RE: Acetylcholinesterase

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From:"Smith, Allen" <>
To:"'Buttigieg Carmen at MOH'" <>
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The old Gomori method works for me. [George Gomori.  MICROSCOPIC
HISTOCHEMISTRY.  U. Chicago Press, 1952, pp. 210-212]

Gomori calls for free floating sections, and I prefer them for this purpose.
Cut frozen sections 20 to 40 microns thick. [Thick sections allow you to 
 follow the nerve for some distance.]
Fix 1 1/2 hr in 10% formalin at 4 C.
Distilled water, 10 min.
Incubate 5 hr. at 37 C in:
	40 mg acetylthiocholine iodide in 20 ml of buffered copper sulfate
Saturated aqueous sodium sulfate, 5 changes, 5 minutes each.
1% ammonium sulfide, 3 minutes.
Tap water 5 minutes.
10% formalin [Gomori says overnight; 2 1/2 hr is good enough.]
Distilled water, 10 minutes.
Hematoxylin, 5 seconds. [I use Ehrlich's acid hematoxylin.]
Distilled water, 3 minutes.
Saturated aqueous lithium carbonate, 5 minutes.
Tap water, 5 minutes.
50% ethanol, 5 minutes.
70% ethanol, 5 minutes.
95% ethanol, 5 minutes.
Absolute ethanol, 3 changes, 5 minutes each.
Xylene, 2 changes, 10 minutes each.
Mount in synthetic resin.  [I use Fisher's Permount.]

Buffered copper sulfate is a pain to make, but it is poisonous enough to
keep a month in the refrigerator.
	22.7 g Glauber's salt [sodium sulfate dodecahydrate]
	22 ml distilled water
	Heat to 37 C and add
	60 mg copper(II) sulfate pentahydrate
	After this dissolves add
	75 mg glycine (free base)
	After this dissolves add
	200 mg magnesium chloride hexahydrate
	Keep this solution at 37 C while adding enough maleate buffer 
	 to bring the pH to 5.15.  This usually requires 2 ml of a maleate
buffer made by dissolving 350 mg maleic anhydride to 6 ml 1 N NaOH.
	 [4% NaOH is 1 N.]

I get my acetylthiocholine iodide from SIGMA [1-800-521-8956].

				-Allen A. Smith
				Barry University School of Graduate Medical
				Miami Shores, FL	

-----Original Message-----
From: Buttigieg Carmen at MOH []
Sent: Thursday, May 04, 2000 3:57 AM
Subject: Acetylcholinesterase

Dear Histonetters

Please help!

One of our pathologists just asked for a new procedure which I have never
before. He wants to stain up nerve fibres and cells containing 
acetylcholinesterase on frozen sections of intestine.
I found a method in an old book where the incubation takes between 7 and 30 
days! Is there anything simpler? 
If anyone has the protcol could you please also send catalogue numbers of
not so common reagents which would certainly be involved.
Thanks for your help.

St. Luke's Hospital
Fax: 0356 224286

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