Home / Resources / Protocols / Protocols for Phalloidin Dyes / Protocol for Phalloidin-TRITC

Protocol for Phalloidin-TRITC

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

In Brief Download the PDF of this protocol

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. For more information on preparing stock solutions, dilutions and reconstitution please see our Molarity Calculator, Dilution Calculator and Reconstitution Calculator.

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 leave 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.