Animals were anesthetized with Fatal-Plus (Vortech Pharmaceuticals, catalog no. 0298-9373-68). Mice were transcardially perfused with cold 1% paraformaldehyde (PFA; Sigma-Aldrich, P6148) for 1 min followed by cold 4% PFA with 0.125% glutaraldehyde (Fisher Scientific, catalog no. BP2547) for 10 min. A peristaltic pump (Cole-Parmer) was used for consistent administration of PFA.Immediately after perfusion, mice were decapitated, and the whole brain was removed and drop-fixed in 4% PFA containing glutaraldehyde for 8 to 12 hours at 4°C. After fixation, the brains were sliced in 250-μm coronal sections using a Leica vibratome (VT1000 S) with a speed of 70 and frequency of 7. The platform was filled with cold 0.1 M phosphate buffer (PB), and the brain was glued (Loctite) perpendicular to the stage and the cerebellum side down. All slices were stored one slice per well in a 48-well plate containing 0.1% sodium azide (Fisher Scientific, catalog no. BP922I) in 0.1 M PB at 4°C. These procedures were performed according to (75). For PBS perfusions, animals were anesthetized with Fatal-Plus. Mice were transcardially perfused with cold 1× PBS for 2 min. Immediately after perfusion, the brain was extracted and dissected into two hemispheres. Each hemisphere was immediately flash-frozen in 2-methylbutane (Sigma-Aldrich, 320404), placed on dry ice, and stored at −80°C.

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