AS ODNs against OPN efficiently decrease OPN protein levels in in vivo mouse wounds. (A) Immunostaining for OPN (brown) reveals expression by wound connective tissue cells at day 1 and 3, which is resolved by 7 d after wounding. (B) Double immunofluorescence of wound tissue at 3 d reveals OPN expression (red) in both F4/80-positive (green; closed arrowheads) and -negative cells (open arrowheads). (C) Western blot analysis of OPN in control- and OPN AS ODN–treated wound tissue confirms the temporal profile of the immunostaining in A, and indicates that AS ODN treatment leads to depletion of OPN. (D) Band intensities were quantified by densitometric analysis on blots from six independent experiments, and values for the OPN/GAPDH ratio are shown (mean ± SEM). (E) Cy3-tagged OPN AS ODNs (red) were applied to wounds to assess to what depths and in which tissues OPN was being depleted (evaluated at 6 h). Dotted line indicates the epidermal/connective tissue interface, and the arrowhead indicates the epidermal wound edge. Inset shows a low-magnification view of the same section. Bars: (A) 50 μm; (B) 10 μm; (E) 100 μm; (E, inset) 200 μm.