Anti neun

After the paraffinized brain slides were dewaxed and rehydrated, the sections were incubated with one of the following two primary antibodies, anti-NeuN (1:500, Servicebio)/anti-Iba1 (1:1000, Servicebio) and anti-NeuN (1:500, Servicebio)/anti-BrdU antibody (1:100, Servicebio) in blocking serum at 4°C overnight. The following day, the sections were incubated with secondary antibodies consisting of Alexa Fluor 488 goat antimouse and Cy3 goat antirabbit for 1 h in the dark at room temperature. The cell nucleus was counterstained with DAPI for 10 min in the dark. The sections were covered with anti-fade mounting medium. Each of the above was washed three times for 5 min with PBS. Iba1-labeled cells were counted in the hippocampal subregions of interest (CA1, CA3, and DG) under an immunofluorescence microscope (Nikon Eclipse C1, Tokyo, Japan). For double labeling of BrdU and NeuN, the images were observed by a confocal microscope (Nikon A1R, Nikon, Japan), and co-labeled BrdU⁺/NeuN⁺ cells were counted and quantified by Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, United States). About 3–5 sections per area per rat (n = 8 rats/group) were analyzed, and the mean number of positive cells in the hippocampus was counted in each image.

We used immunofluorescence staining to detect the rate of neuronal LC3‐positive cell. The slices were then dewaxed, repaired by antigen (EDTA antigen repair solution, PH8.0), sealed by hydrogen peroxide (3% hydrogen peroxide solution), and serum. Then, the slices were incubated with primary anti‐Neun (1:500, Servicebio), LC3b (1:1600, Cell Signaling) in an incubation box overnight at 4°C. Next, the HRP‐labeled secondary antibody was dripped at room temperature to cover the tissue for 50 minutes. Then the tissue was tipped with CY3 (Servicebio) and incubated at dark and room temperature, for 10 min. DAPI was used to dye the nuclei, followed by incubation at room temperature for 10 min. Finally, the slices were sealed after spontaneous fluorescence quenching (Servicebio). Slices were observed under a fluorescence microscope (Nikon Eclipse C1), and images were collected.