P6148

Animals were anesthetized with Fatal Plus (Vortech Pharmaceuticals, Catalog #0298-9373-68). Mice were transcardially perfused with cold 1% paraformaldehyde (PFA; Sigma Aldrich, Catalog #P6148) for 1 min, followed by cold 4% PFA with 0.125% glutaraldehyde (Fisher Scientific, Catalog #BP2547) for 10 min. A peristaltic pump (Cole Parmer) was used for consistent administration of PFA. Immediately following perfusion, mice were decapitated, and the whole brain was removed and drop-fixed in 4% PFA containing glutaraldehyde for 8-12 hours at 4°C. After post-fixation, the brains were sliced in 250 μm coronal sections using a Leica vibratome (VT1000S) with a speed of 70, and frequency of 7. The platform was filled with cold 0.1 M PB buffer, and the brain was glued (Loctite) perpendicular to the stage, cerebellum side down. All slices were stored one slice per well in a 48-well plate containing 0.1% sodium azide (Fisher, Catalog#BP922I) in 0.1M PB at 4°C. Notably, these procedures were performed according to (75 (link)). For PBS perfusions, animals were anesthetized with Fatal Plus. Mice were transcardially perfused with cold 1X PBS for 2 minutes. Immediately following perfusion, the brain was extracted and dissected into two hemispheres. Each hemisphere was immediately flash frozen in 2-methylbutane (Sigma, Catalog#320404), placed on dry ice, and stored at −80°C.

Mice were anesthetized with Fatal Plus (Vortech Pharmaceuticals, Catalog #0298–9373-68), then transcardially perfused, first with cold 1% paraformaldehyde (PFA) (Sigma Aldrich, Catalog #P6148) for 1 min, followed by 4% PFA with 0.125% glutaraldehyde (Fisher Scientific, Catalog #BP2547) for 10 min. To ensure PFA was administered consistently, a peristaltic pump (Cole Parmer, Catalog #EW-07528–10) was used. Brains were extracted, then drop-fixed in 4% PFA with 0.125% glutaraldehyde for 8–12 h at 4°C. At that point, the brains were coronally sliced into 250 μm thick sections using a Leica vibratome (VT1000S) (speed: 70, frequency: 7) in 0.1 M PB cutting solution. For long-term storage, slices were stored in 0.1 M PB with 0.1% sodium azide (Fisher Scientific, Catalog #BP922I) at 4°C, one slice per well in a 48-well plate.

Animals were anesthetized with Fatal Plus (Vortech Pharmaceuticals, Catalog #0298-9373-68). The abdominal cavity and pericardium were carefully opened to expose the heart. Mice were transcardially perfused with cold 1% paraformaldehyde (PFA) for 1 min, followed by 10 min of 4% paraformaldehyde (Sigma Aldrich, Catalog #P6148) with 0.125% glutaraldehyde (Fisher Scientific, Catalog #BP2547). Peristaltic pump (Cole Parmer) was used for consistent administration of PFA. Immediately following perfusion, the mouse was decapitated, and the whole brain was removed and drop fixed in 4% PFA containing glutaraldehyde for 8-12 hours at 4°C. After post-fixation, the brains were sliced in 250 μm coronal increments using a Leica vibratome (VT1000S) with a speed of 70, and frequency of 7. The platform was filled with cold 0.1 M PB buffer, and the brain was glued (Loctite) perpendicular to the stage, cerebellum side down. All slices were stored one slice per well in a 48-well plate containing 0.1% sodium azide (Fisher, Catalog#BP922l) in 0.1M PB. Plates covered in parafilm are stored long term at 4°C. Notably, these procedures were performed according to (Dumitriu et al. 2011 (link)).