The process of Nile Red staining typically involves the following steps: 1. Fixation and Permeabilization: Cells or tissues are fixed using formaldehyde or another fixative to preserve their structure. Permeabilization agents are then used to allow the dye to penetrate the cells. 2. Staining: Nile Red is dissolved in a suitable solvent (often DMSO or ethanol) and applied to the sample. 3. Incubation: The sample is incubated with the dye for a specific period, usually ranging from a few minutes to hours, depending on the sample and experimental conditions. 4. Washing: Excess dye is washed away using a buffer solution to minimize background fluorescence. 5. Imaging: The stained sample is visualized using a fluorescence microscope. Nile Red typically emits bright yellow-gold fluorescence when bound to neutral lipids and red fluorescence when bound to phospholipids.
