Hippocampus Tissue Extraction Protocol

Mice were anesthetized in a CO₂ chamber and then transcardially perfused with 20 mL ice-cold phosphate buffered saline (PBS). The right hemisphere was post-fixed by incubation for 24 h in 4% PFA followed by impregnation in 30% sucrose until the brains sunk to the bottom. Fixed brains were then cut into 30 µm coronal sections with a cryostat (Leica, Deer Park, IL, USA). The contralateral hemisphere was dissected to isolate dHc, which was hemi-sected: half of the dHc was homogenized in radioimmunoprecipitation assay buffer (RIPA; Sigma-Aldrich, Burlington, MA, USA, R0278; 50 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing phosphatase and protease inhibitors (Roche, South San Francisco, CA, USA), was centrifuged for 20 min at 15,000× g, and the supernatant collected. The other half of the brain was used for RNA extraction using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA). A separate cohort of mice was anesthetized and perfused with PBS as described above, and the entire hippocampus was isolated and lysed in 8M urea, 10 mM Tris, 100 mM NaH₂PO4, pH 8.5, supplemented with HALT protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Asheville, NC, USA), as described in [32 (link)].