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Star Republic: Guide for Biologists

Phalloidin staining of filamentous actin

1. Fix 50 ml mid-log phase yeast cells in 4% formaldehyde. Add formadehyde directly to the medium. Incubate 10 minutes at room temperature with gentle agitation.

2. Pellet cells, resuspend in 4% formaldehyde/PBS. Continue fixing at room temperature with gentle agitation.

3. Pellet cells and resuspend in PBS. Incubate 10 minutes at room temperature with gentle agitation.

4. Repeat PBS wash.

5. Resuspend cells in 100 ml Rhodamine-phalloidin (or FITC-phalloidin) in PBS (0.5-3 m). Incubate 20-30 minutes in the dark at 4 C with gentle rocking.

You may need to dry down phalloidin and resuspend in PBS. Calcofluor can also be added at the final concentration of 0.1 mg/ml.

6. Wash cells 3 times with PBS.

7. Stain for nuclei. Or mount and view by fluorescent microscopy.