Heart isolation was performed 7 days after the last 15-minute ischemia, except for the proliferation analysis in AB-treated mice, which was 2 days after the last 15-minute ischemia. For proliferation analysis, sections were pretreated with 2 M HCl for 30 minutes at 37°C and neutralized with 0.1 M sodium tetraborate, followed by primary and secondary antibody incubation. The following primary antibodies were used in this study: rat anti-BrdU (Abcam, ab6326), goat anti-PECAM-1 (R&D Systems, AF3628), mouse anti-αSMA-Cy3 (Sigma, C6198), mouse anti-αSMA (Sigma, A5228), and rat anti-β1 integrin (Millipore, MAB1997), and incubated overnight at 4°C. Secondary antibodies conjugated with fluorophores AF488 (Invitrogen Molecular probes), Cy3 (Jackson ImmunoResearch), and Cy5 (Jackson ImmunoResearch) were incubated in the dark for 1 or 3 hours at room temperature. Cell nuclei were stained with DAPI (Sigma). For hypoxia analysis, sections were stained with fluorophore-coupled antibodies for HP-FITC-MAb (detects pimonidazole adducts) and mouse anti-αSMA-Cy3 (Sigma, C6198) overnight at 4°C. Cell nuclei were stained with DAPI (Sigma).
Hearts with considerable tissue damage and areas of cryosections containing part of the occlusion system were excluded from quantification.
Hearts with considerable tissue damage and areas of cryosections containing part of the occlusion system were excluded from quantification.