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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.
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. |
For Research Use Only. Not for use in diagnostic procedures.