An alternative fixation-processing method for preembedding ultrastructural immunocytochemistry of cytoplasmic antigens: the GBS (glutaraldehyde-borohydride-saponin) procedure.
The use and properties of an alternative fixation procedure for ultrastructural cytochemistry is described that utilized primary fixation of cells with glutaraldehyde with considerable latitude in concentration and time of fixation. This was followed by treatment with sodium borohydride (as described by Weber, Rathke and Osborn (Proc Natl Acad Sci USA 75:1820, 1978)), which increases the accessibility of some cytoplasmic compartments to antibodies, apparently through the reduction of the Schiff bases induced by glutaraldehyde. Through the use of saponin membrane permeabilization, this primary fixation method allows good ultrastructural preservation with accessibility of most, but not all, cytoplasmic structures. Localization of tubulin, clathrin, alpha2-macroglobulin present in lysosomes, vesicular stomatitis virus (VSV) G protein, and myosin are demonstrated. This technique failed to expose the antigenic structure of actin in microfilament bundles or intranuclear antigens. This method should be useful for good preservation of ultrastructure and antibody localization of some, but not all, protein antigens in the cytoplasm of cultured cells, as well as in intact tissue. This technique is especially useful for cytosolic antigens in cultured cells.
D000447:Aldehydes; D000818:Animals; D000941:Antigens / Q000276:immunology; D001894:Borohydrides; D002463:Cell Membrane Permeability; D003593:Cytoplasm / Q000276:immunology* / Q000648:ultrastructure; D005404:Fixatives; D005976:Glutaral; D006651:Histocytochemistry / Q000379:methods; D007120:Immunochemistry / Q000379:methods; D011817:Rabbits / Q000276:immunology; D012503:Saponins
D000447:Aldehydes; D000941:Antigens; D001894:Borohydrides; D005404:Fixatives; D012503:Saponins; D005976:Glutaral