Senescence-Associated β-Galactosidase Staining

SA-β-Gal staining was performed following the previously published protocols (Debacq-Chainiaux et al., 2009 (link); Ma et al., 2020 (link), 2021 (link); Wang et al., 2018 (link)). In brief, the O.C.T-embedded ovarian tissues were cryosectioned at a thickness of 8 μm with a Leica CM3050S cryomicrotome, mounted on Superfrost Plus microslides (VWR) and stored at −80°C. Before SA-β-Gal staining, sections were dried at room temperature (RT), fixed in fixation buffer (2% formaldehyde and 0.2% glutaraldehyde) for 5 min and stained with freshly prepared SA-β-Gal staining solution (1 mg/mL X-gal (Amresco, 0428), 5 mmol/L K₄[Fe(CN)₆], 5 mmol/L K₃[Fe(CN)₆], 2 mmol/L MgCl₂, 150 mmol/L NaCl, 40 mmol/L citric acid/Na phosphate buffer) at 37°C for 2 weeks. Further, the sections were counterstained with Nuclear Fast Red Staining Solution (Beyotime, C0151) to visualize the nucleus. Finally, the sections were dehydrated and sealed with resinous mounting medium. Images were taken with Olympus CKX41 microscope imaging system, and the SA-β-Gal-positive areas were quantified with ImageJ.