2.6. Blood and Tissue Collection

The rats were anaesthetized during euthanasia with 2% isoflurane per oxygen (1 L/min flow rate) via inhalation. Blood was collected into Z-serum clot activator tubes from the abdominal vein. Blood samples were centrifuged at 4000× g for 10 min at 4 °C. Aliquots of the supernatant were stored at −80 °C for biochemical analysis. During euthanasia, the heart was extracted and immediately washed in ice-cold phosphate buffered saline (PBS), dabbed, and weighed. This was then frozen using liquid nitrogen and later stored at −80 °C for further analysis.