Effect of different dietary treatments on aortic calcification at the 12-week time point in male Wistar Kyoto rats. Rats (n = 6 per group) were treated with the following dietary regimens; row 1 represents 12 weeks of normal vitamin K diet (5 μg/g K₁), row 2 represents 12 weeks of the W&K diet (3 mg/g warfarin and 1.5 mg/g vitamin K₁), row 3 represents 6 weeks of normal vitamin K (5 μg/g K₁) after 6 weeks of W&K, row 4 represents 6 weeks of high–vitamin K₁ (100 μg/g) diet after 6 weeks of W&K, and row 5 represents 6 weeks of the high–vitamin K₂ (100 μg/g) diet after 6 weeks of W&K. The thoracic aorta segment (between the aortic arch and the renal branch) was removed immediately after killing the animals in each diet group and fixed in 1% buffered formalin. Subsequently, longitudinal sections of each aorta were stained for mineral by von Kossa stain (column 1), ucMGP (column 2), and cMGP (column 3). Red stain indicates MGP, black stain indicates calcium, and blue indicates cell nuclei. Magnification, × 100. Acquisition was performed using an Axioskop 40 microscope (Carl Zeiss, Sliedrecht, The Netherlands) equipped with Achroplan 10×/0.25 objective lens and using an E-PI 10×/20 aperture. Pictures were taken using a Canon Powershot G5 (Canon, Hoofddorp, The Netherlands) and corrected for contrast and brightness using Adobe Photoshop CS2 for Windows (Adobe Systems, San Jose, CA).