Protocol for Phalloidin-TRITC

This is intended as a guide only - optimization may be needed.

Phalloidin is a phallotoxin isolated from Amanita phalloides, the death cap mushroom. Fluorescent phallotoxins stain F-actin at nanomolar concentrations. They are water soluble and produce low non-specific binding.

Reconstitution

Dissolve the vial contents in 1.5 mL methanol or DMSO. This will yield a solution with a final concentration of approximately 7.3 μM.

This stock solution can be further diluted as required for the intended application, we recommend a dilution of approximately 40X.

Storage

Stock solutions can be frozen for one year when stored at ≤–20°C, desiccated, and protected from light.

Formaldehyde-Fixed Cells

  1. 2X Wash cells with warmed phosphate-buffered saline (PBS), pH 7.4.
  2. Use 3.7% formaldehyde solution (in PBS) to fix cells. Leave for 10 minutes at room temperature (recommend using methanol-free formaldehyde).
  3. 2X Wash cells with PBS.
  4. Place each coverslip in a glass petri dish and extract it with a solution of acetone at ≤–20°C or 0.1% Triton X-100 in PBS. Leave for ~ 5 minutes.
  5. 2X Wash cells with PBS.
  6. When staining, dilute 5 μL Phalloidin-FITC methanolic stock solution into 200 μL PBS for each coverslip. To reduce nonspecific background staining add 1% bovine serum albumin (BSA). Place inside container and eave for 20 minutes.
  7. 2X Wash cells with PBS.

Fixation, Permeabilization, and Phallotoxin-FITC Staining

Prepare a 1 mL solution containing 50 to 100 μg/mL lysopalmitoylphosphatidylcholine and 3.7% formaldehyde. Add approximately 50 μL of Phalloidin-FITC methanolic stock solution Apply staining solution to cells and leave for 20 minutes at 4°C. 3X Wash cells with PBS. View coverslip.

Live cells

Phallotoxins are usually not cell-permeable, but it is possible to label some types of cells. Pinocytosis, microinjections and unknown entry methods by hepatocytes have all been documented. Please conduct a literature search to find an appropriate protocol for your experiments.