Following a 30 min incubation with purified goat IgG (50 (μg/ml) at 25°C and three additional washes with Buffer A, cells were incubated for 60 min with E2-specific antiserum [30] at 1 μg/ml followed by three washes in Buffer A and 60 minute incubation in fluorescently labeled second antibody (1–2 μg/ml).