Nile red staining

The distribution of lipid bodies in oospores was determined using Nile red staining. The Nile red stock solution (1 mM) was prepared by dissolving 100 mg of Nile red powder (Sigma-Aldrich, St. Louis, MO, USA) in 314 ml of methanol. Before staining, 10 μl of stock solutions were diluted 20 × with dimethylsulphoxide (DMSO). An aliquot of 20 μl oospore suspension was mixed with an equal volume of diluted Nile red solution on a microscope slide, and incubated at room temperature for 5 min, followed by two washes with PBS (pH 5.8). The samples were then viewed under an Olympus FluoView™ FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan) with an excitation wavelength of 530 nm and an emission wavelength of 568 nm.