Immunostaining was conducted using primary antibodies against Iba1 (1 μg/ml; Wako Chemicals USA, Richmond, VA), neutrophil elastase (NE) (1:50; Santa Cruz Biotechnology, Santa Cruz, CA), IL-6 (15 μg/ml; R & D Systems, Minneapolis, MN), and iNOS (2 μg/ml; Millipore, Billerica, MA). Brain sections were incubated with phosphate buffer (pH 7.4) containing 2% bovine serum albumin and respective primary antibodies overnight at 4°C. Immunostaining was visualized by either Alexa Fluor (Invitrogen)-conjugated or horseradish peroxidase (HRP)-conjugated secondary antibodies against appropriate immunoglobulin. HRP-labeled immune-complex was visualized by Vectastain ABC kit with the DAB reaction described above. Cellular and tissue architectural changes were determined by cresyl violet staining. Injured neurons were determined by Fluoro-Jade B staining by following the manufacture’s instructions (Millipore). The slides were immersed in distilled water for 2 minutes; then, transferred to 0.06% potassium permanganate for 10 minutes on a shaker table. After rinsing with distilled water, the slides were incubated for 20 minutes in 0.001% Fluoro-Jade B dissolved in 0.1% acetic acid. Slides were rinsed in water, dried at 37°C, dehydrated in xylene, and coverslipped.
