Effect of the high-K₂ treatment (100 μg/g vitamin K₂) on the presence of calcification and MGP at the 12-week time point. Sections were prepared as described in the legend of Figure 2. Longitudinal sections of each thoracic aorta were stained with von Kossa (A) and immunohistochemically with anti–t-MGP (B), anti-ucMGP (C), and anti-cMGP (D) (see “Materials and methods” for details). It is shown that because of the high-K₂ diet cMGP (D) is up-regulated in the calcified area (A; along the elastic fibers). From panel C it can be seen that ucMGP is almost absent. Arrow indicates same area. Red stain indicates MGP, black stain indicates calcium, and blue indicates cell nuclei. Magnification, × 400. A indicates adventitia; M, media; and L, lumen. Details of image acquisition are provided in the legend of Figure 2.