Anti ssea1

Alkaline phosphatase staining was performed with BCIP/NBT Alkaline Phosphatase Colour Development Kit (Beyotime) according to manufacturer’s instructions. For immunostaining, embryos or cells were fixed with 4% paraformaldehyde for 30 min and then permeabilized with 1% Triton X-100 for 30 min followed by blocking with 2% BSA (Sigma). Cells were incubated in primary antibody overnight at 4 °C and secondary antibody at 37 °C for 1 h. Primary antibodies were used that were anti-OCT-3/4 (Santa Cruz), anti-NANOG (Abcam), anti-SOX2 (Cell signaling), anti-SSEA1 (Santa Cruz), anti-SSEA4 (Santa Cruz), anti-E-CADHERIN (BD Bioscience), anti-KLF4 (Stemgent), anti-CDX2 (Biogenex).

IPS cells were seeded on 0.1% gelatin-coated plate. Three days after seeding, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-X-100 in phosphate buffered saline (PBS), and blocked with 1% Bovine serum albumin in PBS. Cells were then incubated at room temperature with anti-Oct4 (Santa Cruz) or anti-Ssea1 (Santa Cruz) antibody for 2 hours and with rhodamine-conjugated anti-rabbit IgG secondary antibody for an additional 1 hour. The cells were washed twice with PBS containing 0.1% Tween20 and stained with Hoechst 33342 (Sigma). Images were captured with an Olympus fluorescence microscope.

Cells were fixed in Mildform^® (Wako) for 30 min and permeabilized in 0.2% Triton-X (Sigma-Aldrich Corporation, MO, USA) in PBS (−) for 10 min. The fixed samples were blocked by incubation in 10% Block Ace (Dainippon Sumitomo Pharma, Osaka, Japan) in PBS (−) for 1 h and incubated with anti-Cdh1 (Santa Cruz Biotechnology, Inc., CA, USA) and anti-SSEA1 (Santa Cruz Biotechnology) primary antibodies diluted in PBS (−) containing 10% Block Ace overnight at 4 °C. The samples were then washed twice with PBS (−) and reacted with TexasRed-conjugated anti-rabbit IgG antibody or PE-conjugated anti-mouse IgM antibody (Santa Cruz Biotechnology) diluted in PBS (−) containing 10% Block Ace for 1 hour in the dark. Samples were counter-stained with DAPI (Vector Laboratories Ltd., Peterborough, England) before microscopic observation. Images were acquired using a fluorescence microscope (Keyence Corporation, Osaka, Japan).