Fluorescence microscopy of microtubules in cultured cells.

Cytoplasmic microtubules are noncovalent polymers of the protein tubulin. In the cells, the main function of microtubules is to provide tracks for organelle transport. Two experimental approaches based on fluorescence microscopy are commonly used to examine organization of microtubules in mammalian tissue culture cells. The first experimental approach involves indirect immunofluorescence staining of chemically fixed cells with tubulin antibody. Fluorescence microscopy of immunostained specimens allows the examination of the distribution of microtubules in the cytoplasm at the moment of fixation. The second experimental approach involves introduction of tubulin subunits covalently labeled with a fluorochrome into the cytoplasm of living cells. Time-lapse fluorescence microscopy of cells containing labeled tubulin subunits allows to examine changes in the spatial organization of microtubules in the cytoplasm and also to directly observe their behavior. In this chapter, we describe preparation of samples for fluorescence microscopy of microtubules.