Sp5 mp inverted confocal microscope

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.

For uptake studies, BMDCs were seeded at a density of 50,000 cells per well into 1.5 glass-bottom 96-well plates from Mat-tek and allowed to grow overnight. Samples were prepared by diluting them to 10 nM concentrations in a total volume of 100 μl BMDC cell medium (RPMI-1640 supplemented with 10% FBS, β-mercaptoethanol and granulocyte–macrophage colony-stimulating factor). Cell uptake was monitored using confocal microscopy (SP5 × MP inverted confocal microscope, Leica) at time points between 0 and 24 h. A sequential z-stack was imaged using a 0.2 μm slice thickness, in the following excitation sequence: first scan: Cy3 excitation/Cy5 emission, second scan: bright field and Cy3 excitation/ Cy3 emission; and third scan: Cy5 excitation/Cy5 emission. To bleach the acceptor dye, region of interest was set to 2.5 × zoom of the initial field of view and laser power for Cy5 excitation was increased to 100%. The slice was bleached for 10 min, until the Cy5 signal was absent. Zoom was reset to 1 and a new z-stack of the initial field of view was recorded.