A1 plus fluorescence microscope

Ovaries were collected at 15 weeks or 20 weeks and fixed with 4% paraformaldehyde in phosphate buffered saline (PBS). Ovaries were dehydrated in alcohol and embedded with paraffin. 7 µm sections were dewaxed and hydrated, then stained with H&E and eventually observed under light microscope. For immunohistochemistry, the sections were also incubated in 3% H₂O₂ for 15 min and then in blocking solution for 45min after dewaxing and hydration. Subsequently, the sections were incubated at 4°C overnight with the following primary antibodies: anti-CD31 (1:250, sc793); anti-VEGF (1:500, sc507) and anti- C/EBP-homologous protein (CHOP) (1:250, sc10790). After washed with PBS for 3 times, the sections were incubated with horseradish peroxidase-conjugated secondary antibodies for 4 h at 37°C. The sections were reacted with 3, 3-diaminobenzidine (DAB). The results were imaged using a Nikon A1 Plus fluorescence microscope (Nikon, Tokyo, Japan).

TUNEL staining (ApopTag Fluorescein in Situ Apoptosis Detection kit, Chemicon) was performed to determine apoptosis level. After dewaxing and hydration, ovarian sections were incubated with 20 μg/ml proteinase K working solution for 15 min at 37°C. The slides were rinsed with PBS for 3 time followed by incubation with TUNEL reaction mixture for 1 h at 37℃. After rinsed with PBS (5 min, thrice), sections were incubated with 4', 6-diamidino-2-pheny-lindole (DAPI, Beyotime, Shanghai, China) for 5 min at room temperature and mounted with aqueous mounting medium(Sigma, St Louris, MO). The results were imaged using a Nikon A1 Plus fluorescence microscope (Nikon, Tokyo, Japan).