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From:"Ronnie Houston" <>
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This technique is commonly used in the diagnosis of Hirschprung's disease by 
looking at ganglion cells in the submucosa, and also to visualise motor end 
plates in skeletal muscle.

Sections: 10 micron cryostat sections of fresh, snap-frozen tissue.

Fixn:     4% formal calcium for  30sec-1min

Incub medium:

Stock A:        acetylthiocholine iodide (Sigma cat# A5751)    10mg
                0.1M acetate buffer pH6.0                      14ml
                0.1M sodium citrate                             1ml
                30 mmol/l copper sulphate                       2ml
                distilled water                                 2ml

Several methods call for the use of tetraisopropyl pyrophosphoramide 
(ios-OMPA;  Sigma cat# T1505),4 mmol/l. This is an inhibitor of butyryl and 
pseudocholinesterases, but, in my experience, unless you were wanting to 
quantify the reaction product, this can be left out. Needless to say, being 
an enzyme inhibitor, it is an extremely dangerous compound to work with.

Stock B:        5mmol/l potassium ferricyanide

Stock solutions A & B can be made in bulk and stored in aliquots at
-20C. Before use, add 1ml of soln B to soln A.

To establish the method in your department, I would suggest following this 

Take several sections through together.
1.   Rinse fixed sections in water.
2.   Incubate at 37C for 1 hour. (this time varies depending on the     
tissue being investigated and the thickness of the section)
3.   Sections should be taken out at 10 minute intervals after about 40 
minutes, and rinsed well in distilled water.
4.   Treat in 1% ammonium sulphide (always use in fume hood) for 1-2 
5.   Wash well in running tap water.
6.   Dehydrate, clear and mount.

A golden brown reaction product indicates the site of enzyme activity.

If you require a darker reaction product, when you have established the 
optimal incubation time for your samples you can treat your samples thus, 
after step 3 above.

4a.  0.05% p-phenylenediamine dihydrochloride(Sigma P1519) in 0.1M phosphate 
buffer pH6.8 for 45 minutes at room temperature.
5a.  Wash in water.
6a.  Treat with 1% osmium tetroxide (always use in a fume hood)
7a.  Wash well in water, counterstain lightly in hematoxylin and blue.
8a.  Wash in water, dehydrate, clear and mount.

It is very important to have thorough dehydration, as areas with high enzyme 
activity seem to be resistant to dehydration.

Reaction product - dark brown to black

If you need any more information, don't hesitate to ask.
Good luck
Ronnie Houston
Cytochemistry & Molecular Pathology
Texas Scottish Rite Hospital for Children
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