Clone g a 5

Immunohistochemistry was carried out on free-floating sections for dorsal and ventral hippocampus under moderate shaking. All washes and incubations were done in 0.1 M phosphate buffer pH 7.4, containing 0.3% bovine serum albumin and 0.3% Triton X-100. The endogenous peroxidase activity was quenched for 10 min at room temperature in a solution of 3% hydrogen peroxide in 30% methanol. After several washes in buffer, sections were incubated overnight at 4 °C with a monoclonal antibody for ionized calcium-binding adapter molecule 1 (Iba1) (diluted 1:2000), a marker of microglia, or a monoclonal antibody for glial fibril acid protein (GFAP) (diluted 1:1000; Clone GA5, Sigma-Aldrich), a marker of astrocytes. Sections were then rinsed in buffer and incubated for 2 h at room temperature with biotinylated goat anti-mouse immunoglobulin G (diluted 1:300; Pierce). After several washes in buffer, sections were incubated for 90 min at room temperature with avidin-biotin-peroxidase complex (diluted 1:250; ImmunoPure ABC peroxidase staining kit). The reaction product was revealed by incubating the sections with 2 mg/mL 3,3-diaminobenzidine (Sigma-Aldrich) and 0.01% hydrogen peroxide in 0.1 M phosphate buffer. Then, sections were dehydrated, mounted on gelatinized slides, coverslipped, and examined with a Leica DMRB-E microscope.