Leadmium green am solution

S. babylonica root tips exposed to different Pb concentrations (0, 1, 10, 50, or 100 μmol/L) for 3, 6, 12, and 24 h were soaked in EDTA solution (Na₂-EDTA, 20 mmol/L) and washed with running water for 15 min. Then, root tips were washed with deionized water 3 times. Afterwards, experimental and control roots were stained using the Pb-specific probe Leadmium™ Green AM solution (Molecular Probes, Invitrogen, Carlsbad, CA, USA) for 90 min at 40 °C in the dark following the manufacturer’s instructions to visualize Pb absorption and distribution [55 ]. Intact cells exhibited green fluorescence due to the Pb-specific probe Leadmium™ Green AM solution. Fluorescence density was analyzed using the “Analyze and Measure” function in Image J software to evaluate the Pb distribution in intact roots. Prepared samples were observed using a Nikon Eclipse 90i confocal laser scanning microscope with an exciter at 488 nm and a barrier at 590/50 nm.

Fresh root tips were immersed in 20 mM disodium ethylenediamine tetra-acetic acid (Na₂-EDTA) for 15 min and then gently rinsed for three times with deionised water. The specimen sections were then immediately immersed in the Cd probe Leadmium™ Green AM solution (Molecular Probes, Life Technologies, California, USA) for 45 min in the dark and then washed three times (5 min each time) with deionised water. A stock solution of Leadmium™ Green AM was made by adding 50 μL of dimethyl sulfoxide (DMSO) to one vial of the dye. This stock solution was then diluted 1:10 with 0.85% NaCl. Roots were immersed in this solution for 90 min in the dark. The sections were examined with a laser confocal scanning microscope (Leica TCS SP5; Berlin, Germany) with excitation and emission wavelengths at 488 and 515 nm, respectively. The fluorescence density of Cd was calculated by selecting the root sections and measuring the total Integrated Density using “Analyse and Measure” function of the Image J software (NIH, Bethesda, MD, USA).