Immunolabeling of Cleared Human Pancreata Provides Insights into Three-Dimensional Pancreatic Anatomy and Pathology
Immunolabeling of Cleared Human Pancreata Provides Insights into Three-Dimensional Pancreatic Anatomy and Pathology
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DOI：
10.1016/j.ajpath.2018.04.002
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发表时间：
2018-07-01
影响因子：
6
通讯作者：
Hruban, Ralph H.
中科院分区：
文献类型：
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作者：
Noe, Michael;Rezaee, Neda;Hruban, Ralph H.
三维可视化病理可以为人类疾病的生物学提供独特的见解。一种快速和易于实施的二苄基醚为基础的技术被用来清除厚切片的手术切除的人胰腺实质。方案适用于新鲜和福尔马林固定的石蜡包埋组织。使用逐渐增加的抗体浓度和离心流来优化抗体向致密胰腺实质中的渗透。使用光片和共聚焦激光扫描显微镜观察抗细胞角蛋白19抗体的免疫标记。该技术成功应用于26个胰腺切片，提供了正常胰腺组织、胰腺上皮内瘤变、管内乳头状粘液性肿瘤和浸润性胰腺导管的三维（3D）图像。腺癌3D可视化突出了难以在二维空间中概念化的过程，例如浸润性癌生长到似乎是预先存在的胰腺导管和小静脉内，以及肿瘤细胞平行于血管的长索的跟踪。将这种技术扩展到福尔马林固定的石蜡包埋组织，打开病理档案，以3D可视化独特的生物样品和罕见疾病。应用免疫标记和清除人类胰腺实质提供了正常胰腺解剖的详细可视化，并可用于表征疾病的3D结构，包括胰腺上皮内瘤变、导管内乳头状粘液性肿瘤和胰腺导管腺癌。
Visualizing pathologies in three dimensions can provide unique insights into the biology of human diseases. A rapid and easy-to-implement dibenzyl ether-based technique was used to clear thick sections of surgically resected human pancreatic parenchyma. Protocols were applicable to both fresh and formalin-fixed, paraffin-embedded tissue. The penetration of antibodies into dense pancreatic parenchyma was optimized using both gradually increasing antibody concentrations and centrifugal flow. Immunolabeling with antibodies against cytokeratin 19 was visualized using both light sheet and confocal laser scanning microscopy. The technique was applied successfully to 26 sections of pancreas, providing three-dimensional (3D) images of normal pancreatic tissue, pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms, and infiltrating pancreatic ductal. adenocarcinomas. 3D visualization highlighted processes that are hard to conceptualize in two dimensions, such as invasive carcinoma growing into what appeared to be pre-existing pancreatic ducts and within venules, and the tracking of long cords of neoplastic cells parallel to blood vessels. Expanding this technique to formalin-fixed, paraffin-embedded tissue opens pathology archives to 3D visualization of unique biosamples and rare diseases. The application of immunolabeling and clearing to human pancreatic parenchyma provides detailed visualization of normal pancreatic anatomy, and can be used to characterize the 3D architecture of diseases including pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasm, and pancreatic ductal adenocarcinomas.