Vevo 770 high resolution imaging system

All protocols were approved by Yale University's Institutional Animal Care and Use Committee, and were performed and administered within National Institutes of Health and ethical guidelines. The mouse vein graft model was performed as previously described.^{8 (link)} Briefly, 12-wk-old C57BL/6 wild type (WT) and eNOS knockout (eNOS KO) mice (The Jackson Laboratory) were purchased. An approximately 2.0-mm segment of the intrathoracic inferior vena cava (WT or eNOS KO) was isolated and implanted into the infrarenal abdominal aorta of a recipient WT mouse. The abdominal aorta was temporarily occluded with atraumatic micro-clamps and a segment corresponding to the length of the vein graft was excised. The vein was sutured into the arterial circulation using 10–0 nylon in continuous fashion. Vein grafts were followed postoperatively using the Vevo 770 High-Resolution Imaging System (VisualSonics). At 3 weeks after surgery, mice were sacrificed and vein grafts were fixed by transcardial perfusion of phosphate-buffered saline (PBS), followed by 10% formalin. Vein grafts were removed and fixed overnight in 10% formalin, followed by 70% alcohol for 24 hours. Tissue was embedded in paraffin and sectioned (5 μm thick). Tissue sections were deparaffinized and either stained with hematoxylin and eosin or processed for immunofluorescence.