Immunohistochemistry staining was performed following a protocol as previously described.40 (link),41 (link),43 (link) Arterial rings were washed with sterile ice-cold PBS and fixed in 2% paraformaldehyde in PBS. The fixed arteries were then transferred in 30% sucrose in PBS until they were processed for embedding in OCT compound. For immunofluorescent staining, 5 μm frozen cross-sections were permeabilized with 0.2% Triton X-100 in PBS, blocked with 10% fetal bovine serum in PBS, and processed for labeling with rabbit anti-eNOS (cat# 610298, BD Bioscience), anti-collagen I(cat#SAB4500362, Sigma-Aldrich), anti-collagen III (cat#AB757P, EMD Millipore), and anti-collagen IV (cat#ab6586, Abcam), respectively. After washing with PBS, the frozen sections were incubated with Cy3-conjugated (red) goat anti-rabbit antibody (Molecular Probes). DAPI was used to counterstain the nuclei. The slides were then examined under an Olympus CX41 fluorescence microscope and images of 4–8 view fields for each slide were captured with a Qcolor3 camera. Elastin contents were measured with Verhoeff staining.31 (link) Fluorescent intensities of elastin, collagen I, III, IV positive area for the whole vessel wall, and the intensity of eNOS for the intima were quantified by using Image-Pro plus 7.0 and ImageJ, respectively.