2.7. ROS Assay: Dihydroethidium (DHE) and 2,7-Dichlorodihydrofluorescein Diacetate (DCFH-DA) Were Used

In vivo, heart tissues of mice were quickly frozen and then sliced. The DHE reagent was added to the slices and incubated at 37°C for 1 h. After washing with PBS, the SAN was observed with a fluorescence microscope.

In vitro, HL-1 cells were seeded at 1 × 10⁵ cells in a cell culture dish for laser confocal microscopy. After culture and treatment, DCFH-DA reagent was added and incubated at 37°C for 1 hour. After washing with PBS, the cells were observed using a confocal laser microscope. The DHE and DCFH-DA reagents were purchased from Beyotime Biotechnology (China).