Cy3 labelled secondary antibody

At the end of the experiment, hypothalamic PVN tissues of rats in each group were obtained for immunofluorescence staining. Tissues were sectioned into 5-μm slices and blocked with 5% normal fetal bovine serum in PBS for 2 h. Thereafter, the slices were incubated with primary antibody against Iba-1 (1:200, GB12105, Servicebio) overnight at 4 °C. The slices were then rinsed with PBS and incubated with Cy3-labelled secondary antibody (1:300, Servicebio) in the dark at room temperature for 60 min. To determine the microglial morphology, the staining imaging was observed with a fluorescence microscope and handled by Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). Three microglial cells per brain slice, three brain slices per animal were measured from the hypothalamic PVN. The parameters of microglial morphology include soma area, processes length, and the number of primary branch projection (ramification). The number of Iba-1-positive cells was also calculated.

At the end of the experiment, the PVN tissues of all rats were obtained, sectioned into 5 μm slices, and blocked with 5% bovine serum albumin for 30 minutes at room temperature. They were then incubated overnight with primary antibody Iba-1 (1 : 200, GB12105, Servicebio) or BDNF (1 : 200; Google Bio#GB11240) or CD206 (1 : 200; Google Bio#GB11062) in a wet box at 4°C. Afterwards, sections were rinsed with PBS and incubated with the corresponding Cy3-labelled secondary antibody (1 : 300, Servicebio) in the dark for 50 minutes at room temperature. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (G1012, Servicebio). Images were captured with a fluorescence microscope and handled by Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). The positive area of Iba-1-positive, BDNF, and CD206 cells was counted in 3 serial sections at 400× magnification. The average of the 3 serial sections was taken. Same method was used to measure the area of DAPI.