Saitoh Sei, Ohno Nobuhiko, Saitoh Yurika, Terada Nobuo, Shimo Satoshi, Aida Kaoru, Fujii Hideki, Kobayashi Tetsuro, Ohno Shinichi
Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan.
Present address: Section of Electron Microscopy, Supportive Center for Brain Research, National Institute for Physiological Sciences, Okazaki 444-8787, Japan.
Acta Histochem Cytochem. 2018 Feb 27;51(1):9-20. doi: 10.1267/ahc.17020. Epub 2018 Feb 21.
Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells of compact and diffuse islets from human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 α (REG1α) was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Immunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. In conclusion, the correlative imaging approach developed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets.
结合对各种生物分子的免疫染色分析以及对单个细胞超微结构特征的研究，是研究正常和病理条件下细胞功能的有力方法。然而，传统方法固定的组织抗原性较弱，这给免疫分析带来了问题。本研究介绍了一种对人胰腺组织标本中致密胰岛和弥散胰岛的同一内分泌细胞进行光镜与电镜关联成像的方法。该方法利用从用戊二醛和四氧化锇固定的环氧树脂包埋标本中获取的连续切片。在用乙醇钠去除环氧树脂、高压灭菌进行抗原修复以及用过氧化氢进行脱渗处理后，对厚环氧树脂切片进行内分泌激素（胰岛素和胰高血糖素）和再生胰岛衍生基因1α（REG1α）的双重免疫荧光染色。将内分泌细胞的免疫荧光图像与从连续超薄切片中获得的相同细胞的电子显微镜图像叠加。免疫荧光图像显示内分泌细胞中分泌颗粒保存良好，而电子显微镜观察显示相同细胞中相应的分泌颗粒和细胞内细胞器。总之，我们开发的关联成像方法可能有助于结合同一人胰岛中内分泌激素的免疫定位来检查超微结构特征。
