4.5. Blood and Tissue Collection

Mice were euthanized by the administration of a high concentration of CO₂ gas. Blood was collected by cardiac puncture from mice under CO₂ anesthesia into chilled tubes containing 10 µL of heparin (1000 USP units/mL). Plasma was separated by centrifuging the blood samples at 3000 rpm for 10 min at 4 °C and then stored at −80 °C until used. Heart tissues were dissected, weighed, snap frozen in liquid nitrogen, and stored at −80 °C until used. A portion of heart tissue was sliced, kept in 10% buffered formalin overnight, and processed for histological studies.