AP activity was detected using an Alkaline Phosphatase Substrate Kit III (Vector, sk-5300), according to the instruction manual. AP-positive colonies under different conditions were counted and analysed by StatView software.
Immunofluorescence staining was performed as previously described [30] (link). Cells were washed in PBS, fixed in 3.7% paraformaldehyde, permeabilised with 0.1% Triton X-100, blocked with blocking solution, and incubated overnight at 4°C with primary antibodies Oct4 (1∶200, sc9081, Santa Cruz), Nanog (1∶200, AB80892, Abcam), or SSEA-1 (1∶200, MAB4301, Millipore). After wash with PBS for three times, cells were incubated with a secondary antibody (1∶200, Goat Anti-Rabbit IgG (H+L) Alexa Fluor 594 111-585-003 Jackson 1.5 mg or Goat Anti-Mouse IgG (H+L) FITC 115-095-003 Jackson 2 mg). Nuclei were stained using Vectashield medium (Vector) added with Hoechst 33342 (Sigma). Fluorescence images were captured using a Zeiss fluorescence microscope (AxionVision Z1).
Immunofluorescence staining was performed as previously described [30] (link). Cells were washed in PBS, fixed in 3.7% paraformaldehyde, permeabilised with 0.1% Triton X-100, blocked with blocking solution, and incubated overnight at 4°C with primary antibodies Oct4 (1∶200, sc9081, Santa Cruz), Nanog (1∶200, AB80892, Abcam), or SSEA-1 (1∶200, MAB4301, Millipore). After wash with PBS for three times, cells were incubated with a secondary antibody (1∶200, Goat Anti-Rabbit IgG (H+L) Alexa Fluor 594 111-585-003 Jackson 1.5 mg or Goat Anti-Mouse IgG (H+L) FITC 115-095-003 Jackson 2 mg). Nuclei were stained using Vectashield medium (Vector) added with Hoechst 33342 (Sigma). Fluorescence images were captured using a Zeiss fluorescence microscope (AxionVision Z1).