Neutral balsam

For immunohistochemical analysis, brain tissue was fixed in 4% paraformaldehyde at 4°C for 24 h, embedded in paraffin, and then cut into 4 µm-sections, hydrated and incubated in 3% hydrogen peroxide at room temperature for 10 min. The sections were washed three times with PBS and then stored at 4°C in PBS supplemented with 3% bovine serum albumin (cat no. B2064, Sigma-Aldrich, Merck KGaA) for 30 min. Sections were then incubated with antibodies against LRP-1 (1:200) or RAGE (1:200) overnight at 4°C, and a negative control was incubated with PBS. These sections were then exposed to biotinylated universal secondary antibodies (1:5,000; cat no. A0208; Beyotime Institute of Biotechnology) for 1 h and subsequently to streptavidin biotin horseradish peroxidase solution (1:1,000) at room temperature for 1 h, following washing with PBS. And subsequently stained with 3,3′-diaminobenzidine solution for 45 sec at 37°C. Sections were counterstained with hematoxylin at room temperature for 20 sec. Graded alcohols were then used to dehydrate dehydrate section, and they were subsequently fixed in neutral balsam (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) at 60°C for 30 min. LRP-1 and RAGE staining was evaluated using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).