Jacobs:Protocol Actin Staining

Materials
5 uL Alexa Fluor 488 Phalloidin in 200 uL Primary Blocking Solution (for each coverslip area)
 * Cells: NIH/3T3 fibroblasts seeded @1,000 to 3,000 cells/cm2 24 to 48 hrs prior to staining
 * Coverslips
 * Kimwipes
 * Aluminum Foil
 * Pipets/Pipet aid
 * Pipet tips/pipetters
 * Aspirator
 * Timer
 * Waste beaker
 * 50 mL centrifuge tube (“formaldehyde waste”)
 * Markers
 * Gloves
 * Phosphate-buffered saline (PBS), pH 7.4
 * 3.7 % Formaldehyde in PBS (use methanol-free formaldehyde)
 * 0.1 % Triton X-100 solution (in PBS)
 * PAP pen (Sigma Aldrich Cat # Z377821)
 * Vectashield mounting media with DAPI – (Vector Laboratories Cat # H-1200)
 * Clear fingernail polish
 * Primary Blocking Solution: PBS + 1% (w/v) BSA + 0.1% (v/v) Nonidet P40
 * Alexa Fluor 488 Phalloidin Solution: protect from light – (Invitrogen Cat # A12379)

Procedure

 * 1) Wash cells 2X with PBS (10 mL per wash) – remove all PBS from around slide after final wash
 * 2) Fix cells: apply 2 mL of 3.7% formaldehyde solution on top of slide for 10 min. @ room temp. Note: formaldehyde is toxic; avoid contact with skin, eyes, etc.; dispose of as hazardous waste, do not pour down sink
 * 3) Wash cells 2X with PBS (10mL per wash) – place waste in the “formaldehyde waste” tube
 * 4) Permeablize cells: apply 2 mL of 0.1% Triton X-100 solution on top of slide for 5 min. @ room temp.
 * 5) Wash cells 2X with PBS (10 mL per wash)
 * 6) Make a PAP pen region the on the slide – see instructions/pattern below
 * 7) Wash coverslip area 1X with PBS (~200 uL should cover area)
 * 8) Actin staining: apply 200 uL of Alexa Fluor 488 Phalloidin Solution for 20 min. @ room temp (Protect from light: for all subsequent steps place aluminum foil over culture dish to prevent photobleaching of the Alexa Fluor 488)
 * 9) Remove the Phalloidin Solution and wash the coverslip area 3X with Primary Blocking Solution
 * 10) Remove the Blocking Solution and add Vectashield mounting medium (25uL per coverslip area)
 * 11) Place a coverslip over the stained area and seal the coverslip by pipetting a bead of clear fingernail polish along the edge of the coverslip
 * 12) Allow the nail polish to dry before viewing on a fluorescent microscope.
 * 13) Remove the slide from the culture dish and dry the bottom of the slide with a Kimwipe
 * 14) Place the slide on the pattern
 * 15) Use a Kimwipe to wipe dry the area in gray (do not disturb the white coverslip area you will be staining)
 * 16) Apply the PAP pen along the black square around the coverslip area
 * 17) Dry culture dish, and place slide back in the dish

Contact

 * History: CMBL – CRJ/JJR, last updated 8/1/07

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