Prb 435p

For alkaline phosphatase (AP) staining, hESCs were fixed with 4% paraformaldehyde (PFA) in PBS for 40 s, rinsed once with PBS and stained using a leukocyte alkaline phosphatase kit (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer's protocol. For immunofluorescent staining, cells were fixed with 4% PFA for 15 min. The fixed cells were washed with PBS and incubated in 0.2% Triton X-100 for 20 min, then washed with PBS and incubated in blocking buffer containing 20% FBS, 10% Glycerol, 100 nM Glycine and 0.1% Triton X-100 for 30 min at room temperature. The cells were then incubated with primary antibodies against Oct4 (Abcam, ab19857), SSEA-4 (Millipore, MAB4304), Nanog (Abcam, ab21624), Nestin (Millipore, MAB5326), Sox2 (Abcam, ab15830), Sox1 (Abcam, ab22572), Pax6 (DSHB, P3U1), Musashi1 (Abcam, ab21628), Tuj1 (Covance, PRB-435P), GFAP (Invitrogen, 13-0300) or O4 (R&D, MAB1326) overnight at 4 °C. The following day cells were washed with PBS and incubated in the appropriate secondary antibodies conjugated to Alexa Fluor 546 or Alexa Fluor 488 (Invitrogen) for one hour at room temperature. Nuclei were counterstained with Hoechst 33342 for 10 min. Images were taken with an Olympus IX71 inverted fluorescent microscope or an Olympus FV10i confocal microscope.