Leaves were cleared with 100% ethanol, re-hydrated and stained with aniline blue (0.05% in phosphate buffer pH8.0) for 24 h.
After incubation, plant material was cleared with 70%% ethanol.
After staining, tissue was cleared with 70% ethanol and stored at 4°C.
Tissues were cleared with 70% ethanol and examined using a Leica M205 FA stereoscope or a Leica DM2500 optical microscope.
Root, shoot and leaf segments from the putative transgenic plants were stained for 24 h at 37°C, cleared with 70% ethanol and observed under a stereomicroscope (MZ8 Leica).
Samples were cleared with 95% ethanol and placed on a circular rotating stage between two polarizing filters, the polarizer and the analyzer, that were oriented at right angles to each other.
For N. benthamiana transient assays, transformed leaves were infiltrated as described above with a solution containing 1 mg ml-1 5-bromo-4-chloro-3-indoyl-β-d-glucuronide (X-Gluc), 50 mm Na2PO4 pH 7.2, 0.5 mm K3Fe CN 6, 0.5 mm K4Fe CN 6 incubated O/N at 37°C and cleared with 70% ethanol (EtOH).
Embryos were dehydrated with ethanol, cleared with xylenes and mounted in DePeX (Electron Microscopy Sciences).
Following fixation, ovaries were dehydrated with ethanol, cleared with Hemo-D xylene substitute, and infiltrated with paraffin.
Slides were counterstained with Mayer's haematoxylin, dehydrated with ethanol, cleared with xylene and finally mounted with Entellan (Merck, Darmstadt, Germany).
