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Application of rapid-frozen substitution fixation method to immunohistochemistry

快速冷冻替代固定法在免疫组化中的应用

基本信息

批准号：
61870001
负责人：
YASUDA Kenjiro
金额：
$ 5.18万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

项目摘要

A rapid-freezing substitution technique is a quite useful tool to study the ultrastructural features of cells in a more life-like state. However, there remained several difficulties in applying the technique to immunoelectron microscopy. In this study, we tried to overcome those difficulties to perform immunolabelling in well- preserved ultrastructure.Frozen rat parotid gland and exocrine pancreas tissue blocks were quenched by smaching against a copper block surface which was pre-cooled with liquid helium. The tissue blocks were freeze-substituted in either of the following media; osmium in acetone, paraformaldelhyde in acetone, and glutaraldehyde in acetone. Paraformaldehyde in acetone and glutaraldehyde in acetone were prepared with our new distillation device. After substitution, trimmed materials were embedded. Ultrathin sections were immunostained with rabbit anti-rat-amylase antibodies and protein A/colloidal gold.High labelling density was obtained from the materials which were fixed by freeze- substitution with glutaraldehyde in acetone or paraformaldehyde in acetone, embedded in Araldite 6005. In materials substituted with Os04 in acetone, embedded in an epoxy resin mixture and etched with sodium metaperiodate solution, labelling density was lowr than the former. In Lowicryl K4M-embedded materials labelling was the ghighest among the mataerials examined so far.The results in this study present an useful and preactical information in the application of immunocytochemistry to rapid-frozen substitution fixed tissues.

快速冷冻替代技术是研究更接近生命状态的细胞超微结构特征的非常有用的工具。然而，将该技术应用于免疫电子显微镜仍然存在一些困难。在本研究中，我们试图克服这些困难，在保存完好的超微结构中进行免疫标记。将冷冻的大鼠腮腺和外分泌胰腺组织块通过与用液氦预冷的铜块表面进行研磨来淬火。将组织块冷冻替代于以下任一介质中；丙酮中的锇、丙酮中的多聚甲醛和丙酮中的戊二醛。丙酮中的多聚甲醛和丙酮中的戊二醛是用我们的新蒸馏装置制备的。替换后，嵌入修剪过的材料。超薄切片用兔抗大鼠淀粉酶抗体和蛋白 A/胶体金进行免疫染色。用丙酮中的戊二醛或多聚甲醛的丙酮中冷冻替代固定的材料，包埋在 Araldite 6005 中，获得高标记密度。环氧树脂混合物和偏高碘酸钠溶液蚀刻，标记密度比前者低。在 Lowicryl K4M 嵌入材料中，标记是迄今为止检查的材料中最高的。本研究的结果为免疫细胞化学在快速冷冻替代固定组织中的应用提供了有用且实用的信息。