Immunofluorescence for human c-Met in HeLa cells

PFA-fixation, Triton permeabilisation after fixation

PFA stocks in the -20°C freezer are 3% PFA in PBS. Dilute 16% PFA stock from TAAB in PBS.
  1. Three rinses with PBS
  2. 3%PFA in PBS for 15min
  3. Two rinses with PBS
  4. 50mM ammonium chloride in PBS for 20min
  5. Rinse three times with PBS. Coverslips may be stored over night.
  6. Before starting with the IF protocol, permeabilise 3-5 min with

    7. 0.2% Triton/PBS

  7. block with 10% goat serum/PBS 30 min
  8. 1st Antibody in 5% goat serum/PBS 20 min

    9. Anti-human c-Met rabbit polyclonal antibody from Santa Cruz (#sc-10)

    diluted at 1:200 in 5% goat serum

  9. wash with PBS 3x5min
  10. 2nd Antibody 20 min

OregonGreen488 labelled anti-rabbit antibody from Molecular Probes diluted at 1:1000 in 5% goat serum/PBS
  1. wash with PBS 3x5min
  2. rinse with PBS, dip into PBS, then millipore, remove excess water by

    3. dipping onto filterpaper and mount on ~50 µl moviol on slides.

  3. remove excess water with filter paper and let the slides dry for at

least a few hours.

14) Long term storage in the dark and in the cold.