Microtubule distribution in cultured cells and intact tissues: improved immunolabeling resolution through the use of reversible embedment cytochemistry

Abstract

To investigate the detailed distributions of microtubules in cultured cells and intact tissues we developed a reversible embedment method for antibody labeling of sectioned material. Fixed tissues were infiltrated with fully polymerized polymethylmethacrylate dissolved in an organic solvent. Evaporation of the solvent left the tissue embedded in hard plastic. After sectioning by conventional methods, the plastic was extracted and sections were processed for indirect immunofluorescence to label microtubules. Clear images of microtubules were observed in sections of cultured epithelial cells, intact chick intestinal epithelium, and dividing sea urchin eggs. Microtubules in the differentiated epithelium of the chick intestine generally paralleled the long axis of the cells and did not focus on a microtubule-organizing center. Mitotic cells of the intestinal epithelium appeared similar to the mitotic cells of epithelial lines in culture. In sections of dividing sea urchin eggs detailed images of spindle and astral fibers were revealed. Immunoelectron microscopic labeling for tubulin was performed on sections of Pt K1 cells using secondary antibodies adsorbed to 20-nm gold particles. Semi-thick sections viewed by high-voltage electron microscopy showed both the overall distribution of microtubules and their detailed interactions with other cellular organelles. Mitochondria were often aligned along labeled microtubules. Reversible embedment cytochemistry should provide a general method for high resolution labeling of cells and tissues with affinity probes.