ELECTRON-MICROSCOPY IMMUNOCYTOCHEMISTRY FOLLOWING CRYOFIXATION AND FREEZE-SUBSTITUTION

This chapter presents methods of immunolabeling after cryofixation. It focuses on postembedding labeling of cells and tissues that have been ultrarapidly frozen (or cryofixed), freeze substituted, and embedded in resins. High-pressure freezing is emphasized as a cryofixation method because it is applicable to the widest range of samples. Moreover, the chapter discusses two techniques that permit low-temperature embedding of material that has been chemically fixed at room temperature. The most crucial variable used in the method is the reactivity of the antibody used. It should have reasonably high titer and ideally, react with antigens after fixation with glutaraldehyde to obtain the best structural preservation. Another important variable is the quality of cryofixation. In the ideal case, one would like to have cells with their water frozen in the vitreous (noncrystalline) state without cryoprotectants. The main reasons to use cryofixation rather than chemical fixation are that fixation is fast (milliseconds) and that cellular components are simultaneously stabilized. It is assumed that cryofixation, compared to conventional chemical fixation, provides images that are more likely to reflect the native structure of living cells. An additional benefit of cryofixation is that it may improve immunocytochemical localization. © 1993, Academic Press Inc.