Alkaline phosphatase–positive cells in aortic adventitial cell cultures were visualized as previously described, but using the Vector Red fluorescent substrate (14 (link),18 (link)). Briefly, aortic mesenchymal cells were cultured on a type I collagen–precoated four-chamber slide (60,000 cells/chamber) for 8 days. During the final 6 days, cells were treated with β-glycerophosphate 5 mmol/L and ascorbic acid 50 μg/mL. Cells were then washed three times with Tris-buffered saline (TBS) (20 mmol/L Tris HCl, 1.5 mol/L NaCl, pH 7.5), fixed with 4% paraformaldehyde in TBS for 4 min, and rinsed three times with TBS. Staining with Vector Red reagent (Vector Red Alkaline Phosphatase Substrate Kit I, catalog number SK-5100; Vector Laboratories) was performed for 1 h in the dark per the manufacturer’s protocol. After washing three times with TBS and twice with distilled water, nuclei were stained with DAPI (Prolong Gold Antifade Reagent, catalog number P36931; Molecular Probes) and the slides coverslipped. Photomicrographs of alkaline phosphatase–positive fluorescent cells (TX2 filter cube), nuclei (DAPI filter cube), and phase contrast images were captured by a Leica DM4000 B fluorescence microscope at ×200 magnification.