Perfusion and Cryosectioning of Mouse Brain

Preparation and staining of mouse brain sections were previously described in [56 (link)]. In brief, the mice were deeply anesthetized with 10 : 1 mixture of ketamine 50 mg/ml (Tekam) and xylazine 20 mg/ml (Seton) (dose 0.1 ml/10 gm i.p.) diluted in 1X phosphate-buffered saline (PBS, pH = 7.4) (1X PBS) and then briefly perfused intracardially (flow rate: 8-10 ml/min for 2-5 min) with 1X PBS. This was followed by 10 min of 4% paraformaldehyde freshly prepared (Sigma-Aldrich) or commercially available 4% formaldehyde (a dilution of 37% formaldehyde solution, Sigma-Aldrich, in 1X PBS); all solutions were adjusted to pH 7.4. To ensure complete tissue fixation, brains were removed carefully and postfixed into the same fixative for 1 h at 4°C and then cryopreserved in 20-30% sucrose/PBS at 4°C in preparation for sectioning. Brains were then embedded in OCT compound (Tissue-Tek®, Ted Pella, Inc.) and sectioned sagittally into 14-20 μm thick slices at -20°C using a Leica CM3050 S cryostat (Leica Microsystems). They were then mounted on glass slides (Fisher Scientific) and stored at -80°C for further use in cresyl and immunofluorescence studies. The second group of brains was gently dissected to isolate the frontal cortex according to the mouse brain atlas [57 ], and the samples were stored at -80°C for a Western blotting assay.