Immunofluorescent labeling of cultured cells

For immunofluorescent staining, cells were grown on coverslips, after which they were fixed in 4% paraformaldehyde for 15 minutes at room temperature (RT). Coverslips were then washed briefly in PBST (90% phosphate-buffered saline (PBS), 10% fetal bovine serum (FBS), 0.05% Triton X-100), permeabilized in permeabilization buffer (90% PBS, 10% FBS, 0.5% Triton X-100) for 15 minutes and blocked in PBST at RT for 1 h. Cover slips were incubated with primary antibody solution at RT for 1 hr. Primary antibodies were diluted in PBST to a concentration of 2 μg/ml. The primary antibodies used were mouse anti-NANOG (MABD24, EMD Millipore), Goat anti-OCT3/4 (sc-8628, Santa Cruz), Rabbit anti-SOX2 (AB5603, Chemicon), and Goat anti-PAX6 (PRB-278P-100, Covance Inc.). The coverslips were then washed three times with PBST at RT for 10 minutes. Next, the secondary antibody diluted in PBST to a concentration of 2 μg/ml was added and the samples were incubated in the dark at RT for 1 h. Secondary antibodies used are donkey anti-rabbit 488 (A-21206, Invitrogen), donkey anti-goat 568 (A-11057, Invitrogen), goat anti-mouse 633 (A-21050, Invitrogen) and rabbit anti-goat 488 (A-11055, Invitrogen). The coverslips were again washed three times with PBST at RT for 10 minutes. Finally, the coverslips were mounted using 3 μl Vectashield mounting medium with DAPI (H-1200, Vectorlabs), after which fluorescence was detected by confocal microscopy (Leica TCS SPE). The same acquisition settings were used for all samples throughout each experiment.