Techniques for Staining Specific Cellular Structures

Microtubules

Microtubules are highly dynamic structures within the cell, and can be very difficult to preserve satisfactorily. Fixation in ice-cold methanol for 10 minutes works well, but distorts their three-dimensional structure. Crosslinking fixatives are, therefore, better suited for confocal work. Formaldehyde, however, does not preserve microtubules well and so glutaraldehyde is the best choice. It is important to warm the fixative to the physiological temperature of the cells (i.e. 37°C for mammalian cells) as microtubules spontaneously disassemble at lower temperatures. Microtubules are also picky about the buffer used for the fixative - they are best preserved in PIPES, pH 6.9 or in imidazole. Following fixation, I have found that permeabilization with 0.5% SDS for 10 minutes consistently yields the best results in terms of uniformity of staining as well as decreased background fluorescence. Numerous tubulin-specific antibodies are available commercially, the best of them being DM1a, a mouse monoclonal antibody. Microtubule preservation may be evaluated by examining their ends located in the cell periphery. In a good fixation, they should appear as long continuous filaments, not as beaded, broken segments. In addition, the astral microtubules of mitotic spindles should be present and distinct.

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