Mouse brains were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) and processed for immunostaining as described previously [31]. TLR3 and HMGB1 were immunostained with TLR3 and HMGB1 antibodies. Microglia were stained with rabbit anti-Iba1 antibody. NADPH oxidase membrane subunit gp91phox was immunostained with monoclonal anti-mouse gp91phox IgG. Activated caspase-3 was immunostained with polyclonal anti-cleaved caspase-3 antibody. Caspase-3 co-labeling with NeuN was performed with caspase-3 and NeuN antibodies. Neurons were stained with MAP2 or NeuN antibody. Astrocytes were labeled with GFAP antibody. Immunolabeling was visualized by using nickel-enhanced 3,3′-diaminobenzidinne (DAB) or Alexa Fluor 488 or 555 dye.
