The levels of ROS from renal tissues were assessed using the dihydroethidium (DHE) fluorescent probe (D7008, Sigma-Aldrich, MO, United States), following the previously described protocols (Fan et al., 2021 (link)). In brief, after frozen sections were incubated with 50 uM DHE for 1 h in the dark at room temperature, the sections were incubated for 10 min with DAPI (1 mg/ml). The cell images were taken with a fluorescent microscope (TE-2000, Nikon, Co., Japan) with an excitation (Ex) wavelength at 525 nm and an emission (Em) wavelength at 610 nm after washing. The values used ImageJ software to capture the red (the oxidized probe) and blue (the reduced probe) fluorescence intensities to calculate the ratio and then standardized to the con group.
The levels of ROS in HK-2 cells were tested by the 2′, 7′- dihydrofluorescein diacetate (DCFH-DA) fluorescent probe (D6883, Sigma-Aldrich, United States), following the manufacturer’s protocols. Briefly, after treating HK-2 cells with 10 uM DCFH-DA in the dark for 1 h, the pictures were detected using a fluorescent microscope (TE-2000, Nikon, Co., Tokyo, Japan) at (Ex/Em) 485 nm/530 nm.
The levels of ROS in HK-2 cells were tested by the 2′, 7′- dihydrofluorescein diacetate (DCFH-DA) fluorescent probe (D6883, Sigma-Aldrich, United States), following the manufacturer’s protocols. Briefly, after treating HK-2 cells with 10 uM DCFH-DA in the dark for 1 h, the pictures were detected using a fluorescent microscope (TE-2000, Nikon, Co., Tokyo, Japan) at (Ex/Em) 485 nm/530 nm.
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