Vector red alkaline phosphate substrate kit

Alkaline phosphatase staining was performed with the Vector Red Alkaline Phosphate Substrate Kit (Vector Laboratories, Burlingame, CA). For immunostaining, cells were fixed in 4% paraformaldehyde with 1% sucrose in phosphate buffered saline (PBS) (Invitrogen) for 15 min at room temperature. The cell membranes were then permeabilized with 0.5% TX-100 in PBS-T (Sigma-Aldrich), blocked in donkey serum (Sigma-Aldrich) and incubated with donkey serum containing primary antibodies including rabbit anti-OCT4 (Merck Millipore), rabbit anti-SOX2 (Abcam, San Francisco, CA) and rabbit anti-NANOG (Merck Millipore) at 1:100 dilutions, washed in PBS-T, and then incubated with Alexa Fluor 594 conjugated goat anti-rabbit secondary antibody (Cell Signaling Technology, Danvers, MA) at a 1:500 dilution. The cell nuclei were counterstained with DAPI. Fluorescent images were taken using a Zeiss or Nikon fluorescence microscope.