Before TTC staining, the isolated heart tissue was perfused with 1X phosphate-buffer saline (PBS; Biosesang, Seongnam, Republic of Korea) several times. Heart tissues from each experimental group were subjected to TTC staining. For this, tissues were incubated in the 1% TTC (Sigma-Aldrich, St. Louis, MO, USA) solution, which was prepared by dissolving TTC in 1X PBS. The incubation was carried out at 37 °C for 1 h in a state shielded from light to prevent photo-degradation [23 (link),24 (link)]. Post-incubation, the tissues were fixed in a 4% paraformaldehyde solution (Biosesang, Seongnam, Republic of Korea) at 4 °C for 4 h. This was followed by sectioning the tissues into 1 mm thick cross-sections for detailed examination. The stained and sectioned heart tissues were then photographed using a digital camera (DIMIS M model, Anyang, Republic of Korea) to document the results of the TTC staining process. The infarcted tissue appears white, while the viable tissue is red. This photographic evidence is crucial for visualizing and analyzing the extent of infarction in heart tissues.
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