Tunel stain, immunohistochemistry, and confocal microscopy
Following perfusion with ice cold PBS and 4% paraformaldehyde (PFA), brains were immersion-fixed overnight in 4% PFA, followed by cryoprotection in two exchanges of 30% sucrose for 48 h, then frozen in OCT (Fisher). Ten-micrometer sagittal sections were cut, then washed with PBS, and permeabilized with 0.1% Triton X-100/0.1% sodium citrate. Tunel reaction was prepared according to the manufacturer’s directions (Roche in situ cell death kit, TMR Red, Cat 12-156-792-910). Sections were immunostained for NeuN or Iba1 by blocking sections with 5% goat serum/0.1% Tween 20/PBS, then incubating overnight at 4 °C with rabbit-anti-NeuN (1:1000, Clone D3S3I, Cell Signaling Technology Cat 12943S) or rabbit-anti-Iba1 (1:1000, Wako Cat 019-19741). Sections were washed with 0.1% Tween 20/PBS, then incubated with goat-anti-rabbit-AF488 (4 μg/ml, Invitrogen, Cat A11008), counterstained with 1 μg/ml DAPI, and coverslipped with Prolong Gold Antifade Mountant (ThermoFisher, Cat P36930). Z-stack images (seven images separated by 1 μm) were captured using a Zeiss LSM 510 laser-scanning confocal microscope at × 40 objective magnification then compressed into a maximum intensity projection with accompanying software. For each region of each mouse, three to six images were taken from two different sagittal sections spaced at least 50 μm apart. The number of DAPI-positive nuclei also positive for NeuN and Tunel were counted by an individual blinded to the conditions.