TTC Staining and Determination of Infarct Size

Following the different treatment methods used, on the 28th day, the hearts were excised under deep anesthesia and the infarct sizes were evaluated. Briefly, the freshly harvested heart tissue was cut into five slices using a rodent heart section mold. To maximize saving the heart tissue, each slice was cut down to a thinner slice (1 mm per slice) and used for TTC staining. The remnants were used for IHC and IF (Supplementary Figure 1). Tissues were then placed in 1% TTC solution (Solarbio, Cat: G3005) and incubated at room temperature in the dark for 15 min. The stained tissues were then photographed under a light microscope (Olympus).

Digital images of the stained sections were captured to assess the changes of infarct size at post-treatment day 28. Morphometric analyses were carried out using NIH Image J software. The infarcted size was calculated according to the formula: infarct size (%) = [sum of (scar circumferential length × thickness of each of the short axis)/sum of (short axis left ventricle length × thickness of the short axis)] × 100%.