | Cell Signaling | 9961 | Citrate or Trilogy |
| CD20 | Mouse | DAKO | M0755 | Citrate or Trilogy |
| CD3 | Rabbit | DAKO | A0452 | Citrate |
| CD34 | Mouse | Cell Signaling | 3569 | Citrate |
| CD4 | Mouse | DAKO | M7310 | Citrate |
| CD45 | Mouse | DAKO | M0754 | Citrate |
| CD68 | Mouse | DAKO | M0876 | Citrate |
| CD8 | Mouse | DAKO | M7103 | Citrate |
| Chromogranin A | Rabbit | DAKO | A0430 | Citrate |
| CK17 | Mouse | DAKO | M7046 | Citrate |
| CK19 | Mouse | DAKO | M0772 | Citrate |
| CK7 | Mouse | DAKO | M7018 | Citrate |
| C-peptide | Rabbit | Cell Signaling | 4593 | Citrate |
| Foxp3 | Rat | e Bioscience | 14-4776-80 | Citrate |
| Ghrelin | Goat | Santa Cruz | SC-10386 | |
| Ghrelin | Rabbit | Abcam | ab85104 | |
| Glucagon | Rabbit | DAKO | A0565 | |
| Glucagon | Mouse | Abcam | ab10988 | Citrate |
| Glucagon | Guinea Pig | Bachem | T-5037 | Citrate |
| Glut-1 | Mouse | Abcam | ab40084 | Trilogy |
| Glut-2 | Rabbit | Santa Cruz | SC-9117 | Not expressed in human islets |
| Insulin | Guinea Pig | DAKO | A0564 | |
| Ki-67 | Mouse | DAKO | M7240 | Citrate |
| Mafa | Rabbit | Novus Biological | NB400-137A | Trilogy |
| Pancreatic polypeptide | Rabbit | Invitrogen | 18-0043 | Citrate |
| Pdx-1 | Guinea Pig | Abcam | ab47308 | Citrate or Trilogy |
| Proinsulin | Mouse | Novocastra | NCL-proin-1G4 | |
| Proinsulin | Mouse | Developmental Studies Hybridoma Bank | GS-9A8 | Citrate |
| Secretagogin | Rabbit | Sigma | HPA 006641 | Citrate |
| Smooth muscle actin | Rabbit | Abcam | ab5694 | |
| Somatostatin | Rabbit | DAKO | A0566 | |
| Synaptophysin | Mouse | DAKO | M0776 | Citrate |
| Fresh Frozen | Host species | Vendor | Cat. # | Comments |
| CD11b | Rat | Abcam | ab6332 | Fix with 4% PF |
| CD11c | Rabbit | Serotec | AHP1226 | |
| CD25 | Mouse | Novus Biological | NB 600-564 | Use overnight incubation for paraffin sections |
| CD94 | Mouse | Abcam | ab61874 | Fix with acetone |
| GAD65 | Mouse | Santa Cruz | SC-130569 | Fix with 4% PF |
| HLA-ABC | Mouse | DAKO | M0736 | Fix with acetone or 4% PF |
Table 2. Primary antibodies.
| Name of the reagent | Company | Catalogue number | Comments |
| Aperio Scanscope CS | Aperio | Digital slide scanner | |
| DAKO Autostainer Plus | DAKO | IHC stains | |
| Label Matrix printing software | BioCarta | LM7UP57 | Slide label software |
| Leica XL Autostainer | Leica | H&E stains | |
| Premium Coverglass | FisherScientific | 12-548-5J | Coverslip |
| Spectrum Information Manager | Aperio | Scanner software | |
| Superfrost Plus slides | FisherScientific | 12-550-15 | Positively charged slides |
| Vegetable steamer | Black and Decker | Various | Antigen retrieval |
| Zebra TLP 3742 | CDWG | TLP3742 | Slide label printer |
Table 3. Specific supplies and equipment.
References
- In’t Veld, P., Marichal, M. Microscopic anatomy of the human islet of Langerhans. Adv. Exp. Med. Biol. 654, 1-19 (2010).
- Matveyenko, A. V., Butler, P. C. Relationship between beta-cell mass and diabetes onset. Diabetes Obes. Metab. 10, 23-31 (2008).
- Saisho, Y. Pancreas volumes in humans from birth to age one hundred taking into account sex, obesity, and presence of type-2 diabetes. Clin. Anat. 20, 933-942 (2007).
- Hruban, R. H. Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions. Am. J. Surg. Pathol. 25, 579-586 (2001).
- Campbell-Thompson, M. Pancreatic adenocarcinoma patients with localised chronic severe pancreatitis show an increased number of single beta cells, without alterations in fractional insulin area. Diabetologia. 52, 262-270 (2009).
- Meier, J. Beta-cell replication is the primary mechanism subserving the postnatal expansion of beta-cell mass in humans. Diabetes. 57, 1584-1594 (2008).
- Rowe, P. A., Campbell-Thompson, M. L., Schatz, D. A., Atkinson, M. A. The pancreas in human type 1 diabetes. Semin. Immunopathol. 33, 29-43 (2011).
- Veld, I. n. ‘. t., P, . Insulitis in human type 1 diabetes: The quest for an elusive lesion. Islets. 3, 131-138 (2011).
- van Belle, T. L., Coppieters, K. T., von Herrath, M. G. Type 1 diabetes: etiology, immunology, and therapeutic strategies. Physiol Rev. 91, 79-118 (2011).
Tags
Staining ProtocolsHuman Pancreatic IsletsIslet AreaEndocrine Cell CompositionProcessing And Staining ProcedureHistopathologyImmunolocalization ExaminationsStandardized ProceduresTransverse SectionsPancreatic HeadPancreas TailSerial SectioningHistopathological CharacterizationIslet Endocrine CellsReplicationT-cell InfiltratesExocrine CompartmentDuctular Components
Cite This Article
Campbell-Thompson, M. L., Heiple, T., Montgomery, E., Zhang, L., Schneider, L. Staining Protocols for Human Pancreatic Islets. J. Vis. Exp. (63), e4068, doi:10.3791/4068 (2012).