4.12. IHC

Well-sliced vaginal tissues were dewaxed with xylene, rehydrated with gradient ethanol, and treated with sodium citrate buffer (10 mM, PH 6.0) for 20 min [42]. After cool-down to room temperature, the tissue was incubated in 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidase. Tissues were then incubated with 5% BSA for 20 min and incubated with primary antibodies against MPO and NE overnight at 4 °C. Tissues were washed overnight with PBS at room temperature, followed by titration with biotinylated goat anti-mouse IgG and SABC. Development with DAB peroxidase, restaining with hematoxylin, sealing with neutral resin, and examination under light microscopy (Olympus BX51; Fulai Optical Technology Co. Ltd., Shanghai, China) at 200× magnification were conducted. Image analysis was performed using Image J.