Acetylcholinesterase
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From: | "Ronnie Houston" <wee_rory@hotmail.com> |
To: | carmen.a.buttigieg@magnet.mt |
Reply-To: | |
Content-Type: | text/plain; format=flowed |
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
procedure.
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
minutes.
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
Dallas
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