2.4. In Situ Detection of Reactive Oxygen Species (ROS)

ROS production was detected using dihydroethidium (DHE, Sigma), a fluorescent probe for detecting intracellular superoxide anion levels. H9c2 cells were stained with 10 μM DHE for 30 min in a dark and humid chamber at 37°C to analyze ROS production in situ. The production of ROS was indicated using red fluorescence, observed by fluorescence microscopy, and subsequently quantified using ImageJ software (NIH) [13].