Alkaline phosphatase staining was performed using 1-Step NBT/BCIP Substrate Solution. The H9 hESCs were incubated with NBT/BCIP substrate solution for 15 min at room temperature, washed three times with phosphate-buffered saline (PBS), and fixed with 4% paraformaldehyde. For the immunofluorescence study, cells were washed three times with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. After washing the cells with PBS three times, cells were permeabilized with 0.1% Triton-X for 15 min at room temperature and blocked by 1% BSA and 2% FBS in PBS containing 0.1% Triton-X for 1 h at room temperature. Subsequently, cells were incubated with primary antibodies, including rabbit polyclonal Oct4 antibody (2 µg/mL), rabbit polyclonal Sox2 antibody (2 µg/mL), and rabbit polyclonal Nanog antibody (2 µg/mL) for 1 h at room temperature. Cells were washed three times with PBS buffer and further incubated with secondary antibodies, including Alexa Fluor™ 488 conjugated goat anti-rabbit IgG H and L (1:500) and Alexa Fluor™ 594 goat anti-mouse IgG (1:500) for 1 h at room temperature, respectively. The cells were mounted in mounting medium (Vectashield), and marker expression was visualized by Zeiss fluorescent microscope.

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