We crosslinked cells in 4% paraformaldehyde for 15 minutes, and washed in 1x PBS three times. To permeabilize the cells, we washed with 1x PBS + 0.1% Triton and then blocked in 1x PBS + 0.1% Triton + 1% BSA for 45 minutes at room temperature. We incubated cells with α-Pou5f1 antibody (Santa Cruz: SC-9081) at 1:100 dilution in blocking solution for 1.5 hours at room temperature and then washed in blocking solution three times. Next, we incubated cells in α-rabbit secondary antibody coupled to GFP (Jackson ImmunoResearch: 111-486-152) at a dilution of 1:1000 in blocking solution for 45 minutes. Finally, we thoroughly washed cells in blocking solution three times, and added vectashield containing DAPI (VWR: 101098-044) to each well.
