2.12 Immunohistochemical assessment of brain microglia/macrophages

To assess microglial/macrophage activation, a separate series of 60μm coronal brain sections were probed with rat monoclonal anti-mouse CD68 (1:200; Bio-Rad), mouse adsorbed biotinylated goat anti-rat IgG antibody (10μg/ml, Vector Labs), followed by ABC Vectastain Reagent and a DAB peroxidase substrate (Vector Labs), according to the manufacturer’s instructions. Digitalized brightfield images captured with a 20× objective (Leica) were processed and analyzed using the Fiji package of ImageJ (http://fiji.sc), as described previously³⁸. All images were processed with the Colour Deconvolution plug-in using the H-DAB vector to remove background and to separate the DAB-only image panel for analysis. CD68+ cells were counted within ipsilateral cortex areas on 3 sections spanning the injury site per brain, and averages were expressed as cells/mm².