Pa1 048

Rosa26^eGFP/L10a mice underwent stereotaxic surgery to infuse retrograde HSV-hEf1α-Cre into the NAc (see above). After waiting three weeks for full expression, mice were transcardially perfused with cold PBS, followed by 10% formalin. In other experiments, mice received ΔFosB overexpression (see above) and were sacrificed and perfused 4–8 weeks following surgery. Brains from all immunostaining experiments were postfixed 24 h in 10% formalin, cryopreserved in 30% sucrose, and sliced frozen on an SM2010R microtome (Leica) into 35-μm sections. Immunohistochemistry was performed using primary antibodies against FosB (ab11959; 1:1000; Abcam), GFP (ab5450; 1:1000; Abcam), and α2AAR (PA1-048; 1:1000; Invitrogen), and secondary antibodies (1:200; Jackson Immunoresearch) conjugated to fluorescent markers (AlexaFluor 488; Cy3; Cy5). Fluorescent images were visualized on an Olympus FluoView 1000 filter-based laser scanning confocal microscope. Intensity of α2AAR signal in individual cells was quantified using ImageJ software by an experimenter blinded to conditions.

Mouse hippocampus was homogenated in RIPA lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1 mM sodium orthovanadate, and protease inhibitors) as previously described [57 (link)]. Proteins were quantified with a BCA assay, separated by SDS-10% PAGE (µg/lane as indicated in the figures) and blotted onto PVDF membranes. Membranes were probed with rabbit α_2A adrenergic receptor polyclonal antibody (1:250, PA1-048, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and with mouse α_2C adrenergic receptor monoclonal antibody (1:500, S330A-80, Invitrogen, Thermo Fisher Scientific) overnight at 4 °C. Membranes were then incubated for 1 h at room temperature with the appropriate horseradish peroxidase-linked secondary antibodies (1:5000, A9044 and A9169, Sigma-Aldrich, St. Louis, MO, USA). Immunoblots were visualized using an ECL (enhanced chemiluminescence) Western blotting detection system. Images were acquired using the Alliance LD6 images capture system (Uvitec, Cambridge, UK) and analysed by UVI-1D software (Uvitec, Cambridge, UK).