Anti zpc

Epithelial cells, oocyte-like cells, and colonies maintained on hAECs were fixed with 4% paraformaldehyde for 15 to 20 minutes at room temperature and permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature. Cells were then blocked with blocking solution for 30 minutes and incubated with anti-OCT4 (rabbit anti-human 1:200; Santa Cruz), anti-NANOG (rabbit anti-human, 1:200; Chemicon, Rolling Meadows, IL, USA), anti-DAZL (goat anti human, 1:500; Santa Cruz), anti-STELLA (goat anti-human, 1:200; Santa Cruz), anti-ZPC (rabbit anti-human, 1:200; Santa Cruz), anti-SCP3 (rabbit anti-human, 1:800; Abcam), anti-GDF9 (rabbit anti-human, 1:200; Millipore), anti-SSEA4 (mouse anti-human, 1:100; Millipore), anti Tra-1-60 (mouse anti-human, 1:100; Millipore), and anti Tra-1-81(mouse anti-human, 1:100; Millipore) antibody for 1 hour at room temperature. Cells were then probed with fluorescein isothiocyanate-labeled IgG (1:200; Santa Cruz) or Rodamine (TRITC)-labeled IgG (1:100; Invitrogen) and incubated at room temperature for another 20 minutes. The slides were then covered with mounting medium (glycerol diluted 3:1 in PBS; Vector Laboratories, Burlingame, CA, USA). Fluorescence images were obtained with a Leica DMI3000 microscope.

IOSE80 cells, oocyte-like cells, and colonies maintained on hAECs were fixed with 4% paraformaldehyde for 15‒20 min at room temperature and permeabilized with 0.1% Triton X-100 for 10 min at room temperature. Cells were then blocked with 5% FBS in PBS for 30 min and incubated with primary antibodies, including anti-NANOG (rabbit anti-human; 1:200; Abcam, Cambridge, UK), anti-EpCAM (goat anti-human; 1:500; Santa Cruz Biotechnology), anti-IFITM3 (rabbit anti-human; 1:200; Novus Biologicals, Littleton, USA), anti-ZPC (rabbit anti-human; 1:200; Santa Cruz Biotechnology), anti-SCP3 (rabbit anti-human; 1:800; Abcam), anti-GDF9 (rabbit anti-human; 1:200; Millipore, Billerica, USA), anti-SSEA4 (mouse anti-human; 1:100; Millipore), anti-Tra-160 (mouse anti-human; 1:100; Millipore), and anti-Tra-1-81 (mouse anti-human; 1:100; Millipore) antibodies for 1 h at room temperature. Cells were then probed with fluorescein isothiocyanate-labelled IgG (1:200; Santa Cruz Biotechnology) or rhodamine (TRITC)-labelled IgG (1:100; Invitrogen, Carlsbad, USA) antibodies and incubated at room temperature for an additional 30 min. Each antibody was detected using corresponding secondary antibodies conjugated to fluorescein isothiocyanate. Fluorescence images were obtained using a Leica DMI3000 microscope or an SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany).