A Microscopic 2,3,5-Triphenyltetrazolium Chloride Assay for Accurate and Reliable Analysis of Myocardial Injury

In this protocol, we present a refined 2,3,5-Triphenyltetrazolium Chloride (TTC)-based method for the accurate and reliable analysis of myocardial infarction (MI) at cellular resolution. This protocol involves a two-step TTC staining procedure performed on infarcted hearts 24 hpost-MI: perfusion, in which a 1% TTC solution is retrogradely perfused through the coronary vasculature via an aortic cannula, and immersion, where the heart is subsequently incubated in TTC solution at 4 °C overnight. After staining, the heart is embedded in OCT compound and cryosectioned transversely into 50-µm thick slices at 100-µm intervals. Following a 24 h fixation process with Zamboni's fixative, a sharp and distinct boundary between viable myocardium (deep red) and necrotic tissue (yellow) becomes discernible under microscopic examination. Notably, this microscopic TTC assay enables MI-detection not only in the acute phase (24 h post-MI) but also across an extended time window beyond 7 days. Because the microscopic TTC assay generates multiple-layer images (80 slices per heart), we developed a robust, color-based semi-automated algorithm that enables rapid and efficient analysis of large image datasets and quantifies the global infarct size within minutes. With these enhancements, the current approach substantially refines the TTC staining process and provides a broadly applicable and valuable tool for laboratories investigating cardiac ischemic injury.