Procedure:

1) 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.

2)   Remove coating solution and block with 1 BSA for 4 hr at 4°C.

3) 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.

4) 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.

5)   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.

6) 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.

7)   The next day, wash five times over 40 min in PBS.   At this time, can immerse in Pierce ‘peroxidase suppressor’ for 30 min at room temperature, then 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.  Wash five times over 40 min in PBS.

5)   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.

Reagent:

PBS-T   (1000 ml)

NaCl                     8 gm

KH2PO4                                 0.2 gm

Na2HPO4                              1.15 gm

KCl                      0.2 gm

Tween 20             1 ml

Thimerosol           0.1 gm