Ten days after the final behavioral test (1-month post alcohol or saccharin intake; Fig. 3), mice (N = 11) were rapidly decapitated, and brains were quickly removed and flash-frozen in cold isopentane (−40°C; 2-methylbutane; Sigma-Aldrich) for use with the multiplex assays described below. Thick coronal sections (0.8–1.0mm) were taken on a cryostat (Leica Biosystems), and using a mouse brain atlas (Franklin & Paxinos, 2001), circular tissue punches (1mm diameter) were collected from the prefrontal cortex (PFC), medial prefrontal cortex (mPFC), nucleus accumbens (Acb), amygdala (AMY), medial hippocampus (mHPC), lateral hippocampus (LHPC), CA1 region of the hippocampus (CA1), lateral entorhinal cortex (LEC) and medial entorhinal cortex (MEC). Tissue punches from each brain region were homogenized with an ultrasonifier in 100μL of buffer (pH7.4) containing 20mmol/L Tris-HCl, 7.8mmol/L Tris-Base, 150mmol/L NaCl, 0.02% Tween-20, and a cocktail of phosphatase and protease inhibitors (HALT) and frozen at −80°C until analysis.
