2.5. Masson's trichrome staining

Masson's trichrome stain applied to differentiate necrotic myocardium (blue cytoplasm) from viable myocardium (red cytoplasm) usually with purple myocardial cytoplasm surrounding a necrotic area. Masson's Trichrome stain (Poly Scientific, Bay Shore, NY) were performed according to the manufacturer's instructions. Briefly, heart tissue slides were placed in staining jar and deparaffinized by submerging into three series of absolute xylene for 4 min. After that, the slides were washed with running tap water for 2 min. Then, slides were treated with the phosphomolybdic acid solution for another 10 min as a mordant and immediately slides were submerged into methyl blue (C.I. 42780, Merck, Germany). Next, slides were washed in running water for 2 min and lastly treated with 1% acetic acid solution for 1 min.