Nile Red Staining for Lipids

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 4 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 4 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: 1. Collect conidia from 3-4 plates per strain and filter through miracloth. 2. Spin down at room temp and resuspend in Approx 500l of distilled water. 3. Adjust concentration of conidia samples to be the same in all samples (eg 1x 104) 4. Place 100l drops on coverslips and place in tray with dampened paper towel and cover until needed. 5. Place a small drop of the stain onto a slide, place coverslip over top. 6. 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).