Biotium, Inc.
Phalloidin Rhodamine conjugate
00027
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Fluorescent dye conjugated phalloidin is a convenient reagent to study actin polymerization and f-actin formation in any cultured fixed cells. This reagent is not suitable for live cell imaging unless delivered in to cell.
Flurescent imaging of f-actin in cultured cells
Human aortic smooth muscle cells and phalloidin-rhodamine conjugate on a glass cover slip
Culture cells on a glass cover slip to a desired confluence. Remove medium, wash cells once with 1x warm PBS and fix with 3% paraformaldehyde solution for 10 min at 37C. Wash three times with 1x PBS and permeabilize with 0.1% -0.5% Triton X-100 in 3% BSA solution prepared in 1x PBS at RT for 5 min. If you use other permeabilizing agents, optimization is necessary. Wash 3 times with 1x PBS and place the cover slip in an humidified chamber. Prepare 100 U/mL phalloidin solution in methanol stock solution. 10 uL of stock solution in 200 uL PBS is required to stain 18x18 mm coverslips. Incubate cells with phalloidin for 10-15 min at RT and wash cells with PBS three times. If you have to perform immunofluorescence staining, you can perform f-actin labeling after secondary antibodies. Mount the coverslip on a microscope slide with an antifade mounting solution.
It is very essential to permeabilize cells sufficiently. If you use saponin, longer time is necessary.
Fluorescent fiber like structures can be seen as a result of conversion of g-actin to f-actin.
23737530
N/A
Simple and dependable method for imaging f-actin
None
Stains f-actin with high specificity. Much better than using actin-specific antibodies.