Bovine serum album

One day after the MWM test, we used a triple fluorescein-labeled method to detect rat hippocampal tissues (n = 7). Rat hippocampal slices were cut into 20-μm thick slices for immunofluorescence staining. The samples were blocked in PBS containing 0.3% Triton X-100 (Sigma-Aldrich, Munich, Germany) and 5% bovine serum album (Beyotime, Beijing, China) for 2 h at room temperature. The samples were then incubated with synapsin-I (Proteintech, 1:100, Cat No. 17785-1-AP), p-CREB (Cell Signaling Technology, 1:100, Cat. no. 9198S), and Neun (Proteintech, 1:100, Cat No. 26975-1-AP) overnight at 4°C. The sections were washed three times with PBS before being incubated for 1 h with a goat anti-rabbit immunoglobulin G-FITC secondary antibody (1:100, Beyotime, P0186). Finally, the sections were stained using 4’,6-diamidino-2-phenylindole (DAPI; Beyotime, P0131). The fluorescence microscopy images were then quantitatively analyzed using the Image Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, United States) using a Nikon Eclipse CI fluorescence microscope.

After neurological status assessment, five rats from each group were sacrificed and their left brains were collected and rinsed with PBS. Sections of the hippocampus (20-mm thick) were sliced for immunofluorescence staining and blocked for 2 h at room temperature with PBS containing 5% bovine serum album (Beyotime, Beijing, China) and 0.3% Triton X-100 (Sigma-Aldrich). Subsequently, sections were incubated overnight at 4° C with an anti-TLR4 antibody (1:100; ab22048; Abcam, Cambridge, UK), anti-MyD88 antibody (1:100; ab133739, Abcam), anti-TRAF6 antibody (1:100; ab40675, Abcam) or anti-Neun antibody (1:100; ab177487, Abcam). After three rinses with PBS, a secondary goat anti-rabbit IgG-FITC antibody (P0186, Beyotime) was applied to sections and incubated for 1 h at room temperature. Finally, after staining sections with 4ʹ,6-diamidino-2-phenylindole (DAPI; P0131, Beyotime) for 10 min, images were captured using a Nikon Eclipse CI fluorescence microscope and analyzed using Image Pro Plus 6.0 (Media Cybernetics, Rockville, MD).