Alexa fluor 488 anti

The cells were plated onto 24-well culture plates before the immunostainings. Cells were fixed with 4% PFA (Fisher Chemical) in PBS for 15 min. The cells were then permeabilized by 0.5% Triton X-100 in PBS for 10 min and treated with Ultravision blocker (Thermo Scientific) for 10 min. Primary antibodies were diluted in 0.1% Tween in PBS, added to the wells, and incubated for 24 h in dark at 4°C on a Stuart SSL4 seesaw rocker. Secondary antibodies, and Hoechst 33342 (Thermo Fisher Scientific) to stain the nuclei, were diluted in 0.1% Tween in PBS and added to the wells. The wells were then incubated in the dark at room temperature (RT) for 30 min on the seesaw rocker.
The primary antibodies used in this study were OCT4 (1:500 goat, Santa Cruz, sc-8628), TRA-1-60 (1:500 mouse, Thermo Fisher Scientific, MA1-023), SSEA (1:1,000 mouse, Millipore, MAB4304), SOX17 (1:500 goat, R&D Systems, AF1924), α-SMA (1:500 mouse, Sigma, A2547), and β-tubulin III (1:500 rabbit, Abcam, Ab18207).
The secondary antibodies used were Alexa Fluor 488 anti-goat (1:500 donkey, Invitrogen, A11055), Alexa Fluor 488 anti-mouse (1:500 donkey, Invitrogen, A21202) and anti-rabbit (1:500 donkey, Invitrogen, A21206), and Alexa Fluor 594 anti-mouse (1:500 donkey, Invitrogen, A21203) and anti-rabbit (1:500 donkey, Invitrogen, A21207).

Antibody staining and DAPI staining (1:1000, Invitrogen/Life Technologies, D1306) were executed using standard procedures. The following antibodies were used: rabbit anti-GFP (1:1000) (Invitrogen, A6455), Alexa Fluor 488 anti-rabbit (1:400) (Invitrogen, A11008), mouse monoclonal antibody directed against Pericardin, EC11 (1:5) (Chartier et al., 2002 (link)) (Developmental Studies Hybridoma Bank), Alexa Fluor 568 anti-mouse (1:400) (Invitrogen, A11031).