Fluorol yellow 088

Transgenic hairy roots and stably transformed M. truncatula (plants of the F1 generation) carrying GUS reporter constructs were stained using 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, as previously described (Gallagher, 1992 ) and visualized by light microscopy.
Microscopic observations of the transgenic roots carrying the ProMtABCG59:NLS-GFP reporter construct and Arabidopsis protoplasts transiently expressing Pro35S:GFP-MtABCG59 or free GFP were performed using laser scanning confocal microscopy (Leica TCS SP5).
For suberin staining, 6-week-old M. truncatula and Nicotiana tabacum roots (10 of each species) were incubated in 0.01% solution of Fluorol Yellow 088 (Santa Cruz Biotechnology, Dallas, Texas, USA) at 70°C for 30 min. Free-hand root sections (from the regions located at least 3 cm above the root tip) were observed under UV-light with fluorescence microscopy (Leica DMI 4000B, Wetzlar, Germany).
For arbuscules visualization, mycorrhizal roots were fixed in 50% ethanol for 3 h, cleared in 20% (w/v) KOH for 3 days at room temperature (21°C–23°C), followed by 0.1 M HCl for 1 h. Then roots were washed in PBS and incubated in WGA-AlexaFluor 488 staining solution (1 µg/ml in PBS and 0.01% v/v Tween 20) in the dark for 16 h. The roots were imaged with laser scanning confocal microscopy (Leica TCS SP5).

Sections were stained for 1 h with 0.005% Fluorol Yellow 088 (Santa Cruz Biotechnology, Texas, USA) [39 (link)] dissolved in 90% glycerol and melted polyethylene glycol 4000 (SERVA Electrophoresis, Heidelberg, Germany). The sections were transferred to the stage of a fluorescence microscope (BX-60 equipped with a DP 73 digital camera; Olympus and viewed in transmitted white light or under incident fluorescent light (filter U-MWB; 450–480 nm excitation; ≥520 nm emission wavelength; Olympus, Hamburg, Germany). The minimum number of biological replicates was three. To confirm the occurrence of a periderm, a minimum of 50 sections through the whole block were examined.