Rhodamine Phalloidin 

Phalloidin is a mushroom-derived toxin which can be used to specifically label F-actins (actin filaments) of the cytoskeleton. It binds to all variants of actin filaments in many different species of animals and plants. Typically, Phalloidin is used conjugated to a fluorescent dye. Phalloidin binds F-actins with high selectivity while Rhodamine provides stable and bright orange fluorescence.

Rhodamine Phalloidin is 1000× stock solution in DMSO which is convenient for labeling, identifying and quantitating F-actins in formaldehyde-fixed and permeabilized tissue sections, cell cultures or cell-free experiments.

The solutions should be stable for at least 6 months if store at -20°C.

Protecting from light, and avoid freeze/thaw cycles.

1. Prepare 1× Rhodamine Phalloidin working solution:

Add 1 μL of 1000× Rhodamine Phalloidin DMSO solution to 1 mL of PBS containing with 1% BSA.

Note: a. Different cell types might be stained differently. The concentration of AMCA Phalloidin working solution should be prepared accordingly.

b. 1% bovine serum albumin (BSA) is used to reduce nonspecific background staining.

2. Stain the cells:

2.1 Wash cells 2-3 times with PBS. Fix the cells in 3.7% methanol-free formaldehyde solution in PBS for 10-30 minutes at room temperature.

Note: Avoid any methanol containing fixatives since methanol can disrupt actin during the fixation process. The preferred fixative is methanol-free formaldehyde.

2.2 Wash the fixed cells 2-3 times in PBS.

2.3 Add 0.1% Triton X-100 in PBS into fixed cells for 3-5 minutes to increase permeability. Wash the cells 2-3 times in PBS.

2.4 Add 100 μL/well (96-well plate) of AMCA Phalloidin working solution into the fixed cells, and stain the cells at room temperature for 20 to 90 minutes.

2.5 Rinse cells gently with PBS 2-3 times to remove excess Rhodamine Phalloidin.

2.6 Run fluorescence microscope or other equipments at desired Ex/Em wavelengths (AMCA Phalloidin, Ex/Em=344/432 nm).