Cbl412

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.

Pig iPSCs were removed off feeder cells twice based on their differences in adherence to the bottom of dish. Then, cells were transferred to low-adhesive 35 mm non-coated plates and cultured in pig iPSC medium without bFGF and hLIF. Aggregated EBs were formed after 5–7 days and transferred onto gelatin-coated tissue culture dishes for differentiation for another 5–7 days. Differentiated cells were fixed for immunofluorescence staining using primary antibodies of three embryonic germ layers, including alpha 1-fetoprotein (AFP; DAK-N150130, DAKO) for endoderm, smooth muscle actin (SMA; ab5694, Abcam) for mesoderm, and β-III-tubulin (CBL412, Chemicon) for ectoderm. The secondary antibodies were the same as those for immunofluorescence staining as described above.