Immunohistochemical Analysis of PVN Markers

After the brain tissue was embedded in paraffin, the part between the optic chiasm and mammillary body was resected in the rostro-caudal direction. The tissue was serially sectioned on a paraffin slicer, and sections that were approximately 1.50 mm from the bregma were obtained [25 (link), 26 (link)]. After dewaxing the slices, 3% H₂O₂ was used to block endogenous peroxidase activity, and 0.01 M citric acid was used to retrieve the antigens prior to antibody incubation. Then, the slices were incubated with primary antibodies overnight at 4 °C [27 , 28 (link)]. The sections were immunohistochemically labelled to identify IL-6 (Bioss, China, 1:100), Gp130 (Santa Cruz, America, 1:20), pSTAT3 (Bioss, China, 1:50), NMDA receptors (Bioss, China, 1:50), and GAD67 (Abcam, England, 1:2000) and then incubated with secondary antibodies (anti-mouse for Gp130 and GAD67; anti-rabbit for NMDA receptors, pSTAT3 and IL-6) for 20 min at room temperature. For each rat, the positive neurons within the bilateral borders of the PVN were manually counted in three consecutive sections, and the average value is reported.