Techniques for Staining Specific Cellular Structures

Actin

Filamentous actin may be efficiently labeled using fluorescently-conjugated phallotoxins - bicyclic peptides isolated from the poisonous mushroom Amanita. The most commonly used member of this family, phalloidin, may be purchased conjugated to a wide variety of fluorescent dyes. Phalloidin selectively binds and stabilizes polymerized, filamentous actin without binding monomeric actin and its non-specific staining is negligible. These properties make phalloidin more attractive than actin-specific antibodies for fluorescence microscopy. Furthermore, phalloidin binds to actin from different species (including plant, animal, and fungal cells) and does not discriminate between different actin isoforms.

Phalloidin is usually purchased as a lyophilized powder and is reconstituted in methanol or DMSO. I find it convenient to prepare it as a 1000X stock of 3 µM in small aliquots and store it below 0°C. For staining, it should be diluted immediately prior to staining into PBS or whatever buffer is being used, and may be included with fluorescent secondaries for double-label experiments. Phalloidin is cell-impermeable, so specimens to be stained must be fixed and detergent-permeabilized. Cells to be stained should be fixed either with aldehydes or cold acetone, but NOT methanol. Methanol fixation destroys the phalloidin-binding site on actin, thereby eliminating staining. For this reason, many people advise that phalloidin be dried down in a vacuum microfuge, then resuspended in buffer - but I have not found this additional step to be necessary. Permeabilization with SDS also eliminates phalloidin binding, so Triton X-100 should be used for permeabilization. Phalloidin-stained specimens should be imaged within a few days of staining as it will dissociate into the mounting medium over time and produce a background autofluorescence that may obscure fine detail.

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