DHE staining

ROS production in primary cells or kidney tissues was evaluated using DHE staining (Invitrogen, D11347). DHE was suspended in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM and diluted for a final concentration of 20 μM in PBS before using. Cells or optimal cutting temperature–embedded kidney specimens were washed three times in PBS. After adding the DHE solution, tissue sections or cells were incubated in a light-protected humidified box at room temperature for 30 min. The slides or cells were stained with DAPI (4′,6-diamidino-2-phenylindole) and imaged immediately with a fluorescence microscope (DMi8, Leica).