Pgrmc2

Paraffin-embedded rat ovary tissues were prepared, followed by deparaffinization and rehydration. After antigen exposed, tissue slides were incubated with 3% H₂O₂ for 10 min and goat serum for 30 min to block non-specific binding. Primary antibodies: ANP (1:100; Abcam, Cambridge, UK), NPRA (1:100; Santa Cruz Biotechnology), NPRC (1:100; Santa Cruz Biotechnology), PGRMC1 (1:50; Cell Signaling), PGRMC2 (1:50; Cell Signaling), PCNA (1:200; Proteintech), p-c-Fos (1:50; Cell Signaling), c-Fos (1:50; Cell Signaling), p-c-Jun (1:50; Cell Signaling) and c-Jun (1:50; Cell Signaling) were incubated overnight at 4 °C. The second antibody was incubated for 40 min after washing with PBS. The signals were visualized with DAB wherein yellowish-brown stain indicated as a positive result. The negative control was obtained by replacing primary antibody with PBS. Images were taken under an inverted microscope.

Frozen tissue sections from control and PCOS rat model were fixed in 4% paraformaldehyde at room temperature following with 20% sucrose for 48 h. The tissues were embedded with OCT and sliced at −20 °C. After fixed with acetone, goat serum was used to block at room temperature for 2 h. The primary antibodies mouse anti-NPRA (1:50; Santa Cruz Biotechnology, CA, USA) and NPRC (1:100; Santa Cruz Biotechnology, CA, USA), rabbit anti-PGRMC1 (1:100; Cell Signaling, Danvers, MA, USA) and PGRMC2 (1:50; Cell Signaling) were incubated at 37 °C for 2 h. Washing with PBS, TRITC-conjugated goat anti-rabbit IgG (1:100; ZSGB-BIO, Shanghai, China) and FITC-conjugated goat anti-mouse IgG (1:100; Sigma-Aldrich, St. Louis, MO, USA) were incubated for 1 h at 37 °C following treatment with DAPI (1:1000; Beyotime, Shanghai, China) for 3 min. The immunofluorescent images were taken with an inverted microscope (Olympus, Tokyo, Japan).
Cells grown on coverslips were fixed with 4% paraformaldehyde for 20 min at room temperature following incubation with 0.1% Triton X-100 for 15 min. After blocking with goat serum, cells were incubated with rabbit anti-PCNA (1:200; Proteintech, Wuhan, China) at 4 °C overnight. TRITC-conjugated goat anti-rabbit IgG was incubated for 1 h and DAPI (1:1000) for 3 min. Immunofluorescent staining was photographed with an inverted microscope.