Mab1924

Cells were cultured in a glass-bottom dish (AGC Techno Glass, Shizuoka, Japan) and fixed with 4% paraformaldehyde for 10 min at 4 °C before being permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 10 min at room temperature (RT). The samples were blocked with 5% normal goat serum in Dulbecco’s phosphate-buffered saline (DPBS; Thermo Fisher Scientific) for 30 min at RT. They were incubated with primary antibodies at 4 °C overnight. After washing with DPBS, the samples were incubated with secondary antibodies conjugated to Alexa 488 or 546 (Thermo Fisher Scientific) for 30 min at RT. After washing with DPBS, the samples were placed in mounting medium with 4',6-diamidino-2-phenylindole (DAPI). The following primary antibodies were used: OCT4 (C-10; Santa Cruz Biotechnology, Dallas, TX, USA), NANOG (ReproCell), Tra1-60 (MAB4360; Sigma-Aldrich), SSEA4 (MAB1435; R&D Systems), SSEA1 (MAB2155; R&D Systems), KLF4 (ab216875; Abcam), PRDM14 (ab187881; Abcam), anti-βIII Tubulin (TUJ1, Promega, Madison, Wisconsin, USA), α-smooth muscle actin (α-SMA; A2547; Sigma-Aldrich), and SOX17 (MAB1924; R&D Systems). Images were acquired using an LSM510 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). All antibodies except for the anti-TUJ1 antibody (1:300) were used at a 1:150 dilution in 5% normal goat serum.