Immunocytochemical visualization of microtubules and tubulin at the light- and electron-microscopic level

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Abstract
An antibody against rat brain tubulin was purified by affinity chromatography and used for the immunocytochemical staining of tubulin-containing structures in tissue-cultured cells at the light- and electron-microscopic level. For this purpose the unlabelled antibody enzyme method (PAP method) was used. Tissue-cultured cells were fixed with glutaraldehyde or formaldehyde, postfixed with acetone and stained with the PAP procedure. With the light microscope, a dense network of darkly stained fibres was visible in non-dividing cells. The fibres converged towards a point in the nuclear vicinity and radiated throughout the cytoplasm towards the cell periphery. Mitotic spindles were also heavily stained in all stages. The cytoplasm of non-dividing and dividing cells contained a variable amount of diffuse staining. The nucleus was unstained. The network could be destroyed by treating the cells with microtubule inhibitors. An intense diffuse staining was visible in cells treated with colchicine or nocodazole. The paracrystalline tubulin precipitates that appeared in cells treated with vinblastine were heavily stained. Preliminary ultrastructural observations were made on sections through cells that were fixed with glutaraldehyde, stained with the PAP procedure and embedded in Epon. The microtubules were seen to be covered with stained PAP complexes. The cortical microfilaments, the 10–nm filaments and the plasma membrane were unstained, as were the contents of nuclei, mitochondria, endoplasmic reticulum, Golgi elements, lysosomes and vacuoles. A variable amount of diffuse staining was visible in the cytoplasm and the membranes of several organelles often showed PAP deposits. This staining was presumed to localize tubulin in non-microtubular form. It is probable that at least part of this staining was immunologically specific, since no PAP complexes were seen in the cells when the antitubulin antibody was omitted from the procedure or when it was replaced by an unrelated antibody solution.