2.4 In situ detection of reactive oxygen species
ROS cellular localization was determined by confocal microscopy in an
Eclipse TE–2000–E2 Confocal Laser Scanning Microscope (Nikon),
essentially as described by Mayta et al. (2018). Discs (110
mm2 in diameter) were collected during the light
period from leaves belonging to the fourth node of 5 different plants
per line, vacuum-infiltrated in the dark with 50 µM DCFDA in 10 mM
Tris-HCl pH 7.5 and 0.1% (v/v) Tween-20, incubated in the dark for 1 h
in the same solution, washed briefly and mounted in water. Imaging was
performed by scanning 5 optical slices (with an interval of ∼1 µm) of
the palisade parenchyma immediately below the epidermis. Fluorescence
intensities were estimated using Fiji software (Schindelin et al.2012), using the z-projections of the different stacks.