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This protocol provides general instructions for labeling F-actin in fixed and permeabilized cells. Actin is one of the most abundant proteins found in cells and can be labeled with a fluorophore very easily when the monomers form a certain type of microfilament, referred to as F-actin. When phalloidin, a molecule that binds F-actin, is conjugated to a fluorophore, it becomes a very useful tool for cell biologists who are using fluorescence microscopy to study their cells. Labeling F-actin can help show the overall shape and structure of the cell and provide context for other fluorescent labels.

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Protocol tip
If you’re using fluorescent phalloidin to visualize actin in your experiment, you’ll get better results if you perm for 20 minutes instead of 15.

What you need

  • Cells that have been fixed and permeabilized (See Fix, Perm, and Block protocol)
  • Phosphate-buffered saline (PBS)
  • Phalloidin conjugated to fluorophore
  • Fluorescence microscope with filter set matched to your fluorophore

F-actin dyes—basic labeling protocol

  • For fixed and permeabilized cells
  • Can be used before or after immunolabeling
  • The volumes given in this protocol are good for a single well in a 6-well vessel or a single 35 mm vessel

1 Prepare 1 mL F-actin staining solution in PBS from conjugated fluorophore. Phalloidins are typically very water soluble and stain F-actin at 100–200 nM concentrations. If you are optimizing for dye concentration, you will need to prepare a staining solution for each concentration you want to test.
2 Remove PBS from fixed and permeabilized cells.
3 Add 1 mL of staining solution.
4 Incubate for 20 minutes at room temperature.
5 Remove dye solution.
6 Wash 3 times with PBS. Learn how to wash 
7 Image cells.
8 Optional: To preserve your sample for long-term storage, you can also mount your sample in mounting medium. Before you start your staining experiment, it’s a good idea to check that your fluorophores are compatible with the mounting medium you want to use.