[Histonet] Cholesterol staining suggestions

[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
42:1539-1554.)

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.
426:377-380).
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
time
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
   kiernan[AT]uwo.ca
   http://publish.uwo.ca/~jkiernan/
   http://instruct.uwo.ca/anatomy/530/index.htm
_______________________________
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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