Figure 1. IMD and its receptor levels in aging-associated vascular calcification induced by VDN in rats. (A) Alizarin red staining for vascular calcium deposition (positive staining: red) (Scale bar=200 μm). (B) SA-β-gal staining for β-galactosidase activity (blue) (Scale bar=100 μm). (C) Immunohistochemistry staining for IMD (Scale bar=200 μm), and (D–F) quantification of (D) calcium deposition-positive staining (n=3), (E) β-galactosidase-positive staining (n=3) and (F) IMD-positive staining (n=4) in the medial layer of rat thoracic aortas. (G–K) Quantitative RT-PCR analysis of mRNA levels of IMD, calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein 1 (RAMP1), RAMP2 and RAMP3 in rat aortas (n=3 in each group). (L) Western blot analysis of protein levels of CRLR and RAMP1, 2 and 3 in rat aortas and (M–P) quantification (n=3). The arrow indicates positive staining. Y=young rats. O=old rats. YV=young+VDN. OV=old+VDN. Data are mean ± SD. *P<0.05, **P<0.01.