Immunofluorescent Staining of iPSC Markers

Immunofluorescent staining was used to examine stem cell markers in iPSC colonies [50 (link)]. Briefly, cells were fixed by 4% paraformaldehyde/PBS for 10–15 min and rinsed with PBS, then permeabilized and blocked with 0.1% Triton X-100/PBS containing 3% BSA for 30 min. After incubation with primary antibodies for 1 h at room temperature or overnight at 4C, the samples were washed three times with PBS and then incubated with secondary antibody for 1 h. The following antibodies were used in the immunostaining: rabbit anti-NANOG (1:100 dilution, Santa Cruz) and rabbit anti-OCT4 (1:100 dilution, Millipore). The cell samples were subsequently incubated with Cy3- or Alexa Fluor 488-labeled secondary antibodies for 1 h. After washing three times with PBS, samples were counterstained with Hoechst 33258 (Invitrogen). Alternatively, the pluripotency of stem cells was examined by Fluorescent Mouse ES/iPS Cell Characterization kit (Cat.#SCR077, Millipore, MA) following the protocol provided by the manufacturer. Fluorescence images were acquired with a Zeiss AxioCam Camera.