Normal blocking serum

The 2nd sections were processed for Iba1-immunohistochemistry. Briefly, after endogenous peroxidase activity was blocked with 0.6 % hydrogen peroxidase, free-floating sections were incubated in 0.01 M phosphate-buffered saline (PBS, pH 7.4) containing 1% normal donkey serum (Jackson ImmunoResearch, West Grove, PA), 0.3 % Triton X-100 (Sigma, St. Louis, MO), and polyclonal rabbit anti-Iba1 antibody (1:10,000, Wako Chemicals USA, Richmond, VA #019−19741) for 65 h at 4 °C. The sections were then incubated in PBS containing Triton-X, normal blocking serum, and biotinylated secondary antibody for 1 h at room temperature, and then in PBS containing avidin-biotinylated HRP complex for another 1 h using the Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA). Subsequently, the sections were incubated for 5 min in 0.05 M Tris buffer (pH 7.2) containing 0.03 % 3′,3′-diaminobenzidine (DAB) as a chromogen (Sigma-Aldrich, St. Louis, MO) and 0.0075 % hydrogen peroxide. All sections were rinsed in distilled water, mounted on slides and were counterstained with FD cresyl violet solution (FD NeuroTechnologies, Inc.). After dehydration in ethyl alcohol, sections were cleared in xylene and cover-slipped in Permount (Fisher Scientific).