H2DCFDA-Based Confocal Imaging of ROS

The H₂DCFDA staining assay was performed according to a previously reported method with slight modifications [32 (link)]. The excised leaves were stained with 10 μM H₂DCFDA (MedChemExpress, Monmouth Junction, NJ, USA) in 10 mM PBS buffer in the dark for 30 min. Images were captured under an Olympus FV3000 confocal laser scanning microscope (Olympus Corp., Tokyo, Japan) with a 488 nm filter. ROS signals were visualized in the range of 501–550 nm, and chlorophyll autofluorescence was detected in the range of 640–735 nm.

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Qi F., Li J., Hong X., Jia Z., Wu B., Lin F, & Liang Y. (2023). Overexpression of an Antioxidant Enzyme APX1 in cpr5 Mutant Restores its Pleiotropic Growth Phenotype. Antioxidants, 12(2), 301.

Publication 2023

H2DCFDA FV3000 confocal laser scanning microscope

Assay Buffer Chlorophyll Confocal laser scanning microscope H2dcfda

Corresponding Organization : Zhejiang University

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Variable analysis

independent variables

H2DCFDA concentration (10 μM)

dependent variables

ROS signals
Chlorophyll autofluorescence

control variables

PBS buffer (10 mM)
Incubation time (30 min)
Imaging with Olympus FV3000 confocal laser scanning microscope
Excitation wavelength (488 nm)
Emission wavelength range for ROS signals (501-550 nm)
Emission wavelength range for chlorophyll autofluorescence (640-735 nm)

controls

No positive or negative controls were explicitly mentioned.

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