Brain Tissue Preparation and Immunohistochemistry

Brains were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) overnight, rinsed in PBS the following day, and were subsequently placed in 30% sucrose in PBS overnight. The cryopreserved brains were then placed in Optimal Cutting Temperature (OCT) compound (Sakura Finetek, Torrance, CA) and frozen on dry ice, after which they were sectioned into 12 µm coronal slices. The following primary antibodies were used: rabbit polyclonal anti-IbaI (1:500; Wako Laboratory Chemical, Osaka, Japan), anti-GFAP (1:500; Agilent Technologies, Santa Clara, CA), anti-cleaved caspase-3 (1:500; Cell Signaling Technology, Danvers, MA). Immunohistochemistry was performed as described previously^{91 (link)}. For biocytin detection, sectioned tissue was stained using the ABC-HRP kit (Vector Labs, Burlingame, CA) and TSA Fluorescein (PerkinElmer, Waltham, MA). Sections were nuclear counter-stained with DAPI (Molecular Probes, Eugene, OR).