Determination of Myocardial Infarct Size

Myocardial infarct size was determined by the double-staining technique using Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich, St Louis, MO, United States) (Bohl et al., 2009). At the end of the experiment, the coronary artery was re-occluded and 1% Evans blue solution was intravenously injected to stain non-ischemic myocardium and determine the area at risk. The heart was cut transversely into 2-mm-thick slices using a heart slicer matrix (Jacobowitz Systems, Zivic-Miller Laboratories Inc., Allison Park, PA, United States). Heart sections were stained with 2% TTC at 37°C for 30 min in darkness. The slices were placed in a solution with 10% formalin at room temperature for 1 day. Infarcted tissue slices were scanned and tissue weights were evaluated by distinguishing the normal myocardium (stained blue) from the area at risk and infarct area (unstained) in a TTC staining assay.