Immunostaining of Cells Adherent to Coverslips

Contributed by Gordon W. Laurie, School of Medicine, University of Virginia, United States

Immunostaining of Cells Adherent to Coverslips


1L PBS-T (1000 ml) 8gm NaCl 0.2hm KH2PO4 1.15gm Na2HPO4 0.2gm KCl 1ml Tween 20 0.1gm Thimerosol


  • Immerse 18 mm2 glass coverslips in EtOH. In the tissue culture hood, individually pull out and carefully flame to sterilize.
  • Allow to cool then place in 35 mm dishes, or in 6 well plates. Coat overnight (4°C) with 3 ml/dish or well of 40 µg/ml BMS made up in ddH2O.
  • Remove coating solution and block with 1% BSA for 4 hr at 4°C.
  • Isolate cells and plate at 6 x 105 cells/ml (3 ml/well). Allow cells to adhere for 1 hr in incubator.
  • Gently pull off media containing non-adherent cells and wash once with media.
  • Pull off media and add 4% formaldehyde (25 ml of 16% ampule stock made up to 100 ml in PBS) to dishes or wells. Fix for 30 min at room temperature.
  • Remove coverslips. Those for immunostaining are placed vertically in two ceramic coverslip holders (need to borrow from Otey lab until we purchase). The remainder can be stored at -70°C.
  • Coverslips in ceramic holders are washed two times in PBS. For washes and incubations, use a 250 ml beaker and a 100 ml volume of wash or reagent. Permeabilize by immersion in PBS-Tween (‘PBS-T’; PBS containing 0.1% Tween 20) for 15 min at room temperature.
  • Wash four times over 5 min with PBS.
  • Block by immersion in PBS containing 1% BSA for 60 min at room temperature
  • Immerse one group of coverslips in preimmune sera and the other in immune sera diluted in PBS/1% BSA. Cover and incubate overnight at 4°C.
  • The next day, wash five times over 40 min in PBS.
  • Immerse in Pierce ‘peroxidase suppressor’ for 30 min at room temperature, then wash several times in PBS.
  • Wash several times in PBS.
  • Immerse in secondary peroxidase-labeled antibody diluted 1/1,000 in PBS containing 1% normal goat serum. Incubate for 60 min at room temp.
  • The next day, wash five times over 40 min in PBS.
  • Place coverslips flat on Parafilm and add Pierce ‘metal enhanced DAB’ diluted 1/10 in peroxide buffer. Allow reaction to go for 5 - 15 min, then replace in ceramic holders to wash two times in water.
  • Dehydrate in 80%, 90% and 2x 100% EtOH, then immerse in xylene (in chemical hood; all 1 min each).
  • Place cover slip cell layer down on a glass slide containing a drop of mounting medium and examine in the light microscope.

This method is based, with permission, on an original protocol available here.