Actin staining shows you the shape and structure of the cell
Actin is one of the most abundant proteins found in cells, and actin filaments can be readily labeled using fluorescent forms of a naturally occurring protein called phalloidin.
Learn about labeled phalloidin and its utility in imaging studies to provide context for other labeled targets.
Actin microfilament labeling in fixed and permeabilized cells
Actin, a protein found in every eukaryotic cell, exists as both monomers and polymers and can be found in many different tertiary forms. Actin plays a variety of roles in cytoskeletal structural support, cell motility, cellular junctions, muscle contraction, and cellular signaling. Given its wide-ranging and diverse roles, it’s not surprising that it is one of the most abundant proteins found in cells. Actin can be labeled with a fluorophore very easily when the monomers form a certain type of microfilament, referred to as F-actin. Phalloidin, a molecule isolated from death cap mushrooms, binds F-actin microfilaments very tightly and with high specificity and—unlike many primary antibodies against actin—does not bind other forms of actin. When phalloidin 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.
Figure 1. Space filling model of phalloidin conjugated to a fluorophore.
Phalloidin conjugates for actin staining
Actin staining with fluorescent phalloidin conjugates can be easily added to an immunolabeling protocol. You can perform immunolabeling either before or after actin staining.
|Figure 2. After fixing, permeabilizing, and blocking, BPAE cells were labeled with ActinRed™ reagent (TRITC-conjugated phalloidin that labels F-actin), and nuclei were labeled with NucBlue™ Fixed reagent (a form of DAPI).|
When you use a fluorescent dye for the first time, it can be useful to test a range of concentrations on your cells. This will help you determine the best concentration to use with your cells under your experimental conditions. In general, it is best to use the highest labeled actin concentration that gives the lowest background so you can get the best signal:background ratio.
If you have trouble visualizing F-actin using a phalloidin-conjugated fluorophore, try optimizing your sample fixation time. You may get better labeling by increasing your fixation time by 5–10 minutes.