Visualizing Aortic Endothelium Localization

Tissues were stored at −80 °C before use and cryosectioned as described33 (link). Mouse en face aortic preparations were fixed in 2% formalin and permeabilized with 0.5% triton. Confocal microscopy was performed with a Leica SP5 MP inverted confocal microscope. Localization of injected LO1 or control IgG3 in relation to endothelium was studied by intravenously injecting phycoerythrin (PE)-conjugated anti-CD31 to identify endothelium (Biolegend, San Diego, CA). Image analysis was undertaken using Volocity 3D Image Analysis Software (PerkinElmer, Massachusetts USA). Ex vivo epifluorescence imaging of rabbit aortae was performed with an Eclipse 90i microscope (Nikon Instruments, Melville, New York).
Further methods for studying mouse, human and rabbit sections are included in the supplementary methods section.