Rabbit monoclonal anti sox2

The cells were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) for 15 min, incubated with 0.1% Triton X-100/PBS for 15 min, and blocked in 5% BSA/PBS for 1 hour. The cells were then incubated with the following primary antibodies overnight at 4°C: mouse monoclonal anti–TRA-1-60 (1:100, MAB4360; Millipore), mouse monoclonal anti–TRA-1-81 (1:100, MAB4381; Millipore), mouse monoclonal anti–SSEA-4 (1:100, 330402; BioLegend), rabbit monoclonal anti-NANOG (1:1000, 5232; Cell Signaling Technology), rabbit monoclonal anti-OCT4 (1:1000, 5677; Cell Signaling Technology), rabbit monoclonal anti-SOX2 (1:1000, 5024; Cell Signaling Technology), rabbit monoclonal anti-DNMT3B (1:1000, 67259; Cell Signaling Technology), mouse monoclonal anti-FLAG (1:1000, F1804; Sigma-Aldrich), and goat polyclonal anti-GATA6 (1:500, AF1700; R&D Systems). The cells were then washed three times with PBS and incubated with appropriate fluorescently labeled Alexa Fluor secondary antibodies (1:1000; Invitrogen) for 1 hour. The cells were then washed three times with PBS, incubated with 4′,6-diamidino-2-phenylindole (DAPI; 0.5 μg/ml; Molecular Probes) for 10 min, and washed once with PBS. Images were acquired using the Leica DMI6000B inverted fluorescence microscope equipped with the Hamamatsu ORCA-R2 charge-coupled device camera and analyzed with the Leica application suite advanced fluorescence software.