Paraformaldehyde fix solution

Dimethyl sulfoxide (DMSO) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Proteinase K was bought from Tiangen Biotech Co., Ltd. (Beijing, China). Antifade Mounting Medium was bought from Boster Biological Technology Co., Ltd. Paraformaldehyde fix solution at a concentration of 4% was obtained from Servicebio Technology Co., Ltd. (Wuhan, China). An RAA fluorescence kit was obtained from Jiangsu Qitian Gene Biotechnology Co., Ltd. (Wuxi, China). A Wizard^® Magnetic DNA Purification System for Food was purchased from Promega Biotech Co., Ltd. (Madison, WI, USA). All primers and probes were synthesized by Sangon Biotech (Shanghai, China). Skim milk was purchased at the local supermarket (Rainbow, China). TOMA was provided by Ningbo International Travel Healthcare Center (Ningbo Customs Port Outpatient Department). The final concentration of TOMA dissolved in 20% DMSO was 1 mg/mL. It was stored at −20 °C in the dark for further use.

Blood was collected from rats under deep anaesthesia, followed by centrifugating at 4°C for 20 min at 1000x g, and serum was collected. After perfused with saline and 4% Paraformaldehyde Fix Solution (Servicebio, G1101), the rats were executed and brain and heart tissues were collected. The brain tissues were divided into three groups: (1) PVN was carefully removed directly from both sides of the third ventricle; (2) brain tissue was frozen after being covered with OCT embedding agent (Servicebio, G6059); and (3) brain tissue was embedded in paraffin. The heart tissues were divided into two groups: (1) 3 mm of myocardial tissue surrounding the infarct region of the left ventricle in rats was carefully removed, and (2) the whole heart tissue was embedded in paraffin.

Mouse vaginal tissues were fixed in Paraformaldehyde Fix Solution (G1101-500ML, Servicebio) for 24 h and embedded in paraffin, and sectioned at 5 μm. Sections were dried at 60 °C for 30 min, deparaffinized and dehydrated in ddH2O; 75% ethanol, 85% ethanol, 90% ethanol, 100% ethanol, and water. Sections of the vagina were stained with hematoxylin (G1140, Solarbio) and eosin (G1100, Solarbio). Cornification of vaginal epithelial cells was identified using PAS staining (G1281, Solarbio). The TUNEL assay was performed on paraffin-embedded sections of the vagina using an In Situ Cell Death Detection Kit (11684817910, Roche) according to the manufacturer’s instructions.