Ab97047

Under deep anesthesia with pentobarbital sodium (intraperitoneal injection at 100 mg/kg), the heart was excised and cut into 2-mm-thick transverse slices, which were then fixed in 10% formalin solution at room temperature for 24 h. The samples were subsequently embedded in paraffin and sectioned (4 µm). Immunohistochemical analysis was performed as previously described (13 (link)). Tissue sections were then deparaffinized using xylene and graded ethanol and were blocked in 10% goat serum (Gibco, Thermo Fisher Scientific, Inc.) for 30 min at 37°C. Samples were incubated at 4°C overnight with primary rabbit polyclonal antibodies against CD31 (ab28364) and VEGF (ab46154; Abcam, Cambridge). Sections were subsequently treated with secondary goat polyclonal secondary antibody against rabbit IgG (ab97047; Abcam). All sections were counterstained with hematoxylin. Immunoreactivity for CD31 and VEGF was measured using a fluorescence microscope and the Image-Pro Plus 4.0 analysis system (Media Cybernetics, Inc.). Capillaries were identified by positive staining for CD31. Results were expressed as the average amount of capillaries per ×10 field. Masson's trichrome stain was performed in the border area to evaluate collagen deposition.