Figure 6
Determination of calcification in rat aortic ring sections. Rat aortic rings were incubated for 2 days in fresh control media followed by calcification media 3.0 mM Pi) for 7 days and fixed with NBF (10%, 15 minutes). Rings were cryosectioned into 5 μm sections. Sections were incubated with Fluorescein-BP (1 µM, 2 hours), permeabilised in KOH (1%, 1 hour), incubated with Alizarin S (0.00005% Alizarin S in 1% KOH, 24 hours), followed by DAPI (500 nM, 5 minutes). Brightfield (A,B) Fluorescence (E,F) Alizarin S (C,D). Merged images (G,H) recorded using Alizarin S, Fluorescein-BP and DAPI channels. Images shown are representative of at least 3 independent experiments yielding comparable results. Scale bars = 250 µm.
