A standard biotin-streptavidin-peroxidase method was used for all immunohistochemical staining. Reagents used in this study were monoclonal antibodies against CD68 (macrophages, 1:200, clone #: FA-11, Cat#: 137002), CD4 (CD4+ T cells, 1:200, clone #: GK1.5, Cat#: 100402), CD8 (CD8+ T cells, 1:200, clone #: 53-6.7, Cat#: 100702), B220 (B cells, 1:200, clone #: RA3-6B2, Cat#: 103202) and CD31 (1:200, clone#: 390, Cat#: 102402) (all above mentioned primary antibodies from Biolegend Inc, San Diego, CA, USA), goat anti-mouse polyclonal antibodies against MMP2 (1:200, Cat#: AF1488, R&D Systems) and MMP9 (1:200, Cat#: AF909, R&D Systems), a biotinylated goat anti-rat secondary antibody (1:400, Cat#: BA-9400, Vector Laboratories, Inc) or rabbit anti-goat IgG secondary antibody (1:400, Cat#: BA-5000, Vector Laboratories, Inc), and streptavidin-peroxidase conjugate (1:200, Cat#: 016-030-084, Jackson ImmunoResearch) (27 (link)–32 (link)). Macrophage accumulation was scored as the grade I to IV, and the densities of CD4+ T cells, CD8+ T cells, B220+ B cells and angiogenesis were quantified as the number of positively stained cells or neovessels per aortic cross section (ACS) (30 (link)). MMP expression levels were quantified as a positively stained area percentage of aortic wall using WinRood 6.5 image software, Mitani Co. Ltd., Tokyo, Japan.
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