AMCA 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 AMCA provides stable and bright blue fluorescence.

AMCA 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× AMCA Phalloidin working solution:

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

Note: 1. Different cell types might be stained differently. The concentration of AMCA Phalloidin working solution should be prepared accordingly.

          2. 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   Optional: 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 AMCA Phalloidin.

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