Plants were immersed in 100 mM sodium phosphate buffer (pH 7.0) containing 0.1% Triton X-100, 1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid cyclohexylammonium salt (Sigma-Aldrich), 2mM potassium ferricyanide, and 2 mM potassium ferrocyanide. Plants subject to vacuum treatment for 10 min and then incubated at 37 °C for 16 h. Tissues were cleared with 30%, 50%, and 70% ethanol for 30 min in each concentration and imaged using an AxioZoom 16, Zeiss binocular microscope.
For cross-sectioning of GUS-stained leaf petioles, after clearing in 70% ethanol, the samples were fixed in FAA solution (3.2% formaldehyde, 5% acetic acid, 50% ethanol) for 30 min and kept overnight at 4°C. The samples were then dehydrated in an ethanol gradient ranging from 50% to 96%, and incubated in 2% eosin overnight at 4° C. After several washes in 96% ethanol, the samples were progressively rehydrated in ethanol/HISTO-CLEAR II (Electron Microscopy Sciences) solution, incubated in 50% HISTO-CLEAR II 50% PARAPLAST PLUS (McCormick Scientific) at 60°C for 2 h, and embedded in 100% PARAPLAST PLUS. Paraffin-embedded samples were cross-sectioned with LEICA RM2155 microtome and imaged using a Leica Leitz Dmrb microscope.
For cross-sectioning of GUS-stained leaf petioles, after clearing in 70% ethanol, the samples were fixed in FAA solution (3.2% formaldehyde, 5% acetic acid, 50% ethanol) for 30 min and kept overnight at 4°C. The samples were then dehydrated in an ethanol gradient ranging from 50% to 96%, and incubated in 2% eosin overnight at 4° C. After several washes in 96% ethanol, the samples were progressively rehydrated in ethanol/HISTO-CLEAR II (Electron Microscopy Sciences) solution, incubated in 50% HISTO-CLEAR II 50% PARAPLAST PLUS (McCormick Scientific) at 60°C for 2 h, and embedded in 100% PARAPLAST PLUS. Paraffin-embedded samples were cross-sectioned with LEICA RM2155 microtome and imaged using a Leica Leitz Dmrb microscope.