C15310069

ESCs were fixed for 10 min with 4% paraformaldehyde (PFA) at room temperature (RT) permeabilized for 10 min with 0.4% Triton X-100-PBS and blocked for 30 min with 10% goat serum in PBST (PBS with 0.05% Tween 20). Incubation with primary antibodies in blocking buffer was performed overnight at 4 °C. After washing with PBST, cells were incubated in blocking buffer with the secondary antibodies for 1 h at RT. Slides were then washed in PBST and mounted with ProLong® Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Images were acquired using a fluorescence microscope (Axioplan2; Carl Zeiss) or a confocal Zeiss LSM700 microscope (Carl Zeiss, Jena) with Zen image acquisition software. Images were processed with Fiji and CC Photoshop software (Adobe). The following primary antibodies were used: goat anti-REX1 (Santa Cruz, sc-50670, 1:50), mouse anti-OCT4 (Santa Cruz, sc-5279, 1:100), rabbit anti-RNF12 (a generous gift from Dr. Ingolf Bach, 1:100), rabbit anti-NANOG (Calbiochem, SC1000, 1:100) and rabbit anti-H3K27me3 (Diagenode, C15310069, 1:500). The following Alexa Fluor secondary antibodies were used: donkey anti-mouse 488 (Thermo Fisher Scientific, A-21202, 1:500), donkey anti-rabbit 488 (Thermo Fisher Scientific, A-21206, 1:500), donkey anti-rabbit 546 (Thermo Fisher Scientific, A10040, 1:500) and donkey anti-goat 647 (Abcam, ab150131, 1:500).

After embryo isolation from the uteri, regions containing the developing germ cells were dissected from E9.5 and E11.5 embryos. E9.5 embryo hindguts and E11.5 embryo trunks were fixed in ice cold 4% PFA for 3 h, followed by consecutive washes in PBS. Tissues were then processed for paraffin embedding using standard histology procedures and 5 µm paraffin sections were dissected with a Cryostat HM 560. Heat-mediated (900 W in a microwave for 20 min) epitope retrieval in citrate buffer pH 6.0 was performed on paraffin sections. After cooling down, sections were blocked with blocking solution (2% BSA, 5% donkey serum in PBS) for 30 min at RT, followed by primary antibody incubation at 4 °C overnight. The next day, slides were washed in PBS and incubated with secondary antibodies for 1 h at RT. Slides were then washed in PBS and mounted with ProLong® Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Confocal imaging was performed on a Zeiss LSM700 microscope (Carl Zeiss, Jena). The following primary antibodies were used: goat anti-OCT4 (Santa Cruz, sc-8628, 1:200) and rabbit anti-H3K27me3 (Diagenode, C15310069, 1:250). The following Alexa Fluor secondary antibodies were used: donkey anti-goat 555 1:400 and donkey anti-rabbit 488 1:250 (both from Thermo Fisher Scientific).