Staining of fungal structures with Wheat Germ Agglutinin (WGA)-AlexaFluor488

In order to stain fungal and oomycete structures within root tissue, Wheat Germ Agglutinin (WGA) conjugated to AlexaFluor488 (Life Technologies GmbH, Darmstadt, Hesse, Germany) was used enabling staining of cell wall components of fungi and oomycetes, such as A. euteiches. Freshly harvested roots were placed in 50% (v/v) ethanol for at least four hours followed by incubation in 20% KOH for 10 min at 90 °C. After washing with distilled water, roots were incubated in 0.1 M HCl for 2 h and then transferred into phosphate-buffered saline (135 mM NaCl, 3 mM KCl, 1.5 mM KH₂PO₄, 8 mM Na₂HPO₄, pH 7.4) containing 0.2 µg ml⁻¹ WGA-AlexaFluor488 for at least 6 h. Stained roots were analyzed using an epifluorescence microscope AxioImager (Zeiss GmbH, Jena, Thuringia, Germany) using the reflector module 09 (EX BP 450-490/BS FT510/EM LP515). Photographs were taken by an AxioCam (Zeiss) and combined using Adobe Photoshop.