Dak n150130

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.

Briefly, after being deparaffinized, rehydrated, and washed in 0.01 M PBS (pH 7.2–7.4), sections were incubated with 3% H₂O₂ for 10 min at room temperature to block endogenous peroxidase, subjected to high pressure antigen recovery sequentially in 0.01 M citrate buffer (pH 6.0) for 3 min, incubated with blocking solution (5% goat serum and 0.1% BSA in PBS) for 2 h at room temperature, and then incubated with the diluted primary antibodies overnight at 4 °C. The following primary antibodies were used for immunocytochemistry: β-III-tubulin (CBL412, Chemicon), SMA (ab5694, Abcam), and AFP (DAK-N150130, Dako). Blocking solution without the primary antibody served as negative control. After washing with PBS three times (each for 15 min), sections were incubated with appropriate secondary antibodies at room temperature for 2 h. Then sections were washed with PBS three times (each for 15 min), and nuclei were stained with Hoechst 33342 (Sigma), placed in Vectashield mounting medium, and photographed with a Zeiss Axio Imager Z1.