On Wed, 2 Feb 2000, Kowsz, Kim P wrote:

> I have tried the PAN reaction (Adams, 1961) on fixed frozen mouse adrenal
> according to the described method.  I tried two fixatives:  formal calcium
> and 10% neutral buffered formalin.  NO STAINING.  I tried adding an
> oxidation step (1% ferric chloride) before the reagent step and still NO
> STAINING.  I do get some blue precipitate but no real tissue staining.  Is
> anyone familiar with this technique that could help me out. 

   I have done this method, just to try it out, and did get
   blue staining of CNS myelin, but the preparations (thickish
   frozen sections) were quite badly damaged and not at all
   pleasing to look upon. This is hardly surprising in view of
   the chemicals used! I didn't use FeCl3 for the pre-oxidation
   (Bayliss-High 1984) but left the unmounted sections for a 
   week in 4% aqueous formaldehyde, following an earlier
   prescription (Adams 1965). The blue colour fades quite
   soon; this is really a pretty crumby method.

> Or offer an alternative stain specifically for sterols 
> and esters.  I have already done Oil Red O, Sudan Black, 
> and Nile Blue.

   PAN seems to be the only truly specific method for cholesterol
   - either free or released from its esters during the reaction.
   There are several less specific techniques. I've not tried any.
   There is also polarizing microscopy: cholesterol (not its 
   esters) exists as very thin elongated birefringent crystals.
   This method dates back to the 1920s. Frozen sections usually
   contain all sorts of other birefringent stuff, notably myelin,
   collagen and dirt that cannot be seen by any other method.
   In degenerating white matter cholesterol crystals are there
   but less conspicuous than the other stuff. (That's how
   I've seen it. Miklossy & van der Loos 1987 painted a rosier
   picture.)

   The Marchi method detects hydrophobic lipids, including
   cholesterol esters and neutral fat, but not the more hydrophilic
   ones such as phospholipids. Pearse's "Histochemistry" describes
   methods for ketosteroids. I've not tried them and they don't
   figure prominently in the literature.
     
   Cholesterol can be specifically detected in electron micrographs
   by decorating it with filipin, but I don't think this method
   has been adapted for light microscopy. 

   Adams CWM 1965. Neurohistochemistry. Amsterdam, Elsevier.
   Bayliss High, O 1984. Lipid Histochemistry. Oxford Univ
     Press & Roy. Microsc. Soc. (Hndbk #6).
   Miklossy J, van der Loos H 1987. Brani Res. 426:377-380. 
   Pearse, AGE 1985. Histochemistry, Theoretical and Applied.
     4th ed. Edinburgh: Churchill-Livingstone, Chap. 17.
 
John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1
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