Dcfh2 da

In situ localization of reactive oxygen species (ROS) was performed in root tips of ten-day-old WT and 35S::TaEPL1 lines using the fluorescent molecule 2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA, Sigma-Aldrich, Burlington, MA, USA), as described by [45 (link)], with some modifications. The effect of the exogenous application of catalase (CAT, Sigma Aldrich, Burlington, MA, USA) on the H₂O₂ levels in the WT and 35S::TaEPL1 lines was also analyzed. Seedlings were placed in 24-well culture plates containing 0.2X MS liquid supplemented with 0 or 250 U/mL of CAT (dissolved in pH7 phosphate buffer) and incubated at 22 ± 1 °C in a growth chamber for 4 h with continuous light. The seedlings were then immersed in 25 μmol DCFH₂-DA in Tris buffer (10 mM Tris, 50 mM KCl, pH 7.2) for 30 min in complete darkness. After rinsing with buffer to remove excess DCFH₂-DA and CAT, the roots were observed and photographed with a fluorescence microscope (Zeiss Axio Imager M2; Carl Zeiss Microscopy, Pleasanton, CA, USA) at 10× magnification using excitation and emission wavelengths of 480 nm and 500–550 nm, respectively. Fifteen seedlings of WT and 35S::TaEPL1 lines were analyzed by treatment; each assay was repeated at least three times.

The H₂O₂ accumulation in leaves was stained by 3,3′-diaminobenzidine (DAB). Details of assay have been described by Zhao et al. (2016a) (link).
The reactive oxygen species (ROS) accumulation was detected using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA, Sigma-Aldrich, United States) according to Zhao et al. (2016a) (link). For DCFH2-DA staining, leaves were treated with 10 μM DCFH2-DA for 20 min and then washed with distilled H₂O. The images were obtained using the confocal microscope (E_x, 488 nm; E_m, 522 nm) (Leica Microsystems, Wetzlar, Germany).

To measure intracellular ROS, the non-fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate was used (DCFH2-DA; Sigma-Aldrich Co., LLC, St. Louis, MO, USA). In addition, 200,000 cells were cultured in a 60 mm plate, pretreated for 24 h with DMEM containing various concentrations of Cr and NAM, washed twice with PBS, and incubated for 30 min in the dark at 37 °C with 5 µM of DCFH2-DA. The cells were then washed 3–4 times with PBS before being incubated with H₂O₂ for 30 min. Then, the cells were washed twice again with HBSS before being examined under a fluorescence microscope using the BioTek Cytation 5 machine, and the mean fluorescence intensity was measured by using ImageJ software. In some experiments, superoxide dismutase (SOD) enzyme activity levels were assayed using a kit and following manufacturer’s directions (SOD Assay kit-WST [Dojindo, Inc., Gaithersburg, MD, USA]).