[Histonet] Osmium & ACE staining

[Tony Henwood]

Sure do,
Method below:

Acetylcholinesterase (ACHE)

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

Cut sections at 5 - 6um and stain with toluidine blue. If the appropriate level is obtained then cut 10 sections for the ACHE stain. 
Solutions

1.	10% buffered formalin cooled to 4oC

2.	Dilute Ammonium Sulphide (about 0.005%)
		Warning: Flammable liquid, Irritant, Toxic stench - see MSDS
20% Ammonium sulphide				20μl
Tap water						20ml

3.	0.1% Silver Nitrate

4.	Stock solution A

 Make up 648ml (for 72 tubes) of solution A:
		
Acetylthiocholine iodide (Sigma A5751)		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 (0.004M) (Sigma T-1505)		 	0.020g
Aliquot 9ml into 10ml tubes, labeled "AA" and store at -20oC

5.	Stock solution B

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

Potassium   ferricyanide 5mM (0.005M)	 
	0.119g/72ml distilled water.
Aliquot into 1ml tubes, labeled "AB" and store at  20oC.

6.	Incubating solution

After defrosting, mix one vial of both stock solution A and B.
Method

1.	Air dry sections for 15 minutes 
2.	Fix sections in 10%NBF for 10 minutes (at 4°C)
3.	Wash well with distilled water
4.	Incubate in substrate solution at 37°C 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

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


Regards

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




-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Norton, Sally
Sent: Sunday, 22 April 2007 3:08 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Osmium


            Hello all,

 

            I was wondering if anyone has a method for doing ACE staining that does not use osmium.

 

            Thank you,

 

            Sally Norton, Histotech

 

            Children's Hospital and Regional Medical Center

            Seattle, Wa


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