Visualization of dimethyl sulphoxide-stabilized tubulin containing structures by fluorescence staining with monoclonal anti-tubulin antibodies.

Abstract

Transmission electron microscopy of numerous tissues and cells has clearly documented the existence of three fibrillar systems characteristic of most, if not all, eukaryotic cells: actin-containing microfilaments, intermediate filaments and microtubules. These structures are assumed to be involved in a variety of cell functions, including the determination of cell morphology, locomotion, and intracellular movements (Dustin 1978). The production of specific antibodies to the microtubule protein, tubulin, has made it possible to identify microtubles in cultured cells or tissue sections by fluorescence immunocytochemistry. This procedure is applicable to a large number of cell types where in spite of their small size, individual microtubules can be detected and the images produced correspond to those seen by transmission electron microscopy (Weber & Osborn et al., 1979). In the case of tubulin, the antigen exists in at least two forms: a soluble 6S tubulin dimer and a polymerized microtubular form. Although antibodies to tubulin are generally directed against the 6S dimer in denaturated or native form, they fortunately bind both to dimer and polymer within the cells (Brinkley et al., 1980). The basic method for the immunocytochemical localization of microtubules consists of fixation followed by treatment with organic solvents at low temperature and the application of the antibody (Weber & Osborn, 1979). For tubulin fixation, cold methanol, formaldehyde, glutaraldehyde, and simple air-drying followed by cold acetone, were used (Brinkley et al., 1980). Improvement in the visualization of cytoplasmic microtubules can be achieved in some cells by treatment with mild detergents in the presence of microtubule stabilizing sulphonium salts prior to fixation (Himes et al., 1977).Dimethylsulphoxide (DMSO) is used for several purposes in cell biology studies apart from its application as a cryoprotectant. The effect of DMSO on microtubules was only studied in vitro. It was found that different concentrations of DMSO are necessary for nucleation and propagation of microtubules. The optimal concentration for the formation of microtubules is between 8 and 10% (Robinson & Engelborghs, 1982). A line of human lung fibroblasts and Tetrahymena piriformis cells were used in the