Subjects were briefly exposed to CO2 and decapitated by guillotine. In Experiments 1 and 3, NAc was dissected from fresh brain on ice and 2-3 samples per treatment condition were pooled for fractionation. All subjects’ CPP expression in the final test was inspected and NAc samples from 2-3 rats with similar CPP expression were pooled per sample tube. The purpose of pooling was to ensure an adequate yield of synaptosomal fraction. The methods of subcellular fractionation were as described previously (e.g., Peng et al., 2014 (link)) and fractions were stored at −80°C until use.
In Experiment 2, brains were extracted and rapidly frozen in powdered dry ice. A series of 500-μm sections were cut using an IEC Minotome cryostat, and NAc core and shell were dissected using a combination of micropunch and microknife under an Olympus dissecting microscope. Tissue lysates were prepared as described previously (e.g., Zheng et al., 2013 (link)) and stored in sample buffer in aliquots at −80°C. The protein content was determined using the BCA reagent kit with bovine serum albumin as a standard (Pierce, Rockford, IL).
In Experiment 2, brains were extracted and rapidly frozen in powdered dry ice. A series of 500-μm sections were cut using an IEC Minotome cryostat, and NAc core and shell were dissected using a combination of micropunch and microknife under an Olympus dissecting microscope. Tissue lysates were prepared as described previously (e.g., Zheng et al., 2013 (link)) and stored in sample buffer in aliquots at −80°C. The protein content was determined using the BCA reagent kit with bovine serum albumin as a standard (Pierce, Rockford, IL).