Immunofluorescence Staining of Pluripotency Markers

Immunofluorescence experiments were carried out following our previously reported protocols [21 (link)]. Briefly, cells growing on the glass slide were fixed with 4% paraformaldehyde for 15 min and permeabilized using 0.25% Triton X-100 diluted in PBS for 10 min at room temperature. To block unspecific epitopes, cells were incubated with PBS containing 1% BSA and 0.1% Tween-20 for 1 h. Cells were then incubated with the following primary antibodies at 4 °C overnight: rabbit anti-OCT4 (5 μg/ml, Abcam, Nanchang, China), mouse anti-SSEA-4 (15 μg/ml, Abcam), rabbit anti-Nanog (1:200, Abcam), rabbit anti-Ki67 (1:100, Abcam), and mouse anti-PCNA (5 μg/ml, Abcam). After that, cells were incubated with secondary donkey anti-mouse or anti-rabbit antibodies conjugated to either Alexa Fluor 488 or Alexa Fluor 568 (Jackson, Nanchang, China). Nuclei were counterstained with DAPI (Thermo Fisher).