Cells in growth media were washed in 10mM Tris, either growing or mating cells were spun down, aspirate Tris and leave cells in 0.5mls Tris. The cells were fixed by adding 2mls 1% paraformaldehyde/PHEM for final concentration of 0.8% for 10min. room temp., washed 1x in PBS, dropped onto coverslips and dried.  After drying, coverslips were soaked in PBS/0.3% Tween for 20min at room temp.  Primary antibody was diluted 1:600 in PBS. coverslip was inverted onto 100ul of PBS/antibody in a weigh-boat and incubated at 4C overnight in a moist chamber.

After overnight incubation coverslips were washed by setting in PBS/0.3% tween for 15min. at room temp. Secondary FITC anti-rabbit antibody was diluted 1:100 in PBS and incubated for one hour at room temp in the dark by inverting coverslip onto 100ul PBS/antibody. Coverslips were washed by setting in PBS/0.3%tween 10 min, room temp.; PBS/0.3%tween/Dapi (0.1mg/ml diluted 1:1000) 10min room temp; PBS/0.3%tween 5 min room temp and finally inverted onto mounting media.