Brains were stored at −70° C until sectioned. On the day of preparation of micro-punch samples, brains were transferred to a cryostat set at −6 to −10° C at least 2 hr prior to sectioning. Sections (300 μm) were obtained and transferred to glass slides that had been pre-cooled in the cryostat. Micro-punch sampling was done on a frozen stage (−25 to −35° C) with an anatomic microscope equipped with a cool microscope lamp. The stereotaxic atlas of Paxinos and Watson (1998) was used to identify the ACB-shell and CeA. Microdissection needles (Fisher Scientific) with an inner diameter of 0.77 mm were used to obtain samples of the ACB-shell and CeA. This inner diameter fits within the entire region and minimizes contamination from adjacent tissue. Punches are taken bilaterally from 2–3 sections. A different fresh sterile micro-punch needle is used between regions and for each animal. After withdrawing the micro-punch sample, a distinct demarcated hole remained; this hole was used to validate the micro-dissection method. All equipment used to obtain tissue was treated with RNAse Zap (Ambion, Inc. Austin, TX) to prevent RNA degradation. A second trained individual independently verified the quality of the micro-punch dissections.
