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Nile red staining of lipids

MM Marion Mussbacher
HS Heike Stessel
TP Teresa Pirker
AG Antonius C. F. Gorren
BM Bernd Mayer
AS Astrid Schrammel
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For Nile Red staining, formalin-fixed cells were washed twice with distilled water, followed by incubation with 60% isopropanol for 5 min at ambient temperature. Thereafter, isopropanol was removed and cells were dried. Then, cells were incubated with Nile Red (1 µM in PBS) for 10 min at ambient temperature and protected from light. After extensive washing, cells were treated with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; 100 ng/ml in PBS) as a nuclear counterstain (5 min; ambient temperature; light-protected). After extensive washing Nile Red and DAPI fluorescence were visualized by an Axiovert 200 M microscope (Carl Zeiss GmbH, Vienna, Austria) using a Green Fluorescent Protein filter (λEx: 450–500 nm; λEm: 528 nm) and a DAPI-filter (λEx: 395 nm; λEm: 460 nm), respectively.

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