Daf fm diacetate

Intracellular NO levels were visualized using DAF-FM diacetate (7'-difluorofluorescein diacetate, Bio-Connect). Seedlings were incubated in the dark for 15 min under gentle agitation in 10 mM Tris-HCl buffer (pH 7.4) containing 50 μM DAF-FM DA and subsequently washed twice for 5 min 10 mM Tris-HCl buffer (pH 7.4). Several roots of all treatments/genotypes were mounted in 10 mM Tris-HCl buffer (pH 7.4) on the same microscope slide. Fluorescence was visualized using a Zeiss Observer Z1 LSM700 confocal microscope (oil immersion, 40x objective Plan-Neofluar N.A. 1.30) with excitation at 488 nm and emission at 490–555 nm. Roots incubated and mounted in 10 mM Tris-HCl buffer (pH 7.4) without DAF-FM DA were used to set background values where no fluorescence was detected. Within experiments, laser power, pinhole, digital gain and detector offset were identical for all samples. Mean DAF-FM DA fluorescence pixel intensity in root tips was determined in similar areas of ~17,000 μm² between epidermis layers using ICY software (http://icy.bioimageanalysis.org/).

Intracellular NO levels were visualized using DAF-FM diacetate (7′-difluorofluorescein diacetate, Bio-Connect). Seedlings were incubated in the dark for 15 min in 10 mM Tris–HCl buffer (pH 7.4) containing 50 μM DAF-FM DA and subsequently washed twice for 5 min 10 mM Tris–HCl buffer (pH 7.4). Several roots of all treatments were mounted on the same slide to allow direct comparison. Fluorescence was visualized using a Zeiss Observer Z1 LSM7 confocal microscope with excitation at 488 nm and emission 520 nm. Roots (0v) incubated and mounted in 10 mM Tris–HCl buffer (pH 7.4) without DAF-FM DA were used as a negative control to set background values. Within experiments, pinhole, gain, laser power and detector offset were identical for all slides. Mean DAF-FM DA fluorescence pixel intensity within the root was determined using ICY software (http://icy.bioimageanalysis.org/).