Immunoperoxidase visualisation of microtubules and microtubular proteins

Abstract

MICROTUBULES are ubiquitous eukaryotic organelles, formed by polymerised tubulin and presumably some additional proteins, which are involved in a wide variety of cell functions (for review see refs 1 and 2). Evidence has been presented for the existence of a dynamic equilibrium between polymerised and non-polymerised microtubular proteins³. Recently immunofluorescence of cultured cells demonstrated a fine cytoplasmic network that was believed to correspond to cytoplasmic microtubules. However, no direct evidence was presented that the antibodies stain individual microtubules, the formaldehyde fixation that was used is not known to preserve microtubules intact and immunofluorescence does facilitate comparison of the total picture of the microtubular apparatus with its appearance in histological and ultra-structural sections (horizontally or vertically) through the same cell. Moreover, the inadequate resolution of the light microscope does not allow an accurate localisation of the non-polymerised tubulin and the study of its interaction with various cellular organelles. We have therefore developed an immunochemical technique described here that could simultaneously be used for the localisation of microtubules and tubulin in whole cells and in histological and ultrastructural sections, after adequate fixation with glutaraldehyde.