RE: [Histonet] ACE

From:Tony Henwood

You could try this:

Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at  Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318

http://www.histosearch.com/homepages/TonyHenwood/default.html
http://us.geocities.com/tonyhenwoodau/index.html



ACETYLCHOLINESTERASE
Principle:

The diagnosis of Hirschsprung's disease is by showing the absence of
ganglion cells in the distal portion of the large intestine.

Hirschsprung's disease may be divided into 3 main groups:

The first and most common, comprises those patients with aganglionosis as
far as the rectosigmoid junction (short segment disease).

The second group of patients has aganglionosis extending beyond the
rectosigmoid junction but not involving the small bowel (long segment
disease).

The third group which accounts for 2-14% of all Hirschsprung's disease
patients have aganglionosis extending into the small bowel, sometimes as far
as the duodenum or stomach (total colonic aganglionosis).

In both long and short segment disease the appearance in the
Acetylcholinesterase preparations will show an increase in nerve fibres
present in the muscularis mucosae and this is accompanied by an absence of
ganglion cells and an increase in nerve fibres and nerve trunks in the
submucosa. This general picture varies with the age of the patient and many
clinical factors need to be taken into consideration before a conclusion can
be reached.
Section Requirements:
Cryostat sections (5-6 m) on poly-L-lysine slides
Cut sections at 5m and stain with toluidine blue. If the appropriate level
is obtained then cut 10 sections for the AChE stain. Cut and stain another
tol blue at the end.   (To be discussed with the Pathologist)
Solutions:

1. 10% buffered formalin cooled to 4oC

2. Dilute Ammonium Sulphide (about 0.005%)
20% Ammonium sulphide		25 ul
Tap water			50 ml

3. 0.1% Silver Nitrate

4. Incubation Medium A.

 Make up 648m/ (for 72 tubes) of medium A:

Acetylthiocholine iodide				3640mg
Sodium Acetate (0.06M)			 	3.719 g
Acetic Acid (0.1M)			 		0.082ml
Sodium Citrate (0.1M)			 	1.058 g
Copper Sulphate (0.03M)			 	0.540 g
Add distilled water up to			 	633.7ml
OMPA (Tetraisopropyl Pyrophosphoramide) (0.004M)		0.020g
Aliquot medium A Into 9ml tubes and store at -25oC

5. Incubation Medium B

Make up 72ml of medium B (for 72 tubes):

Potassium - ferricyanide 5mM (0.005M)	 0.119g/72ml distilled water.
Aliquot into 1ml tubes and store at -25 C.

Incubation solution:
After defrosting, mix one vial of both Incubation medium A and B.
Method:
1. Air dry sections for 15 minutes 
2. Fix sections in formol-saline for 10 minutes (at 4C)
3. Wash well with distilled water
4. Incubate in substrate solution at 37C for 60 minutes (mix A+B)
5. Wash briefly in distilled water
6. Treat with dilute ammonium sulphide for 30 seconds ( in fume hood)
7. Wash in tap water then rinse in distilled water
8. Treat with 0.1% silver nitrate for 1 minute at room temperature
9. Wash in distilled water
10. Counterstain with haematoxylin for 30 seconds
11. Rinse slides in water
12. Dip in blueing solution for 1 minute
13. Rinse quickly in distilled water
14. Dehydrate, clear and mount
Results:
Nerve fibres, ganglion cells, and other tissue containing
acetylcholinesterase are stained dark brown to black. (Beware - erythrocyte
membranes also contain endogenous acetylcholinesterase).

References:  
Karnovsky and Roots, J. Histochemistry and Cytochemistry, 1964, Vol 12, p
219-221. 
Filipe and Lake, Histochemistry in Pathology, 2nd Ed, 1990, p463.
 


-----Original Message-----
From: Lee & Peggy Wenk [mailto:lpwenk@covad.net]
Sent: Friday, 12 December 2003 9:22 PM
To: Histonet
Cc: Sharon Scalise
Subject: [Histonet] ACE


Need help with the ACE (acetylcholine esterase) procedure for Hirshsprung
Disease.

Two choices:

1. Can someone tell me a source of ethopropazine? We are no longer able to
obtain it through Sigma/Aldrich. We did order it from another company, but
now cannot get the stain to work.

OR

2. Can someone share with me their procedure, which does not include this
chemical.

Thank you.

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073


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