Atherosclerosis Quantification and Immunohistochemistry

One hour prior to sacrifice, all mice were intraperitoneally (i.p.) injected with the hypoxia-specific marker pimonidazole (100 mg/kg, hypoxyprobe Omni HP3 kit, Hypoxyprobe Inc., Burlington, MA, United States). Mice were euthanized with a pentobarbital overdose (100 mg/kg i.p.), and blood was withdrawn via the right ventricle for flow cytometry, absolute white and red blood cell counts (Coulter Ac.T diff, Beckman Coulter, United States), and total cholesterol analysis. Mice were perfused via the left cardiac ventricle with PBS containing sodium nitroprusside (0.1 mg/ml; Sigma-Aldrich, Seelze, Germany). Aortic root and all organs were subsequently excised and fixed in 1% PFA overnight, processed, and paraffin-embedded.

Aortic roots and arches were serially sectioned and stained with hematoxylin and eosin (H&E, Sigma) for plaque area and lipid core content quantification. Plaque stage was quantified on plaque characteristics such as foam cells (early), fibrous cap (intermediate), and necrotic core (advanced). Five consecutive H&E sections at 20 μm intervals were analyzed blindly using computerized morphometry (Leica QWin V3, Cambridge, United Kingdom) and averaged per mouse. Sections within this 100 μm interval were used for the remaining immunohistochemical stainings. If appropriate, antigen retrieval was performed at pH6 (Dako REAL target retrieval, Dako). Atherosclerotic plaques were characterized for macrophage content (MAC3 + area/total area, BD Cat# 553322, RRID:AB_394780). Hypoxia was detected in the aortic roots, using a rabbit polyclonal antibody (clone 2627, Cat# HP MAb-1, RRID:AB_2801307) directed against pimonidazole derivates, formed in vivo specifically in hypoxic but living cells (% pimonidazole/total plaque area). Liver inflammation was quantified as percentage CD45 + cells/total visible area (BD Cat# 553076, RRID:AB_394606).