Can someone help me with the protocol for the choleterol staining using filipin (which’s a fluroscent antibiotic from sigma). I have been trying it for quite some time now but doesn’t seem to work.
Please specify the excitation - emission wavelengths.
Filipin is a fluorescent polyene antibiotic commonly used to stain unesterified cholesterol in cell membranes. So if your cholesterol is esterified, it will not work.
A generalized protocol may be used as below
Fix Cells: Wash the cells with PBS twice to remove any media.
Fix the cells with 4% paraformaldehyde in PBS for 15-20 minutes at room temperature.
Wash the cells three times with PBS to remove residual fixative.
3. Filipin Staining
Prepare Filipin Solution: Dissolve Filipin complex in PBS or DMSO to a final concentration of 50-200 µg/mL. Use freshly prepared or store aliquots at -20°C for up to a week to avoid degradation.
Stain Cells: Incubate the fixed cells with the Filipin solution for 30-60 minutes at room temperature in the dark (Filipin is light-sensitive).
Wash Cells: After staining, wash the cells three times with PBS to remove excess dye.
4. Imaging
Mount Coverslips: Mount the coverslips on glass slides using an appropriate mounting medium that preserves fluorescence.