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.
- 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
|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.|