Aorta histology, von Kossa staining, and quantitative analysis.

Descending thoracic aortas from mice were harvested and fixed in 10% formalin for 48 hours and then transferred to 70% ethanol for storage at 4°C. Fixed aortic segments were embedded in paraffin and sectioned at 5 μm thickness. Sequential sections were stained with hematoxylin and eosin as well as Masson’s trichrome staining. Images were acquired using an Olympus BX51 microscope. Aortic diameter and wall thickness were determined by 10× images using ImageJ. Number of cells in the vascular media was determined using object count in ImageJ. Calcification of the descending thoracic aortas was detected by von Kossa staining of 5-μm longitudinal cryosections as previously described (87). Calcification was visualized as distinct black deposits of calcium using an BX51 microscope (Olympus) coupled with imaging software (Cellsens). Additional methods are described in the Supplemental Material (see supplemental material; available online with this article; https://doi.org/10.1172/JCI147116).