Gold labeling of microtubules in cleaved whole mounts of cortical root cells.

Abstract

A method is described for localizing microtubules using gold-labeled antibodies in combination with anti-tubulin. Cortex cells of Equisetum hyemale are broken open while still in buffer, after initially being attached to poly-L-lysine-coated grids. Thus, the cytoplasm becomes accessible to the antibodies. After application of the antibodies, the cleaved cells are post-fixed, stained, dehydrated, and critical point-dried. Different fixation procedures are compared: fixation in paraformaldehyde, in glutaraldehyde, and in glutaraldehyde followed by a sodium borohydride reduction step. All three methods result in good labeling of the microtubules, with low backgrounds. However, organization of the cytoplasm is best preserved in cells fixed in glutaraldehyde without sodium borohydride treatment. The method is highly suitable for studying the membrane-bound cytoskeleton because detergent extraction and/or embedding are avoided.