2.6. Immunohistochemistry and Amyloid Plaque Staining

Coronal brain sections (30 μm) were stained with the ABC Peroxidase Staining Kit (Pierce) by following the manufacture's protocol. All sections were blocked by 3% hydrogen peroxide in PBS for 20 minutes. Sections were incubated at room temperature for 30 minutes with 5% serum and 0.3% Triton X-100 in PBS and then incubated overnight at 4°C with GFAP antibody (1 : 4,000; AB5541; Millipore). Secondary biotinylated antibody was used at a dilution of 1 : 1000 (Vector Laboratories). After washing 3 times with PBST, sections were immersed into ABC reagent for 30 min. After the GFAP staining, amyloid plaques were detected with commercial kit of Congo Red (Sigma-Aldrich) by following the instruction. Sections were immersed in Mayer's Hematoxylin Solution for 5 min and then rinsed in tap water for 5 min. After being incubated in Alkaline Sodium Chloride Solution for 20 min, the sections were placed in filtered Alkaline Congo Red Solution for another 20 min. All sections were rinsed with absolute ethanol for 3 times and cleared in xylene before being mounted.