Harris uni core 2

One day after the last RI test, brains were rapidly removed after cervical dislocation and then sliced to 1mm thick coronal sections using a mouse brain slicer matrix (Alto Stainless Steel Coronal 1.00mm Brain Matrix, CellPoint Scientific) on ice. The DRN was removed using a 2-mm punch biopsy tool (Harris Uni-Core-2.00, Electron Microscopy Sciences) in ice-cold PBS. Samples were collected into 1.5 mL tubes, immediately frozen on dry ice, and stored at −80 °C until cDNA synthesis. For RNA extraction and cDNA synthesis, DRN samples were homogenized in TRIzol Reagent (Invitrogen) and then chloroform was added to isolate RNA. The RNA layer was processed with RNeasy Micro Kit (Qiagen) to extract total RNA and the total RNA concentration was measured using Nanodrop (Thermo Fisher Scientific). cDNA was synthesized from 500 ng of total RNA by reverse transcription using qSCRIPT cDNA SuperMix (Quanta Biosciences). Quantitative PCR reaction was conducted with Perfecta SYBR Green (Quanta Biosciences). Analysis was done using the ΔΔ Ct method and samples were normalized to Gapdh.

One hour after the last RI test, brains were rapidly removed after cervical dislocation and sliced to 1mm thick coronal sections using the mouse brain slicer matrix (Alto Stainless Steel Coronal 1.00mm Brain Matrix, CellPoint Scientific) on ice. The medial prefrontal cortex, ventral striatum, hypothalamus, and DRN were removed using 2 mm punch cannula (Harris Uni-Core-2.00, Electron Microscopy Sciences) in ice-cold PBS. Samples were collected into 1.5 mL tubes, immediately frozen on dry ice, and stored at −80 °C. To measure brain IL-1β level, 100 μL extraction solution (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 % Triton-X100, and 1 μg/mL protease inhibitor cocktail [Complete tablet; Roche Diagnostics] in distilled water) was added to each frozen brain sample, and samples homogenized on ice. Then, samples were agitated for 90 min at 4 °C, centrifuged at 956 × g for 20 min at 4 °C, and the supernatants were collected into new tube. They were stored at −80 °C until ELISA assays were performed. For multiplex ELISA analysis, the DRN sample was collected the same as described above for IL-1β measurement, except the tissue was homogenized with ProcartaPlex cell lysis buffer (ThermoFisher) and left on ice for 30 min before centrifugation and collection of supernatant.