Some applications of cryosubstitution in ultrastructural studies of the cell nucleus

Cryofixation followed by cryosubstitution, without the use of any chemical fixatives, was carried out on cultured mouse P815 cells. The principal aim of our work, which was to show that these techniques provide excellent morphological preservation of cellular and in particular nuclear components, was demonstrated. All nuclear structural components, nucleolar or nucleoplasmic, were clearly revealed using this technology. The cells were cryofixed by impact freezing onto a copper mirror cooled with liquid nitrogen or helium, cryosubstituted in acetone and embedded in either Lowicryl K11M or 1R White acrylic resin. Ultrathin sections were contrasted using either the usual uranyl acetate-lead citrate double staining, a differential staining for nuclear nucleoprotein structures, or the silver staining revealing nucleolar organizer regions. In view of the absence of conventional fixatives, the specimens prepared in this way would offer to be material of choice for ultrastructural identification of intra-nuclear antigens, especially those sensitive to conventional fixatives such as, for example, aldehydes. Advantages and differences of these techniques with regard to more conventional electron microscopic procedures are discussed.