Male Apoe−/− and Apoe−/−AMPKα2−/− mice were fed a western diet containing 21% milk fat and 0.15% cholesterol for 10 weeks starting at 8 weeks of age. Similarly, 8-week old male Apoe−/−AMPKα2sm+/+ and Apoe−/−AMPKα2sm−/− mice were placed on western diet for the initial 6 weeks to establish aortic lesions. In the presence of western diet, mice were treated with 50mg/kg/day pravastatin for an additional 4 weeks. Saline solution was used as solvent control. Mice were sacrificed and blood was collected. Mice were then perfused via the left ventricle with 5 ml PBS followed by 10 ml 4% paraformaldehyde. Brachiocephalic arteries (BA) were carefully dissected and fixed overnight in 4% paraformaldehyde prior to embedding in optimum cutting temperature compound (OCT; BDH Laboratory Supplies).
Details of materials and experimental procedures are in the Methods section in the Online Data Supplement.