2.6. Heart Tissue Homogenization and Protein Collection

Another set of experiments was performed on another group of rats to assess the biochemical parameters. At the end of the I/R protocol, the heart was rapidly excised, and then the whole ventricular tissue was collected, quickly frozen in liquid nitrogen and stored at −80 °C until analysis. One hundred milligrams of tissue from each organ was isolated and then homogenized by a hand homogenizer on ice. One hundred milligrams of heart tissue was homogenized with 1 mL of homogenization buffer (20 mM Tris HCl pH6.8, 1 mM Na₃VO₄, 5 mM NaF and cOmplete™ Protease Inhibitor Cocktail). The tissue homogenate (100 mg/mL tissue extract) was centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatants were collected and frozen for further analysis.