[Histonet] Filipin staining

From:"Sarka Lhotak"

Hi Jennifer,
I've done some Filipin staining on cells and tissues. Filipin stains
free cholesterol which can be found in membranes, i.e. plasma membrane
and intracellular membranes, such as endoplasmic reticulum, membranes
of vesicles et., and you can find free cholestrol also in extracellular
crystals in the necrotic cores of lesions. 

Staining is done on frozen sections fixed with formalin or
paraformaldehyde and rinsed with PBS (or cells treated similarly). You
have to prepare the staining solution FRESH. I buy Filipin from Sigma,
dissolve a little crystal of it in 5 microlitres of DMSO, then add 500
microlitres of PBS and use it for staining immediately. Stain for 1-2
hrs, rinse with PBS, then in distilled water and mount in Permafluor
(Shandon). Observe in fluorescence microscope under UV light (same
configuration as for DAPI or Hoechst). It bleaches quickly, you have to
photograph it fast.

Hope it helps. Let me know if you have more questions.

Sarka Lhotak, PhD

McMaster University, Hamilton, ON   

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