Immunostaining and immunofluorescence

For histological analysis, the aortic roots were sliced into 5 μm serial cryostat sections in the aortic valve plane. Cryosections were fixed in 4% paraformaldehyde for 30 min and rinsed in the tris‐buffered saline (TBS). Non‐specific binding sites were blocked using an avidin/biotin blocking kit, followed by incubation of the sections in 1% BSA (Sigma) and 5% normal goat serum in PBS. The slides were incubated overnight at 4°C with an anti‐mouse SMA antibody (1:200) for smooth muscle cells, an anti‐CD68 antibody (1:200) for macrophages, an anti‐Foxp3 antibody (1:50) for regulatory CD4⁺ T cells (Tregs) and anti‐IL‐17A and anti‐CD4 antibodies (1:50) for T cells. The slides were rinsed and incubated with secondary antibodies. The processed sections were visualized using an Olympus microscope (IX71; Olympus Corporation, Tokyo, Japan) and a fluorescence microscope (Olympus Microscope BX‐51; Olympus Corporation) or a confocal microscope (Nikon, Tokyo, Japan). The means from 10 serial cryosections/tissue from eight samples per group were recorded. Macrophages and smooth muscle cells were quantified by assessing the percentage of the total plaque area that was positive for each marker. CD4⁺ T cells and Tregs were assessed by counting the number of positively stained cells.