Von Kossa staining and calcium concentration measurement

The aorta was stained for calcification with Von Kossa [9]. Tissue sections were incubated with 1% silver nitrate solution under ultraviolet light for 30 min followed by incubation with 5% sodium thiosulfate for 10 min to remove the unreacted silver. Aortic sections were counterstained with nuclear fast red, photographed blindly by two investigators using Axioplan 2 Imaging (Carl Zeiss MicroImaging, Inc., Thornwood, NY). The calcium content in thoracic aortas was measured using the o-cresolphthalein complexone method (Sigma-Aldrich, St. Louis, MO) [9], which was quantified by normalized concentration to protein concentration (μg/mg protein) determined by Bradford protein assay.