Masson’s Trichrome staining

Mouse hearts were fixed by retrograde perfusion via the aorta with 4% paraformaldehyde (diluted in PBS) for 10 min at room temperature. The hearts were then incubated in increasing concentrations of sucrose dissolved in PBS (10% for overnight, 20% for 8 h, and 30% for overnight) on a horizontal rotator at 4 °C before being embedded in TissueTek OCT compound. Hearts were then cryosectioned at 10-µm slices using a Leica vibrating microtome (Model No.: CM3050S) at five different levels of the heart from the ligation site to the apex at 300-µm intervals. Ten consecutive slices were made for each level. Heart sections were then stained using a Masson’s Trichrome Staining kit (Sigma, Catalogue No.: HT15) according to the manufacturer’s instructions. Fibrosis areas were analyzed with ImageJ software using the Colour Deconvolution for Masson’s Trichrome Stain tool. The scar size for each the 5 different levels was calculated as the percentage of fibrotic area to the total area of the left ventricle section. The heart’s total scar size was then calculated as the mean of the scar sizes at the 5 levels and reported in Fig. 4.