Brain perfusion and fixation

Mice and rats were anesthetized with an isoflurane (1–3%)/oxygen vapor mixture and perfused through the ascending aorta with 0.1 M PBS, followed by 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4). Brains were collected, post-fixed overnight in 4% PFA in 0.1 M PBS and then cryoprotected in 30% sucrose in 0.1 M PBS (pH 7.4) for 72 h at 4°C. The cryoprotected brains were embedded in Tissue-Tek OCT compound (Finetek, Torrance, CA). 30–40 μm coronal sections were cut on a cryostat (Leica Biosystems, Wetzlar, DE) and collected directly onto slides and allowed to dry overnight at room temperature. Slides were stored at −20°C until processing.