2.8 Confocal microscopy
Al-morin fluorescence in the cross-sections of different root zones was examined according to Klug et al. (2011). The two-day-old plants treated with 5 μM Al for 2, 6, 24, and 72 h were washed with distilled water to remove hydroponic solution from the root surface, and 10-mm of root tips were excised, and immediately embedded in 5% (w/v) low-gelling point agarose (Fluka, Switzerland) at 35 °C. The embedded root tips were sectioned using a razor blade and incubated with 30 µM morin (Sigma, USA) for 1 h under darkness. Confocal imaging of Al-morin fluorescence was performed using a Leica Laser Scanning Confocal Microscope SP5 (Leica Microsystems, Germany) equipped with a 20× water immersion objective. The excitation wavelength was set at 405 nm, and the emission was detected at 490-510 nm. Images were processed with LAS AF software (Leica Microsystems, Germany) and were quantified using Image J software (NIH, USA).