Ckx41 microscope imaging system

Sudan Black B staining (SBB staining) was performed using a previously published protocol (Georgakopoulou et al., 2013 (link)). In short, OCT-embedded and snap-frozen tissues were cryosectioned at a thickness of 10 μm with a Leica CM3050S cryomicrotome. Frozen sections were taken out of the refrigerator at −80°C, placed at RT for a few minutes and fixed in 1% (w/v) formaldehyde/PBS for 5 min at RT, and then rinsed gently with distilled water three times, incubated in 50% ethanol and 70% ethanol for 5 min each in turn. Next, frozen sections were stained in Sudan Black B solution (0.7 g SBB in 100 mL 70% ethanol) for 5 min at RT, and then rinsed quickly in 75% ethanol to remove excess staining solution, rinsed with distilled water three times. Finally, sections were left to stain in nuclear fast red solution for 3 min, washed in distilled water and mounted with glycerol. Images were taken with Olympus CKX41 microscope imaging system, and Image Pro Plus was used to quantify the Sudan Black B positive area.

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.

Oil Red O staining was performed using a previously published protocol (Huang et al., 2022 ). In short, OCT-embedded and snap-frozen tissues were cryosectioned at a thickness of 10 μm with a Leica CM3050S cryomicrotome. Frozen sections were taken out of the refrigerator at −80°C, placed at RT for a few minutes and soaked in the PBS solution for 5 min. After filtering Oil Red O staining solution (Sigma-Aldrich) through a 100 μm filter, frozen sections were stained in 60% Oil Red O solution for 8–10 min. To avoid background staining, wash the slides with 70% ethanol for a moment, and then rinsed with running tap water and counterstained with hematoxylin. Images were taken with Olympus CKX41 microscope imaging system, and Image Pro plus was used to quantify the oil red O positive area.