Ovarian Tissue Immunohistochemistry and Immunofluorescence

To evaluate protein localization and intensity, immunohistochemistry and immunofluorescence were performed according to previously described methods [26 (link), 27 (link)]. Ovarian tissue slides were dewaxed in xylene and graded series of ethanol solutions. Slides were then fixed in 4% paraformaldehyde and blocked with 3% bovine serum albumin (BSA) at 37°C. The sections were incubated overnight at 4°C with antibodies against p-IRE1α (1: 200, ab124945, Abcam, Cambridge, UK), XBP1 (1: 100, WL00708, Wanleibio, Shenyang, China), p-PI3K (1 : 200, AF3242, Affinity, USA), p-AKT (1: 200, 4060T, CST, USA), p-p53 (1: 200, ab33889, Abcam, UK), p-NF-κB (1: 200, 3033S, CST, USA), and cleaved-caspase-3 (1: 200, 66470-2-Ig, Proteintech, USA). All secondary antibodies were diluted (1 : 2000) and incubated at 25°C for 2 h. The sections were processed according to the avidin-biotinylated-peroxidase complex and DAB staining techniques, followed by observation under an optical microscope. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (1 : 2000, C1002, Beyotime, China) for 30 min and photographed using an Olympus laser scanning confocal microscope (FV3000).