HeLa cells grown on coverslips were washed twice with PBS and permeabilized with 0.2% Triton X-100 in PBS for 2 min at room temperature. Following 2 PBS washes, the coverslips were fixed with 3.7% formaldehyde and subsequently blocked in 2% skim milk powder in PBS-T (PBS containing 0.1% Tween 20) for 2 hrs. For detection of gp210, ab15601 was diluted 1:500-1:1000 in 2% skim milk powder in PBS-T and incubated on the coverslips for 1 hour at RT. Following incubation with primary antibodies, coverslips were washed three times with PBS-T over a period of 45 min. Antibody binding was detected with donkey anti-rabbit antibodies conjugated to Cy3 at a dilution of 1:2500.
