A1 laser

Confocal imaging was performed on a Nikon A1 laser scanning confocal attachment on an Eclipse Ti microscope stand (Nikon Instruments, Melville, NY) using a 40x/1.3 Plan Fluor oil-immersion objective. The confocal pinhole was set at 1 Airy unit, to produce an optical section of approximately 0.5 um. MitoTracker Green and MitoSOX Red (Life Technologies, Grand Island, NY) were imaged sequentially and emission was collected with standard fluorescein and rhodamine emission filters. Single optical sections were collected for each cell shown.

The ovarian paraffin sections were deparaffinized, rehydrated, and subjected to high temperature (95–98 °C) antigen retrieval with 0.01% sodium citrate buffer (pH 6.0) for 16 min. After that, the sections were washed with PBS on a shaker for 5 min, and then blocked with 1% normal donkey serum in PBS for 1 h at room temperature and incubated with primary antibodies for 12–16 h at 4 °C. The antibodies used were listed in Table S1. Subsequently, ovarian sections were rinsed thoroughly with PBS and incubated with Alexa Fluor 488- or 555-conjugated donkey secondary antibodies for 1 h at 37 °C. Then slides were rinsed in PBS, counter-stained with Hoechst 33342 for 5 min. Finally, sealed the sections in an anti-fade fluorescence mounting medium with coverslips. Immunohistochemistry was performed using Histostain™-SP Kits (PV-9001, ZSGB-BIO, China) and DAB peroxidase substrate kits (ZLI-9017, ZSGB-BIO, China) according to the manufacturer's protocols. Sections were examined and photographed using Nikon 80i or Nikon A1 laser.