一种用于透射电子显微镜和免疫细胞化学的快速冷冻、冷冻置换细胞的制备方法。

A method for preparing quick-frozen, freeze-substituted cells for transmission electron microscopy and immunocytochemistry.

作者信息

Nagele R G, Kosciuk M C, Wang S M, Spero D A, Lee H

出版信息

J Microsc. 1985 Sep;139(Pt 3):291-301. doi: 10.1111/j.1365-2818.1985.tb02645.x.

DOI:10.1111/j.1365-2818.1985.tb02645.x

Abstract

A quick-freeze, freeze-substitution method is described which employs glutaraldehyde as well as osmium tetroxide (OsO4) in a 'double-fixation' protocol comparable to that used for conventional transmission electron microscopy. Cultured cells are quick-frozen in Freon 22 and freeze-substituted in an ethanolic solution of glutaraldehyde. Specimens destined for TEM are postfixed in OsO4 in acetone, embedded in Epon-Araldite, and sectioned. This method yielded ultrastructural preservation which was comparable to that obtained from methods employing OsO4 alone as a freeze-substitution fixative. However, if glutaraldehyde is used alone as a freeze-substitution fixative, specimens can be processed for immunocytochemistry without additional treatment with permeabilizing agents.

摘要

本文描述了一种快速冷冻、冷冻置换方法，该方法在“双重固定”方案中使用戊二醛以及四氧化锇（OsO4），这与传统透射电子显微镜所使用的方法类似。培养细胞在氟利昂22中快速冷冻，并在戊二醛的乙醇溶液中进行冷冻置换。用于透射电子显微镜观察的标本在丙酮中的OsO4中进行后固定，包埋在环氧树脂-阿拉尔代特中，然后切片。该方法产生的超微结构保存效果与仅使用OsO4作为冷冻置换固定剂的方法相当。然而，如果单独使用戊二醛作为冷冻置换固定剂，则标本可以不经透化剂额外处理而直接用于免疫细胞化学。