Triton x 100

The microglial cells were fixed with methanol, after several washes in 0.1 M PBS, cells were permeabilized in PBS containing 0.3% Triton X-100 (ZSGB-BIO, Beijing) for 30 min, rinsed in PBS and then pre-incubated with 10% goat serum in PBS for 60 min at room temperature. The cells were then incubated in a solution of OX-42 antibody diluted in PBS overnight at 4°C. Then TRITC -conjugated goat anti-rabbit (1∶200 dilution; ZSGB-BIO, Beijing) were applied for 2 h at room temperature. Nuclei were stained with the fluorescent nucleic acid dye DAPI (1∶500 dilution; Vector, USA). After several washes, outgrowth cells were visualized using fluorescence microscope (Olympus IX70, Japan), adapted with a Mercury lamp (Olympus, Japan). The images were processed with the Image-pro Plus program.

As a biological control, DEAD box polypeptide 4 (Ddx4) (Fujiwara et al., 1994 (link); Castrillon et al., 2000 (link)), an evolutionarily conserved germ cell-specific RNA helicase, was detected in oocytes from the IVG system. Three oocytes were assessed in both groups. Germinal vesicle (GV) oocytes that failed to mature after oocyte IVM were fixed in 4% paraformaldehyde (Servicebio, Wuhan, China) for 30 min, and then transferred to drops of 0.5% TRITON X-100 (ZSGB-BIO, Beijing, China) for 30 min. Oocytes were blocked in 3% BSA diluted in phosphate-buffered saline (PBS; pH 7.4, Gibco). One hour later, oocytes were incubated with anti-DDX4 antibody (1:200; ABCAM; Cambridge, UK) overnight at 4 °C followed by incubation with Alexa Fluor 647 goat anti-mouse IgG (1:500; Thermo Fisher, Carlsbad, CA, USA) for 1 h. Cell nuclei were stained with DAPI dihydrochloride (Invitrogen). Confocal images were acquired using a Leica Corp. TCS SP8 confocal system (Leica Corp. Microsystems, Heidelberg, Germany).