Evos fl auto cell imaging system fluorescence microscope

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 10 min, and blocked with 3% bovine serum albumin for 30 min at room temperature. After blocking, cells were incubated overnight at 4 °C with primary antibodies. Primary antibodies are NANOG, TRA-1-81 (1:250, Stem cells); KRT8, KRT18, KRT19, AP-2γ, FOXG1 (1:200, Abcam); ALDH1A3, AP-2α, AFP (1: 200, Santa Cruz); Brachyury (T) (1:50, Novus); P63 (1:100, Genetex); p-SMAD1/5/9 (1:200, Cell Signaling); CDH1, Desmoplakin (1:500, BD Biosciences). Alexa 594-conjugated secondary antibody (1:400, red, Molecular Probes, Eugene, OR) or an Alexa 488-conjugated secondary antibody (1:400, green, Molecular Probes) was used to visualize the staining. Following three washes with PBS, slides were mounted with the VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA). Prior to mounting, slides were incubated with 2 μM 4′,6-diamidino-2-phenylindole (DAPI) fluorescence (Molecular Probes) for 10 min at 37 °C to stain the nuclei. The fluorescence images were taken using the EVOS FL Auto Cell Imaging System fluorescence microscope (ThermoFisher Scientific, NY, USA).