I wanted to applaud you for the strong safety emphasis in your method.
while the NCCLS format requires us to include this information in our methods,
some folks just provide superficial information.
nice job.
Vinnie Della Speranza
Manager for Anatomic Pathology Services
Medical
University of South Carolina
165 Ashley Avenue Suite 309
Charleston,
SC 29425
Ph: 843-792-6353
fax: 843-792-8974
>>> "Morken,
Tim" <tim9@cdc.gov> 08/02/02 07:28AM >>>
Ernestine wrote
<<I am in need of staining procedure for
Acethycholinessterase
(ACHE>>
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