Microglial immunohistochemistry

Microglial morphology was characterized in the striatum at the level of the anterior commissure. Striatal sections were mounted, and microglia were immunolabeled with anti-Iba1 (RRID: AB_2636859, Abcam ab178846). Brain sections were permeabilized for 15 minutes in methanol containing 3% hydrogen peroxide and then blocked with 1× TBE, 0.05% (v/v) Tween-20, and 2% (v/v) normal serum. Sections were incubated for 3 days in primary antibody (1:1000) with appropriate controls in blocking buffer at 4°C. They were then washed and incubated for 24 hours with biotinylated secondary antibody prior to washing, labeling with streptavidin-HRP conjugate (Vector Labs, RRID:AB_2336827), and detection using 3,3’-diaminobenzidine per the manufacturer’s instructions. Reactions were quenched with water and then washed twice in 1× TBE prior to coverslipping with fluoromount G (Southern Biotech).