Oil Red O Staining of Ovarian Tissue

Oil Red O (ORO) staining was conducted as previously described (Jing et al., 2023 (link)). In brief, O.C.T-embedded ovarian tissue sections (8 μm thickness) were taken out of the refrigerator to air-dry and fixed with 4% PFA for 20 min following washes with PBS for three times. Then, the sections were rinsed in 60% (v/v) isopropyl alcohol for 5 min. Without washing, the sections were promptly stained using 60% ORO solution for 11 min. To remove the background staining, the slides underwent a 10-s wash with 70% ethanol. Subsequently, the slides were rinsed and counterstained with Harris hematoxylin to visualize the nucleus once the color was suitable for microscopic observation before being mounted with 80% Glycerol/PBS. Images were captured with Olympus CKX41 microscope imaging system, and the ORO-positive areas were quantified with ImageJ.