Cell surface and cytoskeletal antigens in cerebral cell cultures after chloroform-methanol delipidation.

Abstract

Fixation and in situ total delipidation of nerve cells in monolayer cultures have been used for immunofluorescence visualization of cellular antigens. Cells grown on poly-L-lysine-coated coverslips are exposed to a chloroform/methanol (2/1 by vol) solvent mixture for 15 min at -65 degrees C. This step results in the fixation of cells and is accompanied by the complete extraction of cellular lipids. About 3.2% of the total 35S-methionine-labeled trichloroacetic acid-insoluble cellular material is extracted into the organic solvent mixture, demonstrating that aqueous soluble proteins are not significantly extracted under these conditions. A total loss of tetanus toxin binding sites, presumably reflecting the removal of polysialogangliosides from the cell surface, is observed. The accessibility of antibodies against tubulin and against intermediate filaments after this treatment has been investigated. Visualization of these cytoskeletal elements by indirect immunofluorescence and by phase microscopy suggests that the removal of cellular lipids does not affect the apparent cytoarchitecture and that extraction with these organic solvents does not interfere with the staining of tubulin and intermediate filaments, the latter being exclusively present in nonneuronal cells. The advantage of this method of cell fixation and lipid extraction for immunofluorescence of cells in monolayer culture is discussed.