•Nile Red is a dark purplishred powder (Sigma N3013), the stock solution must be prepared in acetone (250mgml) and kept in a tightly sealed, lightproof container at 4 degrees.•This stock solution must be diluted 100x before use, best done in 50mM TrisMaleate (this stock solution can keep for a few months at 4 degrees) and polyvinylpyrrolidone (PVP) (23% wv). It is mixed using a magnetic stirrer (can take a while). This buffer plus the PVP will only keep for a couple of days, so it is best made fresh on the day storing it in a lightproof container at room temp.
Nile Red staining for lipids Making the stain: • Nile Red is a dark purplish-red powder (Sigma N-3013), the stock solution must be prepared in acetone (250mg/ml) and kept in a tightly sealed, lightproof container at degrees Preparing the stain for use: • This stock solution must be diluted 100x before use, best done in 50mM Tris/Maleate (this stock solution can keep for a few months at degrees) and polyvinylpyrrolidone (PVP) (2-3% w/v) It is mixed using a magnetic stirrer (can take a while) This buffer plus the PVP will only keep for a couple of days, so it is best made fresh on the day storing it in a lightproof container at room temp Using the stain: Collect conidia from 3-4 plates per strain and filter through miracloth Spin down at room temp and resuspend in Approx 500µl of distilled water Adjust concentration of conidia samples to be the same in all samples (eg 1x 104) Place 100µl drops on coverslips and place in tray with dampened paper towel and cover until needed Place a small drop of the stain onto a slide, place coverslip over top View under the Nikon optiphot microscope with EFD-3 episcopic fluorescence attachment and the B-2A filter (excitation at 450-490nm, 505nm dichroic mirror, 520nm barrier filter)