Ultrastructural immunolocalization of polyamines in HeLa cells subjected to fast-freezing fixation and freeze substitution.

Abstract

Polyamines have been localized at the ultrastructural level in HeLa cells subjected first to fast-freezing fixation (FFF)-freeze substitution (FS) and then to an immunocytochemical method combining anti-polyamine antibodies and immunogold labelling. Polyamines were found in both the cytoplasm and the nucleus and, in the latter, particularly over the dense chromatin area. To our knowledge, this is the first example of the electron microscopic localization of a hapten after FFF-FS. For the preservation of fine ultrastructural details, this FFF-FS method is not only adequate but also greatly reduces, if not totally eliminates, any leeching-out and redistribution of the polyamines during the preparation of the sample. For the preservation of antigenicity in situ during FS, epoxy resin was more effective than hydrophilic LR white resin, probably due to the solubility of polyamines in the latter.