Take care to keep wells hydrated at all times. Drying out will lead to non-specific staining.

  • Thaw plates and permeabilize with 0.1% Triton-X 100 in PBS
    1. Incubate at RT 10’
    2. Use about 1 ml per well if not-rocking and 0.75 ml if on the rocker for all steps
    3. Wash wells 3X with PBS
  • Block endogenous peroxidases with H2O2
    1. Make up 3% H2O2 fresh
    2. Add to wells and incubate for 10’ at RT
    3. Wash 3X with PBS (quick washes)
  • Block in PBS + 1% BSA and 2% horse serum from the host of the secondary antibody (use 20 ml per ml)
    1. Add 750 ml to 1 ml of block to each well
    2. Incubate at RT for 1 hr on a rocker
  • Bind colonies with CK8/18 primary antibody (Vector Labs CK8/18)
    1. Make up aCK8/18 at 1:750 in PBS + 1% BSA, use 1 ml per well
    2. Incubate overnight at 4()o()C, humidify by placing moist paper towels under the lids