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Journal of Histochemistry and Cytochemistry, Vol. 46, 847-854, July 1998, Copyright © 1998, The Histochemical Society, Inc.

ARTICLE

Immunoelectronmicroscopy of Soluble and Membrane Proteins with a Sensitive Postembedding Method

Jorge E. Moreiraa,c, Valerie Dodaneb, and Thomas S. Reesea,c
a Laboratory of Neurobiology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland
b National Institute of Neurological Disorders and Stroke and Laboratory of Cellular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland
c Marine Biological Laboratory, Woods Hole, Massachusetts

Correspondence to: Jorge E. Moreira, Departamento de Morfologia, Faculdade de Medicina (FMRP-USP), 14049-900 Ribeirao Preto, Sao Paulo, Brazil.

The application of immunoelectronmicroscopy to soluble proteins is limited because soluble proteins can redistribute during fixation. Fixation may also adversely affect the recognition of proteins associated with membranes. We show here how displacements of soluble proteins can be prevented and antigen sensitivity improved by freeze-substitution immunocytochemistry. The usefulness of this method for soluble cytoplasmic proteins is demonstrated for the twitchin protein in Aplysia muscle and the kinesin motor proteins in squid giant axons, in which the sizes of various cytoplasmic pools of kinesins are estimated. The utility for membrane proteins present in small numbers of copies is demonstrated by labeling a glutamate receptor subunit in mouse cerebellar cortex and the ZO-1 protein in tight junctions between MDCK cells. Thus, freeze-substitution immunocytochemistry can show the native distribution of both soluble and membrane proteins labeled with polyclonal antibodies and, at the same time, can reveal structural features comparable to those in chemically fixed or osmium freeze-substituted samples.

(J Histochem Cytochem 46:847–854, 1998)

Key Words: freeze-substitution, immunoelectron microscopy, soluble proteins, membrane proteins


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