Cells were fixed with 4% paraformaldehyde in PBS for 15 minutes, washed 3x with PBS, and blocked in PBS with 2% BSA (Sigma); 0.1% Triton X-100 and 2% low fat milk for 30 minutes at room temperature. Primary antibodies include POU5F1, NANOG, BRACHYURY (Santa Cruz; 1:100, 1:100, 1:300); TRA1-81 (1:100, Stemgent); MAP2, AFP, NESTIN, SMA (Millipore; 1:100, 1:400, 1:2000, 1:10,000); PAX6, NESTIN, OLIG1, GALC, A2B5 (Chemicon; 1:200, 1:200, 1:200, 1:200, 1:500). Fluorescence conjugated secondary antibodies were used according to manufacturer’s protocol (Life Technologies, Molecular Probes). Images were obtained with IX51 Olympus and Nikon Eclipse Ti microscopes.
