Harris uni core micro punch

After brain extraction, target regions were immediately dissected out on an ice plate according to the Bregma zero coordinates: Amygdala (AMY, −1.05~−2 mm), dorsal striatum (dST, +1.04~−1.46 mm), hippocampus (HIP, −1.5~−2.18 mm) and prefrontal cortex (PFC, +1.54~+1.98 mm) [30 ] by a 1.0-mm Harris Uni-Core micro-punch (Electron Microscopy Sciences; Hatfield, PA 19440, USA). Bilateral punches tissues were pooled from each mouse. Each tissue sample was quickly cryopreserved in liquid nitrogen and stored at −80 °C until assay.

The mice were euthanized, and the brains were rapidly extracted and sliced into coronal sections (0.4 mm thick) using a brain matrix followed by either preparation of synaptosomes from the striatum as described above or extraction of tissue from specific brain regions. Tissues from dStr, NAc, ventral tegmental area (VTA), and substantia nigra compacta (SNc) were extracted using a 1.0 mm Harris Uni-Core micropunch (Electron Microscopy Sciences). Synaptosomes and brain tissues were solubilized in the lysis buffer containing 50 mM Tris-HCl pH 7.4, 0.5% sodium deoxycholate, 1% NP-40, 150 mM NaCl, 2 mM EGTA, 2 mM EDTA, and phosphatase and protease inhibitors. Lysates were cleared by centrifugation for 15 min at 16,000 × g at 4°C. Aliquots of lysates (30 µg of protein for striatal synaptosomes, dStr, and NAc; 50 µg for whole MB, VTA, and SNc) were denatured in sample buffer at 37°C for 30 min. Lysates were resolved by 7.5% SDS-PAGE, transferred to nitrocellulose (Li-COR), and probed with appropriate primary and secondary antibodies conjugated to far-red fluorescent dyes (IRDye-680 or IRDye-800) followed by detection using Odyssey Li-COR system. Quantifications were performed using ImageJ software.

After the behavioral (day 25) and social defeat stress (day 18) tests were completed (Figure 2), the mice were terminated by decapitation under ether anesthesia. The amygdala and dentate gyrus regions of the dorsal hippocampus were punched out bilaterally using a 1-mm Harris Uni-Core micropunch (Electron Microscopy Sciences, Hatfield, Pennsylvania, United States).