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A method for preparing quick‐frozen, freeze‐substituted cells for transmission electron microscopy and immunocytochemistry
Author(s) -
Nagele R. G.,
Kosciuk M. C.,
Wang S. M.,
Spero D. A.,
Lee H.
Publication year - 1985
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1985.tb02645.x
Subject(s) - fixative , glutaraldehyde , osmium tetroxide , immunocytochemistry , transmission electron microscopy , fixation (population genetics) , acetone , araldite , electron microscope , ultrastructure , staining , chemistry , materials science , chromatography , pathology , anatomy , biology , biochemistry , nanotechnology , organic chemistry , medicine , optics , layer (electronics) , physics , adhesive , gene
SUMMARY A quick‐freeze, freeze‐substitution method is described which employs glutaraldehyde as well as osmium tetroxide (OsO 4 ) 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 OsO 4 in acetone, embedded in Epon‐Araldite, and sectioned. This method yielded ultrastructural preservation which was comparable to that obtained from methods employing OsO 4 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.