Sodium citrate

Rats under deep anesthesia were subjected to cardiac perfusion with PBS followed by 4% paraformaldehyde. The intact TNC was then immediately isolated and postfixed with 4% paraformaldehyde at 4 °C. After dehydration using graded concentrations of sucrose solution (20% and 30%), the tissues were sectioned using a cryostat (Leica, Japan) into 15-μm slices, which were placed on carrier slides. Antigen repair with sodium citrate (Beyotime, Beijing, China) and blocking with 10% goat serum (Boster, Beijing, China) were then executed. For fluorescence colabeling experiments, the primary antibodies were mixed and diluted with 1% PBS and were subsequently incubated with the sections at 4 °C overnight. After incubation with the fluorescent secondary antibody and counterstaining of the nuclei with 4’,6-diamidino-2-phenylindole (DAPI), a confocal laser scanning fluorescence microscope (Zeiss, Germany) was used to acquire images, which were analyzed with ImageJ.

For immunohistochemistry, the sections were first deparaffinized in xylene and then rehydrated in gradient alcohol and then boiled for 10 min with sodium citrate (Beyotime, China) for antigen retrieval. After washing in PBS buffer (3 × 10 min), the Abcam ABC HRP Kit (ab64264) was used and all procedures were carried out according to the instructions. The sections were incubated with primary antibodies GFAP (Merck, MAB360, 1 : 250) and Iba1 (Wako Chemicals, 019 19741, 1 : 200) at 4°C overnight. After incubation, the sections were washed in PBS which was performed according to the ABC-IHC Kit manufacturer's protocol. Then, brain slices were stained for 2 min using diaminobenzidine (DAB). Finally, the sections were stained with hematoxylin, dehydrated with ethanol and xylene, and covered with coverslips. Sections were imaged by using a Leica microscope and analyzed with Image-Pro plus 6.2 software.

After anesthetized, rats were perfused transcardially with 4% paraformaldehyde. The PAG tissue was isolated immediately following perfusion, then post-fixed in 4% paraformaldehyde at 4 °C for 24 h, and subsequently transferred to sucrose with increasing concentrations (20–30%) until it sank. Afterward, the PAG was sliced transversely at 10 μm using a freezing microtome (Leica, Japan), and the tissue sections were collected and secured on glass slides. After undergoing antigen retrieval with sodium citrate (Beyotime, Shanghai, China), the sections were incubated with 10% goat serum (Boster, Wuhan, China) at 37 °C for 30 min, then incubated overnight at 4 °C with the primary antibodies diluted in PBS. The next day, the secondary antibody was employed at 37 °C for 1.5 h. Whereafter, the sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, China) at 37 °C for 10 min. The images were captured under the confocal laser scanning fluorescence microscope (ZEISS, Germany). Image-J was used to analyze the fluorescence intensity. The analysts were blinded to the experimental groups. Detailed antibody information used in immunofluorescence staining is shown in Table 2.