Hsp70

Immunostaining was performed using the SP kit (Zhongshan Goldenbridge Biotechnology). Wax seal tissue slices were dewaxed, rehydrated, and then boiled in a 0.01 mol/L citrate buffer for 15 min. The activity of endogenous peroxidase was blocked by a hydrogen peroxide of 0.3% for a period of 10 min. The samples were incubated with rabbit anti‐p‐AKT (1:50; Rui Ying Biological, Suzhou, China), p85 (1:50; Rui Ying Biological), p‐p85 (1:50; Rui Ying Biological), ARNT (1:50; Boster, Wuhan, China), MDR1 (1:50; Boster) antibodies, mouse anti‐AKT (1:100; Boster), or HSP70 (1:50; Beyotime Biotechnology) antibody at 4°C overnight. Then, the samples were incubated with the secondary antibody at room temperature for 1 h. The immune reaction was revealed by a DAB (3,3′‐diaminobenzidine) kit. After stained with hematoxylin, the tissue preparations were dehydrated and mounted. Sections exposed to nonimmune sera were used as negative controls. The staining score is as follows: 0, no staining; 1+, minimum staining; 2+, moderate to strong staining of at least 20% of cells; 3+, strong staining of at least 50% of cells. 0 or 1+ stained cases were classified as negative, and those with 2+ or 3+ staining were considered positive.

Total protein was extracted from the ovarian tissue using a lysate containing phenylmethanesulfonyl fluoride [PMSF] (Beyotime, Shanghai, China) according to the manufacturer's instructions and detected using a BCA protein assay kit (Tiangen Biotech, China). The sample amount was 50–55 μg of protein per sample. Samples were separated by electrophoresis on polyacrylamide gels and subsequently transferred to nitrocellulose membranes. The membranes were incubated in 5% skimmed milk powder for 1.5 h, washed three times with Tris-buffered saline Tween-20 (TBST) (pH 7.6), and incubated with the primary antibodies [monoclonal anti-actin (1:1,000; Beyotime), rabbit antibody GH (1:300, bs-6579R; BIOSS), rabbit antibody GHR (1:300, bs-10661R; BIOSS), and monoclonal mouse antibody Hsp70 (1:300 BOSTER)] in primary antibody dilution buffer (Beyotime) at 4°C overnight. After washing with TBST, the membranes were incubated with anti-rabbit/mouse IgG antibody (1:2,000; CWBIO, Beijing, China) for 2 h at 37°C, followed by washing with TBST. Next, the membranes were immersed in a high-sensitivity luminescence reagent (BeyoECL Plus; Beyotime), exposed to film using a FusionCapt Advance FX7 (Beijing Oriental Science and Technology Development Co., Ltd., Beijing, China), and analyzed using Ipp 6.0 (Image Pro-Plus 6.0; Media Cybernetics).