Pelling:Protocols/Triple Stain

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This is a very general triple stain guideline with immunochemistry for Actin, Microtubules and DNA

Reagents Needed

Buffers 
Wash Buffer, Paraformaldehyde, Triton X-100, PBS (Room Temp and 4°C).
DABCO Mounting Medium 
DABCO is found in the fridge and may have formed a gel. Good idea to keep 5-10 mL in a 15mL falcon tube which can be placed in the water bath during the staining procedure. This should help liquefy the DABCO by the the time you need it in the last steps.
Cells 
Assuming cells are low density and grown in a 35mm dish or one well of a 6-well plate – scale up volumes as necessary.

STEP 1 – Fixing and Permeabilizing

  1. Warm 25mL PBS, 2mL Paraformaldehyde, 2mL Triton-X, 20mL Wash Buffer to 37°C in water bath for 15 mins.
  2. Place dish/plate on gel pack that is warmed to 37°C.
  3. Discard media into waste beaker
  4. Wash and discard with warm PBS 3x.
  5. 2mL of warm paraformaldehyde for 10min.
  6. Wash and discard with warm PBS 3x. OPTIONAL - Store in Wash Buffer in fridge for later use or go to step 7.
  7. 2mL of warm TX for 3min.
  8. Wash and discard with warm Wash Buffer 2x.
  9. 2mL of warm Wash Buffer for 15min.

STEP 2 – Actin Staining with Phalloidin AlexaFluor-XYZ (XYZ = 488 or 546)

  1. Make 200μL of Phalloidin-AlexaFluor at a concentration of 1:100 in room temp Wash Buffer in a microcentrifuge tube.
  2. Discard Wash Buffer from cells.
  3. Gently add Phalloidin-Alexa to cells and incubate in the dark for 20min.
  4. Wash and discard with warm Wash Buffer 2x.
  5. 2mL of warm Wash Buffer for 15min.

STEP 3 – Microtubule Staining with Primary and Secondary Antibodies

  1. EVERYTHING from now on must be done on ice at 0-4°C. Get a bucket of ice.
  2. Make up 200μL of monoclonal mouse alpha-tubulin primary antibody at a concentration of 1:200 in cold Wash Buffer in a microcentrifuge tube and store on ice.
  3. Discard wash buffer from cells.
  4. Wash and discard with cold PBS from the fridge 3x.
  5. Gently add primary antibody to cells and incubate in the dark on ice for 15min (some antibodies require 30min or even overnight in fridge).
  6. Wash and discard with cold Wash Buffer 2x.
  7. 2mL of cold Wash Buffer for 15min.
  8. Make up 200μL of rabbit-anti-mouse secondary antibody conjugated to AlexaFluor-XYZ at a concentration of 1:200 in cold Wash Buffer in a microcentrifuge tube and store on ice.
  9. Discard wash buffer from cells.
  10. Gently add secondary antibody to cells and incubate in the dark on ice for 15min (some secondary antibodies require 30min).
  11. Wash and discard with cold Wash Buffer 2x.
  12. 2mL of cold Wash Buffer for 15min.

STEP 4 – Staining Nuclei with DAPI

  1. Make up 200μL of DAPI at a concentration of 1:500 in cold PBS in a microcentrifuge tube and store on ice.
  2. Wash and discard with cold PBS from the fridge 3x.
  3. Gently add DAPI to cells and incubate in the dark on ice for 10min.
  4. Wash and discard with cold PBS from the fridge 2x.
  5. 2mL of cold PBS on ice until you are ready to mount.

STEP 5 – Mounting Cells

  1. Discard PBS.
  2. Add 10-50μL of DABCO antifade reagent to the middle of the coverslip. Avoid Bubbles!
  3. With tweezers pick up a glass coverslip and gently place the coverslip down onto the culture dish.
  4. Work quickly to avoid letting the cells dry out too much.
  5. Put the lid back on the dish and parafilm together to prevent too much dehydration. Store the sample in the fridge, in the dark overnight, and image within ~ 1 week.