Normal goat serum (ngs)

Paraffin-embedded temporal cortex sections (obtained from Netherlands Brain Bank) from a 92-year-old control male were hydrated, rinsed in phosphate-buffered saline (PBS) for 10 min, and treated with 1.5% hydrogen peroxide in PBS for 1 hour at 37 to quench endogenous peroxidase activity. After being washed in PBS containing 0.5% triton X-100 (PBST) (3*5 min), the sections were treated with microwaves (700 W) in 0.05 mol/L citrate-buffered saline (pH 6.0) for 2×10 min for antigen retrieval. Subsequently, the sections were washed in PBST (3×5 min) and incubated in 5% normal goat serum (Vector Laboratories, Burlingame, CA) in PBST for 1 hour at 37 to block nonspecific staining. Then the sections were incubated with primary antiserum of rabbit anti-CARS (Novus biological, NBP1-86624, at 1:200 dilution) in PBST containing 5% normal goat serum for 24 hours at 4 . Amplification was performed with biotinylated goat anti-rabbit IgG (1:200; Vector Laboratories, USA) and avidin-biotin-peroxidase complex (1:200; Vector Laboratories). Finally, the immune complex was visualized by incubation with 0.05% 3, 3'-diaminobenzidine (Sigma-Aldrich) in PBST containing 0.03% H2O2 for 10 min. The sections were mounted in Tris-HCl buffer containing 0.5% gelatin and dried overnight at room temperature. Photographs were collected using an Olympus BX52 microscope (Olympus, Japan).