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Figure 5 Cell death and cytokine expression in sclerotic aortic valves of apolipoprotein E (ApoE)–/– mice. (A) Hearts were harvested from 8-week-old wild-type mice (n = 3) and 97- to 108-week-old ApoE–/– mice (n = 3) and snap-frozen in OCT compound. Frozen sections (4 µm) were stained for MCP-1, PDGF, VEGF, and SDF-1 using the avidin-biotin complex technique and vector red substrate (red). Nuclei were counter-stained with hematoxylin. Arrows indicate positive cells. Scale bar = 20 µm. (B) Apoptotic cell death was detected by TUNEL staining. Frozen sections (4 µm) were fixed with 4% paraformaldehyde and permeabilized. TUNEL staining (green) was performed according to the manufacturer’s specification. Arrows indicate apoptotic cells. Scale bar = 20 µm. (C) Co-localization of TUNEL-positive cells and cytokine expression in the sclerotic aortic valve of 104-week-old ApoE–/– mice. Immunofluorescence staining for chemokine or cytokines (red) was performed on the frozen sections after TUNEL staining. Scale bar, 20µm.