Filipin staining of cells

From LipidomicsWiki

FILIPIN STORAGE AND STAINING

Filipin is unstable (the stability varies somewhat from batch to batch). The stock is prepared in DMSO at the concentration 50 mg/ml and stored in the freezer (-20°C) under argon (nitrogen can also be used). Always protect from light + exposure to oxygen. When reconstituting the stock, do even not open the filipin powder vial; just inject the DMSO through the rubber cork and argonate the stock through a needle. For use, draw small working aliquots from the stock bottle (again with the needle through the cork) in order to avoid freezing/thawing the stock all the time. Store the working aliquot under argon/nitrogen, too.

For filipin staining no separate permeabilization of cells is needed (should actually be avoided) as filipin itself permeabilizes the cells. For filipin staining only, dilute 1:100-1:500 in PBS and incubate 15 min at room temperature, wash 3x 5 min and mount. For combining with antibody stainings, again no separate permeabilization, just add the filipin 1:100 in the blocking solution (10% FBS usually), then incubate 30 min at +37°C and proceed with antibody stainings normally.

The detection is in the UV range (360/460 nm) but the fluorescence is very bright. If not, it’s usually due to dead filipin. Depending on the microscope objective, the bleaching may be a problem. Using anti-fading reagents (dabco) in the mounting medium may help a little.

Refs:

Hölttä-Vuori M., Tanhuanpää K., Möbius W., Somerharju P. and Ikonen, E. 2002: Modulation of cellular cholesterol transport and homeostasis by Rab11. Mol. Biol. Cell 13: 3107-3122

Linder M. D., Uronen R.-L., Hölttä-Vuori M., van der Slujis P., Peränen J., and Ikonen E. 2007: Rab8-dependent recycling promotes endosomal cholesterol removal in normal and sphingolipidosis cells. Mol. Biol. Cell 18:47-56