Mab4301

Cells or tissue sections were washed twice in PBS, fixed in freshly prepared 3.7% paraformaldehyde for 30 min at 4°C, washed once in PBS and permeabilized in 0.1% Triton X-100 in blocking solution (3% goat serum plus 0.1% BSA in PBS) for 30 min at room temperature, then washed once in PBS, and left in blocking solution for 2 h. Cells were incubated overnight at 4°C with primary antibodies against Oct4 (sc5279, Santa Cruz), Nanog (ab80892, Abcam), SSEA-1 (MAB4301, Millipore), βIII-Tubulin (CBL412, Chemicon), alpha 1-fetoprotein (AFP; DAK-N1501, Dako), alpha smooth muscle actin (α-SMA; ab5694-100, Abcam) and Nestin (MAB353, Millipore). Then cells were washed three times (each for 15 min) with blocking solution, and incubated for 2 h with secondary antibodies at room temperature. Goat Anti-Mouse IgG (H+L) FITC (115-095-003, Jackson) and Goat Anti-Rabbit IgG (H+L) Alexa Fluor^® 594 (111-585-003, Jackson), diluted 1:200 with blocking solution, were used. Samples were washed, and counterstained with 0.5 mg/ml Hoechst 33342 (H1398, MP) in Vectashield mounting medium. Fluorescence was detected and imaged using a Zeiss Axio-Imager Z1 fluorescence microscope.

Cells were washed twice in PBS, then fixed in freshly prepared 3.7% paraformaldehyde in PBS (pH 7.4) for 30 min at 4 °C, washed in PBS for one time and permeabilized in 0.1% Triton X-100 in blocking solution (3% goat serum plus 0.1% BSA in PBS) for 30 min at room temperature, then washed in PBS for one time, and left in blocking solution for 2 h. Cells were incubated overnight at 4 °C with primary antibodies against Oct4 (sc5279; Santa Cruz), Nanog (ab80892; Abcam), SSEA-1 (MAB4301; Millipore), βIII-tubulin (CBL412; Chemicon), alpha 1-fetoprotein (AFP; DAK-N1501; Dako), alpha smooth muscle actin (α-SMA; ab5694-100; Abcam), γH2AX (05-636; Millipore), TRF1 (TRF12-S; Alpha Diagnostic) and Zscan4 (AB4340; Millipore). Then cells were washed three times (each for 15 min) with blocking solution, and incubated for 2 h with secondary antibodies at room temperature. Goat Anti-Mouse IgG (H + L) FITC (115-095-003; Jackson) and Goat Anti-Rabbit IgG (H + L) Alexa Fluor^® 594 (111-585-003; Jackson), diluted 1:200 with blocking solution, were used. Samples were washed, and counterstained with 0.5 μg/ml Hoechst 33342 (H1398; MP) in Vectashield mounting medium. Fluorescence was detected and imaged using a Zeiss Axio-Imager Z1 fluorescence microscope.

Immunofluorescence staining was performed as previously described.³⁰ Cells growing on slides were fixed with 4% paraformaldehyde and were permeabilized by 0.5% Triton X‐100 (in DPBS) for 15 min at room temperature. The cells were blocked in 5% bovine serum albumin (BSA) in DPBS for 1 h at room temperature and incubated with the primary antibodies against OCT4 (1:500, Santa Cruz, SC‐5279), NANOG (1:500, Cosmo Bio, RCAB001P), SSEA1 (1:100, Millipore, MAB4301) in BSA/DPBS buffer overnight at 4℃. The samples were washed three time in DPBS and incubated with fluorochrome conjugated secondary antibodies Alexa Fluor 594 donkey anti‐mouse IgG (Thermo Fisher, A21203), or Alexa Fluor 594 donkey anti‐rabbit IgG (Thermo Fisher, A21207) in BSA/DPBS buffer for 2 h at room temperature. The cells were washed three times in DPBS and DNA was labelled with DAPI (1 µg/ml, Merck Millipore) in DPBS. The stained cells were observed using an LSM 880 microscope (Zeiss) with a Plan Neofluar 63×/1.4 Oil DIC objective.