Ernestine wrote <>

Here's one for you.

ACETYLCHOLINESTERASE IN THE DIAGNOSIS OF HIRSCHSPRUNG'S DISEASE

PRINCIPLE:
The presence of acetylcholinesterase is indicative of the presence of neural
processes. Hirschsprung's Disease is diagnosed by the absence of ganglian
cells. This stain is a series of replacement reactions which ends in the
deposition of copper ferrocyanide at sites of acetylcholinesterase activity.
The product is made darker by exposure to osmium tetroxide.
SPECIMEN:
Cryostat sections cut at 10 um and mounted on poly-l-lysine coated slides or
Plus slides. Sections are air-dried and then fixed for 30 seconds in 4%
formaldehyde in 0.1M calcium acetate (formol-calcium).
REAGENTS:
CAUTION:	Most of the chemicals used in this technique are highly
dangerous!!

Formaldehyde is a suspected carcinogen and a strong irritant
Osmium tetroxide (OsO4) vapors and solutions tain skin black and are very
toxic and damaging to the eyes, skin and respiratory tract.  Do not breathe
vapors.  The effects of contact may be delayed.  Material is strong
oxidizer.  Wear goggles and gloves when working with Os04.  Handle material
under a hood.
Glacial acetic acid  is an irritant and can cause deep burns
Dimethylformamide is toxic and carcinogen
Copper Sulfate is toxic
Acetyliocholine is irritant
Potassium Ferricyanide is immediately dangerous to life and health. Can also
be absorbed through intact skin
N,N-Dimethyl-M-Phenylenediamine Dihydrochloride is highly toxic
Ethopropazine HCL is harmful to CNS
Calcium Acetate is harmful
For further information, please refer to MSDS
.

1.)	4% Formaldehyde IN 0.1M Calcium Acetate (Formol-Calcium)
40% Formaldehyde (AP 270)	...................100.00 mL
Dried Calcium Acetate (AP 120)...................15.8  gm
Distilled Water to 1 liter 
The pH is approximately 6.8 as made, and requires no adjustment and no
marble chips are needed.
2.)	0.1M Acetate Buffer, pH 6.0
0.1N Acetic Acid	50.00 mL
0.1M Sodium Acetate	950.00 mL
3.)	0.1N Acetic Acid
Glacial Acetic Acid, concentrated (AP 5)...................5.75 mL
Distilled Water	...............1000.00 mL
4.)	0.1M Sodium Acetate
Sodium Acetate Trihydrate (AP 660)	13.61 gm
Distilled Water	1000.00 mL
5.)	0.1M Sodium Citrate
Sodium Citrate (AP 700)	29.41 gm
Distilled Water	1000.00 mL
6.)	30 mM Copper Sulfate
Copper Sulfate, anhydrous (AP 210) ..............4.79 gm
Distilled Water	..............1000.00 mL

7.)	4 mM Ethopropazine
Ethopropazine (AP 255)	1.4  gm
Distilled Water.............................1000.00 mL
8.)	5 mM Potassium Ferricyanide
Potassium Ferricyanide (AP 585)	..............1.65 gm
Distilled Water	..............1000.00 mL
9.)	0.05% P-Phenylene Diamine Dihydrochloride IN 0.05M Phosphate Buffer,
pH 6.8
p-phenylene diamine dihydrochloride (AP 235).........5.00 mg
0.05M Phosphate buffer, pH 6.8	...............10.00 mL
Prepare just before use and the solution must be colorless.
0.05M PHOSPHATE BUFFER, pH 6.8
0.05M Sodium Phosphate dibasic (Na2HPO4) (AP 730)..........10.0 mL
0.05M Potassium phosphate monobasic (KH2PO4) (AP 615)	.........10.0 mL
	1.)	0.05M SODIUM PHOSPHATE DIBASIC  (Na2HPO4)	
		7.1 gm of Sodium Phosphate dibasic dissolved in 1 liter of
distilled water.
	2.)	0.05M POTASSIUM PHOSPHATE MONOBASIC  (KH2PO4)
		6.8 gm of Potassium Phosphate Monobasic dissolved in 1 liter
of distilled water. 
10.)	1% Osmium Tetroxide
2% Osmium Tetroxide (Ask from E.M.)	50.00 mL
Distilled water	50.00 mL
11.)	Harris' Hematoxylin ( see Histology manual p H702. B11) 
12.)	Incubation Medium
1.	Solution A. 
Acetylthiocholine iodide (AP 15)	5.0 mg
0.1M Acetate buffer, pH 6.0	6.5 mL
0.1M Sodium citrate	0.5 mL
30 mM copper sulfate	1.0 mL
Distilled water	1.0 mL
4 mM ethopropazine	0.2 mL

2.	Solution B.  
Add 1.0 mL 5 mM Potassium Ferricyanide just before use.
A and B can be made in bulk and stored in aliquots at -20oC .
CONTROL:
Neural tissue:  Brain, nerve or spinal cord
PROCEDURE:
1.	Cut frozen tissue block at 10u and collect sections on poly-L-lysine
coated glass slides.
2.	Air-dry sections for 30 minutes and circle tissue sections with a
glass scribe.
3.	Fix sections in 4% formaldehyde in 0.1M Calcium acetate
(formol-calcium) for 30 seconds.
4.	Rinse the fixed sections for 10 seconds in tap water.
5.	Wipe around tissue sections, place in moist chamber and cover the
section with the above Incubation Medium and incubate at 37oC for 1 hour.
6.	Wash briefly in tap water.
7.	Wipe around tissue sections, place in moist chamber and apply with
freshly prepared 0.05% p-phenylene diamine dihydrochloride in 0.05M phospate
buffer, pH 6.8 for 45 minutes at room temperature.
8.	Wash in tap water.
9.	Wipe around tissue section, place in moist chamber and cover with 1%
Osmium tetroxide for 10 minutes at room temperature (go to E.M. Lab.).
10.	Wash well in tap water, counterstain lightly with Harris'
hematoxylin for 10 seconds depending on the intensity of the stain.
11.	Wash, dehydrate thoroughly, clear and mount.

RESULTS:
Nerve fibers and cells containing acetylcholinesterase:	dark brown to black.
NOTE:
1.	Do not counterstain too heavily.
2.	Thoroughly dehydrate because areas with strong activity are
resistant to dehydration.
3.	Beware of areas of RBC's which appear to have nerve fibers lying
over.  This is due to the acetylcholinesterase of the RBC membrane.

REFERENCE:
	M. Isabel Filipe and Brian D. Lake, Histochemistry in Pathology, 2nd
Edition, Churchill Livingstone, New York 1990, p. 211 and 463.


-----Original Message-----
From: ErnieMiddleton@aol.com [mailto:ErnieMiddleton@aol.com]
Sent: Thursday, August 01, 2002 9:53 PM
To: histonet@pathology.swmed.edu
Subject: ACHE


Hi,
I am in need of staining procedure for Acethycholinessterase (ACHE).  I need

this ASAP.  Thank you.

Ernestine Middleton HT\HTL, BS,MPH
Pathology Manager
111 E. 210 Street.
Bronx, NY
718-920-4157
Fax# 718-547-1920