Histochemical GUS staining of Arabidopsis plants

Seedlings were fixed in cold 90% acetone for at least 30 min at 4 °C. Acetone was removed, and the material was washed twice with rinse solution (0.5 M Na₂HPO₄, 0.5 M NaH₂PO₄, 0.1 M K₃Fe(CN)₆, 0.1 M K₄Fe(CN)₆). The rinse solution was removed, and stain solution added (rinse solution complemented with 2 mM x-Gluc (5‐bromo‐4‐chloro‐3‐indolyl‐beta‐D‐glucuronide). Samples were then gently vacuum-infiltrated for 30 min and then incubated at 37 °C, in the dark, for 30 min or 24 h. Samples were then washed in water and cleared in chloral hydrate (8 g choral hydrate, 3 mL 100% glycerol, 1 mL dH₂O) for at least 45 min, before mounting in chloral hydrate.

Embedding primary roots GUS stained for sectioning was performed using Technovit 7100 according to the manufacturer’s instruction (Electron Microscopy Science). Sectioning was performed using a Leica UC7 (Ultra-microtome). Images were obtained with a DM6000 microscope (Leica).