15' in -20 degree 100% MeOH

1 x 10' wash in PBS

1 x 60' block in goat serum

1 x 60' primary antibody (rabbit-anti-PAR-1 in 1% BSA, 10% serum in PBS) -- optionally incubated 4 hours @ 16 degrees C

1 x 10' wash in PBS

1 x 10' wash in PBS + 0.5% Tween

1 x 10' wash in PBS

1 x 1 hr secondary antibody (1:00 to 1:200)

1 x 10' wash in PBS

1 x 10' wash in PBS + 0.5% Tween

1 x 10' wash in PBS

Reference: Guo, S. and Kemphues, K., 1995, Cell 81:611-620