F-actin staining with phalloidin protocol

Preparation of phalloidin stain (to be done before culture fixation or during 10min period of permeabilization) 

USE GLOVES WHEN HANDLING PHALLOIDIN, it’s a poison

Purchase Rhodamine-Phalloidin from MOLECULAR PROBES®
It comes in methanol.  Store it at –20ºC (no need to aliquot it)

Methanol interferes with phalloidin’s binding to filamentous actin, so you will have to evaporate off the methanol prior to using it. To do that do the following:

Use a final dilution of 1:2
Therefore if you are going to need 40μl of solution to add to your slides, take 20μl of the phalloidin from the stock bottle in the –20ºC freezer, and quickly put it into a small GLASS test tube (they work better than plastic).  The phalloidin will try to get out of your pipette as you are doing this due to the methanol that is currently in it.

Next, evaporate off the methanol using air blowing along the inside of the test tube and using the heat from your hand on the outside of the tube. This will take 2-5 minutes usually.

When completely evaporated down, you will probably see an empty tube (you need ALL OF THE METHANOL GONE for this to work well). Keep this in the dark.

Then add your 40 μl of buffer: TPBS/BSA → this buffer consists of a PBS (1x) and Tween-20 (0.1%) solution with BSA dissolved into it (1%).
Vortex it well
Place this on ice until use

Preparation of fluorescence fixative (3% paraformaldehyde in PBS – pH 7.3)
Add 3.75g paraformaldehyde to about 75 mL dH2O in a 125 mL Erlenmeyer flask with a stirring bar.  Place on a hot plate in fume hood and carefully place thermometer into the solution.  Bring to temp. between 59-60 oC (do not let the temp. rise above 60 oC!).  Wait for 10-20min for solution to clear and if necessary finalize clearing with 0.1M NaOH added drop by drop (should only require 2-3 drops maximum).  When solution is clear, take off heat and cool to room temperature.  Add the following constituents to complete the fixative:

• 12.5mL 1.5M NaCl
• 2.5mL 0.2M Na/K PO4, pH 7.3
• 0.42mL 1.5M KCl

• Bring volume to about 100mL with dH2O and adjust to pH 7.3
• Transfer solution to a graduated cylinder and bring to final volume of 125mL with dH2O Cover top of cylinder tightly with parafilm and mix thoroughly by gently inverting several times
• Staining of keratinocytes (cultured on a 3.2cm x 1.4cm silicone membrane to a confluence of approximately 80%)

• Wash culture with PBS buffer (80µL) 2x
• Fix culture at temperature of 4oC with 45µL 3% paraformaldehyde solution for 10min
• Wash with 80µL PBS 2x
• Permeabilize cells in 4 oC using 45µL Triton X-100 (0.2%; in PBS) for 10min
• Wash with 80 µL PBS 2x
• At room temperature, add the 40 µL of phalloidin stain solution and let sit for 30 min
• Wash very, very well with PBS buffer, there is no such thing as over washing with this stuff (5 or 6 washes with 80µL volumes of PBS will suffice)
• View under fluorescent microscope with the TRITC  filter cube