Ssea4

Reprogrammed cells were fixed in 4% paraformaldehyde (Merck) for 20 minutes at room temperature, washed twice with phosphate-buffered saline, and then permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 1 hour at room temperature. The cells were blocked for 1 hour with 5% bovine serum albumin and 2% goat serum in phosphate-buffered saline solution. Samples were incubated at room temperature for 1 hour with primary antibodies against antistage-specific embryonic antigen 4 (SSEA4) (STEMCELL Technologies), octamer-binding transcription factor 4 (OCT4) (Chemicon), sex-determining region Y-box 2 (SOX2), and NANOG homeobox (NANOG). The secondary antibodies used were AlexaFluor 564 mouse antibodies (Molecular Probes, Invitrogen), incubated at room temperature for 1 hour. The nucleus was stained with 4′,6-diamidino-2-phenylindole (VYSIS). Cells were visualized using a confocal laser scanning microscope (LSM 710; Carl Zeiss) with objective lens ×63 in oil immersion, having a numerical aperture of 1.4. Argon laser of 590 nm was used to excite the surface marked with secondary antibodies, and the emission was measured at 617 nm. Image analysis and colocalization studies were carried out using the ZEN 2008 system confocal software (ZEN, version 2.5).

The cells were fixed with 4% paraformaldehyde, incubated at room temperature for 30 min, followed by blocking for 2 h using 1% bovine serum albumin. The cells were washed twice with 1× PBS before addition of primary antibodies of the pluripotency markers, TRA-1-60, TRA-1-81, SSEA-4 or OCT4 (Stemcell Technologies) at 1:100 dilutions and incubated overnight at 4 °C. A FITC-conjugated rabbit anti-mouse antibody (Merck Millipore) was added and the mixture was further incubated for 1 h at room temperature. Nuclei were counterstained with DAPI (Gibco) and observed under an inverted fluorescent microscope.