Immunohistochemical analysis of murine aortic tissues

Mouse aortic tissues were harvested and embedded in optimal cutting temperature (OCT) compound, and aorta segments at 6 μm were prepared as described previously.^{16 (link),17 (link)} The serial sections were stained with antibodies for macrophages (Mac-3, 1:900, 553322, BD Biosciences, San Jose, CA), T cells (CD4, 1:90; 553043, and CD8, 1:100, 01041D, BD Biosciences), major histocompatibility complex class-II MHC-II) (1:250, 556999, BD Biosciences), smooth muscle cell (SMC) (α-actin, 1:750, F3777, Sigma-Aldrich), CD31 (1:1500, 553370, BD Biosciences) and neutrophils (Ly6G, 1:200, BE0075, BioXcell, West Lebanon, NH). CD4⁺ and CD8⁺ T cells, and CD31⁺ microvessels were counted blindly and presented as numbers of total cells per mm² of lesion area. Ly6G⁺ neutrophils were counted and presented as number of cells per section. MHC-II-positive and Mac-3-positive areas were measured using computer-assisted image analysis software (Image-Pro Plus; Media Cybernetics, Bethesda, MD) and presented as positive area per mm² of lesion area. Media SMC accumulation was graded according to the grading keys described previously.^{32 (link)}