Ssea3

LICs, iPSC and X-02 were seeded, separately, in 24-well plates with each well receiving 10 to 20 clones. After culturing for 3 days, the supernatant was removed, and the cells were washed 2 times with PBS and fixed with 4% PFA for 20 min at room temperature. After washing 2 times with PBS, the cells were permeated with PBS containing 0.5% Triton X-100 for 15 min and blocked with PBS containing 2% BSA + 5% normal goat serum for 1 h at room temperature. The cells were then incubated with primary antibodies including NANOG, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 (all were purchased from Abcam, USA) at 4°C overnight. On the next day, the cells were washed 3 times with PBS, and secondary antibodies, Alexa Fluor ® 555 R goat anti rabbit IgG, Alexa Fluor ® 555 goat anti-mouse IgG, and Alexa Fluor ® 555 goat anti-mouse IgM (Invitrogen, USA), were added and incubated at room temperature for 1 h. Subsequently, the cells were washed with PBS 3 times, stained with 1 μg/ml DAPI for 5 min, washed 3 times again with PBS, mounted, observed and imaged under a fluorescence microscope.