Rabbit polyclonal anti oct4

Cells were grown on coverslips and then fixed with 4% paraformaldehyde for 30 minutes. Cells were blocked with 5% goat serum and 5% bovine serum albumin (BSA) at room temperature, and then incubated with primary antibodies: rabbit polyclonal anti‐OCT4 (1:100; Santa Cruz Biotechnology; sc‐9081), mouse monoclonal anti‐TRA‐1‐81 (1:100; Santa Cruz Biotechnology; sc‐21706), mouse monoclonal anticardiac troponin T (cTnT) (1:100; Abcam; Ab8295) and rabbit polyclonal anti‐α‐actinin (1:100; Abcam; Ab137346) at 4°C overnight. After washing with PBS, the slices were incubated with secondary antibodies: chicken antirabbit IgG Alexa Fluor 594 (1:200; Invitrogen; A21442), chicken antimouse IgG Alexa Fluor 488 (1:200; Invitrogen; A21200), goat antirabbit IgG Alexa Fluor 488 (1:200; Invitrogen; A32731) and goat antimouse IgG Alexa Fluor 594 (1:200; Invitrogen; A21145) at room temperature for 1 hour. Then the slices were rinsed in PBS three times and mounted with DNA‐specific 4′,6‐diamidino‐2‐phenylindole (DAPI; H1200; Vector Lab). Fluorescence was evaluated by a confocal laser scanning microscope (Leica, Wetzlar, Germany).

The cells in 48 plates were washed with 1× -concentrated phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature, washed twice with PBS, and incubated for 10 min at 37°C in blocking buffer (PBS containing 10% normal goat serum). Then, they were incubated overnight in a humidified chamber at 4°C with one of the following: 1:500 dilution of a rabbit polyclonal anti-Mvh antibody (Abcam); 1:150 dilution of rabbit polyclonal anti-Oct4 (Santa Cruz Biotechnology); 1:200 dilution of a rabbit polcyclonal anti-Dazl antibody (Abcam); 1:100 dilution of rabbit polyclonal anti-GFRA1 (ABclonal). After washing twice with PBS, the cells were incubated at 37°C for 30 min with a 1:150 dilution of tetramethylrhodamine isothiocyanate (TRITC) conjugated secondary antibody (goat anti-rabbit IgG, then incubated at 37°C for 20 min with 500 ng/mL 4′,6-diamidino-2-phenylindole (DAPI; Sigma). The cells were subsequently mounted in anti-fade mounting medium. Images were obtained using a Leica DMI3000 B microscope and a Leica DFC550 digital camera.