Immunofluorescent Staining of Cells

 

  1. Grow cells on cover slips (make sure they are glass because plastic will interfere with the fluorescence).
  2. At desired confluence or after treatment, remove medium and wash cells 3X with PBS (room temperature).
  3. Add 4% paraformaldehyde in PBS ~ you can also use ISH fix w/o glutaraldehyde ~ and leave on cells for 10 min.  You can also fix in methanol for 10 min.
  4. Remove fix and wash cells 3X in PBS.  Add PBS to store at 4 C until further processing.
  5. Permeabilize cells for 10 min with 0.1% Triton-0.1% deoxycholate in PBS.  You can also use 0.05% saponin in PBS for 10 min.
  6. Wash with PBS 2X.
  7. Incubate fixed, permeabilized cells with filtered 1 - 5% BSA in PBS for 10 min to block.  If you have high background use 5%.
  8. Prepare primary antibody solutions in filtered 1% BSA/PBS.  Cut parafilm into squares and fold up the edges.  Place in a humid chamber.  Add 150-200ul of antibody solution onto the parafilm.  Invert the cover slip onto the drop.  Cover chamber and place at 4 C overnight.

 

For rhodamine-labelled phalloidin ~ incubate with 100X diluted for 1 hr at RT in the dark.

 

Day 2.

 

  1. Wash cells with PBS 3X.
  2. Prepare second antibody in appropriate buffer at appropriate dilutions.  Start at the recommended dilution and go from there).  Remember that secondary antibodies are labeled with fluorescence which is very sensitive to light.  Do reactions in the dark or with minimal light.
  3. Add drops of secondary antibody onto clean squares of parafilm as before.  And incubate for 1 hr.  ** Note:  phalloidin does not take a secondary antibody!
  4. Wash cells with PBS 3X.
  5. Put drops of VECTASHIELD Mounting medium (from Vector Labs) onto glass slide.  This is a special mounting medium that preserves fluorescence.  Carefully place glass cover slips with cells onto the drop.  DO NOT MOVE THE COVER SLIP TOO MUCH.  CELLS WILL COME OFF.  Remove excess mounting medium with a Kim wipe or pipette.  Permanently seal cover slip to the slide with nail polish.  This is because the aqueous mounting medium does not harden and the cover slips will slide off.
  6. Store the slides at 4 C in the dark.