Conjugation: AF594

Platform: Ex/Em=590/617 nm

Sample Types: Formaldehyde-fixed, permeabilized tissue sections, cell cultures or cellfree experiments

Validation Methods

Immunocytochemistry (IC), Immunofluorescence-Tissue (IF-Tissue)

Storage

This product is lyophilized powder. Stored at -20°C and protect from light for 1 year.

Shipping

Shipped with gel ice packs. Immediately store the product in a standard freezer at -20°C upon receipt.

Staining fixed cells:

The following protocol describes the staining procedure for adherent cells grown on glass coverslips or 8-well chamber slides. Phalloidins also can be used to stain fixed frozen or paraffin tissue sections, as well as yeast and fungi.

1. Wash cells 3 times with PBS.

2. Fix cells on ice with 4% formaldehyde solution in PBS for 15 minutes.

Note:  Methanol can disrupt actin during the fixation process. Therefore, it is best to avoid any methanol containing fixatives or other solvent-based fixatives. The preferred fixative is methanol-free formaldehyde.

3. Wash cells 3 times with PBS.

4. Permeabilize cells with 0.5% Triton X-100 in PBS at room temperature for 10 minutes.

5. Wash cells 3 times with PBS.

6. Dilute 1-5 μl fluorescent phalloidin stock solution in 200 μl PBS for each cover slip or chamber to be stained. Place the staining solution on the coverslip for 20 minutes at room temperature.

Note：Staining volume can be adjusted according to the sample. To avoid evaporation, keep the coverslips inside a covered container and the chamber slides covered during the incubation.

7. Wash 2-3 times with PBS.

8. Image using fluorescence microscopy.

Note：Fluorescent phalloidins are photostable enough to image in PBS, but for best results we recommend mounting with antifade mounting medium. Staining living cells Fluorescently-labeled phalloidin is not cell-permeant and has therefore has not been used extensively with living cells. However, living cells have been labeled by pinocytosis or unknown mechanism. In general, a larger amount of stain will be needed for staining living cells. Alternatively, fluorescent phalloidins have also been injected into cells for monitoring actin distribution and cell motility.