Immunofluorescence staining

To identify and localize IL-6 protein in coronary arterioles, transverse sections of the mouse heart were stained using markers of endothelial cells, vascular smooth muscle cells, and macrophages. Freshly isolated hearts were embedded and frozen in OCT and sectioned at 5 μm. Slides were incubated with blocking solution (10% donkey serum in PBS) and permeabilized (0.1% Triton X-100 in PBS). Primary antibodies to IL-6 (goat polyclonal 15 micro g/ml, AF-406-NA; R&D), the endothelial cell marker, von Willebrand factor (vWF; rabbit polyclonal, 1:1,000, ab6994; Abcam), smooth muscle α-actin (rabbit polyclonal, 1:800, ab5964; Abcam), or macrophage marker CD68 (rat monoclonal, 1:1000, ab53444; Abcam) were used for sequential double immunofluorescence staining. Secondary antibodies were conjugated with the fluorophores FITC or Texas red. Sections were mounted in an anti-fading agent (Slowfade gold with DAPI; Invitrogen), and then the slides were observed and analyzed with a fluorescence microscope (IX81; Olympus) with a x40 objective (0.90 numerical aperture). For negative controls, primary antibodies were replaced with goat polyclonal IgG (Abcam), rabbit polyclonal IgG (GeneTex), and rat monoclonal IgG (Abcam) isotype controls at the same concentration [28]. The specificity of the primary antibody was confirmed as the absence of immunofluorescence staining signals in the IL-6^-/- mice.