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Some applications of cryosubstitution in ultrastructural studies of the cell nucleus
Author(s) -
Schack MarieLouise,
Fakan Stanislav,
Villiger Werner
Publication year - 1991
Publication title -
biology of the cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.543
H-Index - 85
eISSN - 1768-322X
pISSN - 0248-4900
DOI - 10.1016/0248-4900(91)90085-2
Subject(s) - uranyl acetate , biology , ultrastructure , osmium tetroxide , staining , nucleus , cryofixation , biophysics , nucleoprotein , cell nucleus , nucleolus organizer region , silver stain , nucleolus , microbiology and biotechnology , electron microscope , biochemistry , anatomy , genetics , gene , optics , physics
Summary— 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 miror 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 revealling 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.