The coronal sections (40 μm) of mouse brains were cut using a cryostat (Leica CM3050S) and stored at −20°C in cryoprotective storage solution [300 g of sucrose, 10 g of polyvinylpyrrolidone, 500 ml of phosphate buffer (0.1 M), 300 ml of ethylene glycol, and up to 1000 ml of ddH2O] until use. Upon use, the sections were washed three times with PBS and incubated with 3% H2O2 for 15 min to inhibit endogenous peroxidases. After three washes with PBS, the sections were blocked for 1.5 hours with blocking buffer (0.3% Triton X-100 + 5% goat serum + 2% bovine serum albumin in PBS) at room temperature, followed by primary antibodies overnight at 4°C. Primary antibodies used include mouse monoclonal anti-6E10 (SIG-39320, Covance, Princeton, NJ, USA), rabbit polyclonal anti-Iba1 (019-19741, Wako, Richmond, VA, USA), mouse monoclonal anti-GFAP (MAB360, Millipore, Darmstadt, Germany), rat monoclonal anti-CD68 (ab53444, Abcam, Cambridge, MA, USA), rat monoclonal anti-Trem2 (#237916, R&D Systems, Minneapolis, MN, USA), and rabbit polyclonal anti-Tom20 (Sc-11415, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

For immunohistochemical staining, the signal was visualized with biotinylated immunoglobulin G (IgG), followed by streptavidin-conjugated horseradish peroxidase (VECTASTAIN ABC Kit, Zhongshan Jinqiao, Beijing, China), and finally reacted with hydrogen peroxide (DAB Kit, Zhongshan Jinqiao). Images were captured using a Leica SCN400 scanner. The GFAP- and Iba1-positive cells and the number of Aβ plaques in the PFC and hippocampal DG region were counted blindly using ImageJ Pro Plus software.

For immunofluorescent costaining, the sections were labeled with fluorescent secondary antibodies as follows: Alexa Fluor 488 AffiniPure donkey anti-rat IgG (712-545-150, Jackson ImmunoResearch, West Grove, PA, USA), TRITC AffiniPure goat anti-rabbit IgG (111-025-003, Jackson ImmunoResearch), Cy5-conjugated donkey anti-mouse IgG (715-175-150, Jackson ImmunoResearch), Alexa Fluor 594 AffiniPure goat anti-mouse IgG (115-585-003, Jackson ImmunoResearch), and FITC AffiniPure goat anti-rabbit IgG (111–095-003, Jackson ImmunoResearch). The images were acquired using a Nikon confocal microscope (Nikon, Melville, NY, USA).

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