Cf 568

Immunofluorescent assay was performed as previously described.13, 14, 17, 18, 19 Briefly, frozen tissue sections were labeled with appropriate isotype IgG control or antibodies as indicated in the figures and visualized with appropriate secondary antibodies conjugated with CF 488A or CF 568 (Sigma) for the unconjugated primary antibodies. Sections were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma). Images were examined and acquired with SP5 confocal microscopy with Leica TCS Sp5 software (Leica) at room temperature. Positively stained areas for the respective antibody in a given image were highlighted and quantified (percentage over the atherosclerotic lesion area) by computer‐assisted quantification using the Axiovision software. Two experienced investigators blinded to the treatments each tissue had received performed this analysis. Three sections were analyzed per aortic roots (or per mouse) and averaged.

Detailed information on the primary antibodies used in this work is listed in Supplementary Table 5. The second antibodies used in this study were Alexa Fluor 647 (dilution 1/100, anti-mouse A31571, anti-rabbit A31573, anti-rat A21247, anti-goat A-21447; Thermo Fisher Scientific), Atto 647 N (dilution 1/100, anti-mouse 50185-1ML-F; Sigma-Aldrich), CF568 (dilution 1/100, anti-rabbit 20098, anti-rat 20092, anti-mouse 20105; Biotium), and Alexa Fluor 488 (dilution 1/100, anti-mouse A21202; Sigma-Aldrich). Dyomics 654 (Dy654)-conjugated secondary antibodies were custom-made by conjugating Dy654 N-hydroxysuccinimidyl (NHS) ester (654-01; Dyomics) to different IgG antibodies respectively (anti-mouse 715-005-151, anti-rabbit 711-005-152, anti-rat 712-005-153; Jackson ImmunoResearch).