The superoxide anion (O2
•–) was detected as described by
Yamamoto et al. [40] using dihydroethidium (DHE), a reduced
form of ethidium bromide, which is non-fluorescent
and can passively cross the membrane of live cells. Once in
the cell, it is oxidized to give a fluorescent dye that binds to
nearby DNA. The production of O2
•– in lupin roots was
observed following the staining of roots with 10 μM DHE in
100 μM CaCl2, at pH 4.75 for 12 h. After a shorter time of
incubation with DHE (1 h), the differences between the
NO-treated and untreated roots were very faint, so we prolonged
the incubation time to 12 h. After washing, the roots
were observed using a fluorescence microscope Zeiss Axiovert
200M (model LSM510 filter set no. 9, excitation 450–
490 nm, emission 520 nm or more), magnification 50× and
photographed using a digital camera (AxioCam, Zeiss). An
argon laser (488 nm) was used for excitation, with emission
at 565–615 nm. Fluorescent and confocal microscopy experiments
were repeated three times with similar results