Nb100 58842

Cells were cultured on cover glasses in 24‐well cell culture plates, fixed with 4% PFA at room temperature for 10 min, and washed thrice with PBS. Rabbit anti‐E‐cadherin (Cell Signaling Technology, 3195, 1:200), mouse anti‐N‐cadherin (Invitrogen, 333900, 1:200), rabbit anti‐Nanog (Novus Biologicals, NB100‐58842, 1:300), and mouse anti‐Rex1 (made in‐house, 1:500) were added as the primary antibodies prepared with 0.3% Triton X‐100 and 10% goat serum in PBS at 4°C overnight. Then, cells were washed thrice with PBS. Goat anti‐rabbit Alexa 647 (Abcam, ab150079, 1:500) and goat anti‐mouse Alexa 568 (Invitrogen, A11004, 1:500) were added as the secondary antibodies prepared with PBS at room temperature for 1 h. Then, cells were washed thrice with PBS. Nuclei were stained with DAPI prepared in PBS at room temperature for 5 min. Image collecting was conducted with Zeiss LSM 800.

Cells were cultured on coverslips. fixed with 4% paraformaldehyde and then blocked in PBS with 5% BSA and 0.05% Triton-X-100. Cells or tissue sections were incubated overnight at 4 °C with various primary antibodies, including anti-POU5F1 (ab184665, Abcam), anti-Sox2 (MAB2018R-100, R&D), anti-Nanog (NB100-58842, Novus Biologicals), anti-Nanos3 (ab70001, Abcam), anti-DDX4 (ab27591, Abcam), SCP3 (ab 15,093, Abcam) or anti-DAZL (NB100-2437, Novus biologicals). Next, the cells were stained with Cy3 fluorescent dye-conjugated secondary antibodies (Jackson) and 4,6-diamidino-2-phenylindole (DAPI; Invitrogen), while the tissue sections were stained with haematoxylin. The cells just were stained with Cy3 fluorescent dye-conjugated secondary antibodies (Jackson) and 4,6-diamidino-2-phenylindole (DAPI; Invitrogen) as control.