Electron microscope demonstration of tubulin in cilia and basal bodies of rat tracheal epithelium by the use of an antitubulin antibody.

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Abstract
It was previously demonstrated that cytoplasmic microtubules and the microtubules of cilia, flagella and sperm tail contain tubulin. Although the morphology of cytoplasmic microtubules and that of axonemes differs in cells from which they were isolated, the tubulin of the 2 structures shares physical and chemical properties. In some mammalian tissues, such as tracheal epithelium, cilia and basal bodies are difficult to isolate and characterize. The use of an enzyme-labeled immunoglobulin probe would facilitate identification and in situ localization of such proteins. Tubulin prepared from porcine brain by ion-exchange chromatography and from rat brain by the method of cyclic polymerization and depolymerization, with subsequent disk gel electrophoresis with SDS [sodium dodecyl sulfate], were injected i.v. into rabbits. The animals were intermittently bled and the antisera extracted. The specificity of the antisera was proved by indirect immunofluorescence staining of the mitotic spindle, specific blocking of spindle staining by purified tubulin and not by other proteins, staining of 3T3 [mouse fibroblast] cytoplasmic microtubules, single line on immunoelectrophoresis, failure of control antisera to show any of these, and precipitation of antibody with all tubulin preparations and not with actin. EM of ciliated cells of the tracheal epithelium (stained with antitubulin by the indirect enzyme-labeled antibody method) showed that the basal bodies, outer doublets and central pair of the cilia contain tubulin. This indicates that tubulin in microtubules of cilia and basal bodies of rat tracheal epithelium is antigenically similar to tubulin extracted from cytoplasmic neurotubules of brains from the same species and from a different mammalian species. No other axonemal strucutures stained with the antitubulin. Three different preparations of tubulin from pigs and rats were used to immunize rabbits. All elicited similar antisera which gave identical staining patterns. The specificity of the staining was demonstrated by the absence of staining with immune serum absorbed with purified tubulin, the absence of staining with preimmune serum, and the absence of staining if any of the reagents were omitted during the staining reaction.

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