Histological Analysis of Vaginal and Uterine Tissues

After the experiments, the separated vagina and uterus were fixed with 10% formaldehyde at 4 °C for 48 h and embedded in paraffin. Paraffin blocks were cut into 5 μm sections and stained with hematoxylin and eosin (H&E) or tartrate-resistant acid phosphatase (TRAP) according to a standard protocol [35 (link)]. The vaginal epithelial-layer thickness was measured on Eclipse Ti2 (Nikon) using NIS-Elements BR 5.01 software (Nikon Instruments Inc., Seoul, Republic of Korea). Immunostaining was conducted using a standard protocol. Vagina and uterus sections were incubated with a primary antibody (Table 1) overnight at 4 °C. For immunohistochemical staining, a 3,3′-diaminobenzidine peroxidase substrate detection kit (Vector Laboratories, Inc., Newark, CA, USA) was used to detect immunoactivity, and the tissues were stained with hematoxylin by counterstaining. Images were obtained using a microscope (Eclipse Ti2; Nikon).