A simple fixation procedure for immunofluorescent detection of different cytoskeletal components within the same cell

In recent studies on the cytoskeletal organization of T51B rat liver cells by indirect immunofluorescence microscopy, we have been unable to achieve double-staining of microtubules and intermediate filaments within the same cell. In acetone-fixed cells, microtubules were poorly preserved, and two out of three monoclonal antibodies tested did not stain them properly. In formaldehyde-fixed cells, the monoclonal anti-cytokeratin produced an incomplete staining pattern against a diffuse background. We have now developed a fixation protocol which includes simultaneous fixation and extraction with formaldehyde and nonionic detergent in the present of microtubule stabilization buffer. Although developed for a specific purpose, it is of general application as it yields excellent preservation of all cytoskeletal components tested so far, without masking antigenic determinants. The procedure is both simple and fast and will, therefore, be valuable for efficient processing of samples from large-scale experiments, such as the screening for cytoskeletal changes during longterm treatment of cells with drugs or carcinogens.