We used immunostaining to detect the expression of androgen receptor (AR), AKT, and phosphorylated AKT (p-AKT). The IHC was performed using 5-mm sections of paraffin-embedded ovarian tissue. The tissue was deparaffinized in xylene, hydrated in a serial grading of alcohol solutions, incubated in an antigen recovery solution (sodium citrate buffer 0.01 Mol/L, pH 6.0) in a pressure cooker, and boiled for 5-6 min. Then, following the manufacturers' instructions of SP kit (ZSGB-BIO, Beijing, China) and DAB kit (ZSGB-BIO, Beijing, China), the samples were incubated overnight at 4°C with the primary antibody and rinsed with tris-buffered saline with Tween (TBST) three times before incubation with 2nd antibody (antibodies used were: AR, 1:250, rabbit monoclonal, cat#:ab133273, Abcam; AKT, 1:300, rabbit monoclonal, cat#:4691, Cell Signaling Technology; p-AKT, 1:100, rabbit monoclonal, cat#:4060, Cell Signaling Technology). The negative controls were incubated without the primary antibody. After rinsing with TBST, the slides were examined by 2 independent pathologists. The expression levels of the target protein were scored based on the percentage of cells that stained positive (0: no positive cells; 1: ≤ 10% positive cells; 2: 11‒50% positive cells; 3: 51‒90% positive cells; and 4: ≥ 91% positive cells). Scores of 0 and 1 were considered low expression level, and scores of 2‒4 were considered high expression level. To better measure the expression of AR, TissueQuest software (TissueGnostics) was used, and results were given as the percentage of tissue stained positive per millimeter squared of total specimen area. The positive value was 1% or more.

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