Re: [Histonet] Cholesterol staining suggestions

From:"John A. Kiernan"

The only specific method for cholesterol that
doesn't involve a nasty acid is the cholesterol
oxidase-DAB method (Emeis et al 1977 Histochem. J.
9:197-204). It can also detect cholesterol esters
if the sections are first treated with cholesterol
esterase. It's rather more than a "stain" but should
be OK for research. It can also be used at the EM
level (eg Pelletier & Vitale 1994 J Histochem Cytochem

Regarding the traditional stains for cholesterol:
According to Adams, Pearse and Bayliss High, the
perchloric acid-naphthoquinone method is the most
specific and sensitive one for cholesterol and its
esters. I've tried it once or twice; it works but
it's rather rough on the sections. The blue colour
fades after a few hours. An older, simpler method
is that of Schultz (here summarized from Bayliss High's
1984 "Lipid Histochemistry" p.40-41). I've not tried
it myself. 

Frozen sections, after formal-calcium fixation,
air-dried onto slides.
1. Oxidize sections in 2.5% iron alum in 0.2M acetate
   buffer, pH 3 for 4 hours at 37C.
2. Wash, 3 X 20 min in the acetate buffer.
3. Rinse in distilled water. 
4. Treat with 5% formalin in tap water 
   (ie 2% formaldehyde), 10 min.
5. Drain and allow to dry.
6. Apply a drop or two of Schulz reagent (see below),
   and then a coverslip. Examine with microscope.
Result. Cholesterol blue-green, fading quickly.
Schulz reagent: Add 1ml glacial acetic acid dropwise to
1 ml conc. sulphuric acid, with agitation. It becomes hot
and syrupy; let it cool.

Cholesterol and its esters are birefringent with crossed
polars. This property was discovered by B. Doinikow (1913)
Deutsche Z. Nervenheilk. 46:20-42) and it has been exploited 
to trace degenerating myelinated axons in frozen sections of 
human brain (Miklossy & Van der Loos 1987 Brain Res.
I tried to reproduce their results in the 1990s but the
sections contained great quantities of birefringent dirt
(completely invisible with ordinary illumination). The dirt
didn't consist of tiny needle-like crystals like the ones 
described by Doinikow and shown in the colour photos in
Miklossy's paper. My material had been stored for a long
in formalin before cutting, so perhaps lipids everywhere
had changed into some sort of birefringent junk.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
Gayle Callis wrote:
> I had an inquiry about what staining method is best for cholesterol but
> after looking at the methods (PAN) in the literature I have on hand, I am
> not sure I want to deal with this type of staining protocol.  Would it be
> better to do an immunostain?  If so, this would be on murine tissue.
> Any suggestions are welcome for the grad student asking about cholesterol
> Gayle Callis
> Research Histopathology Supervisor
> Veterinary Molecular Biology
> Montana State University - Bozeman
> PO Box 173610
> Bozeman MT 59717-3610
> 406 994-6367
> 406 994-4303 (FAX)
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