•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.
Trang 1Nile 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 500µl of distilled water
3 Adjust concentration of conidia samples to be the same in all samples (eg 1x
104)
4 Place 100µl 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)