[Histonet] ?Quicker Copper Stain

[Lee & Peggy Wenk]

Below is our procedure. In a 60 degree C oven/incubater/waterbath, it takes
a little over 1 hour. With a microwave, it will be done in about 10 mintues.

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073

- - - - - - - - -

COPPER - RHODANINE

PREPARED BY: Peggy A. Wenk, BS, HTL(ASCP)SLS

PURPOSE:
This stain demonstrates increased amounts of copper, but may not be specific
for copper. Copper is normally found in small amounts in the body. Abnormal
increased deposits of copper can be found in Wilson disease, and in
cirrhotic liver. In Wilson disease, copper accumulates in certain organs,
such as the liver and brain, causing scarring and cirrhosis. In cirrhotic
livers, the scarring may trap the copper, rather than the copper causing the
scarring.

PRINCIPLE:
The rhodanine may be specific for the protein to which the copper is bound.
It may be demonstrating other metals. This stain is more sensitive than the
rubeanic acid method, but may not be as specific. Hematoxylin is used as a
counterstain.

FIXATION:
Any well fixed tissue.
10% neutral buffered formalin preferred.

TECHNIQUE:
Cut paraffin sections at 10 um.

CONTROL:
Section of tissue with copper

QUALITY CONTROL:
1. 	Sections should be 6-10 um thick, to locate minimal deposits.

EQUIPMENT:
Balance, Erlenmeyer flasks, graduated cylinders, magnetic stirrer, forceps.

CAUTION:
Follow standard safety procedures when preparing stains.
RHODANINE (5(p-dimethylaminobenzylidine) should be kept away from sources of
heat. 	Can violently decompose at elevated temperatures. May be harmful if
swallowed.
HEMATOXYLIN is incompatible with oxidizers & alkalies. Store separate from
them.
AMMONIUM HYDROXIDE may cause severe skin and eye burns. Vapors are
irritating 
	to eyes and respiratory tract. Harmful if swallowed or inhaled.

REAGENTS:

STOCK RHODANINE SOLUTION
Rhodanine (5(p-dimethylaminobenzylidine)) (C12H12N2OS2)		0.2 g
Absolute ethanol, reagent		100.0 mL
Stir together several hours to make saturated solution. Store at room
temperature. Stable 4-6 months. 

WORKING RHODANINE SOLUTION
Stock rhodanine solution		3.0 mL
Distilled water		40.0 mL
JUST BEFORE USE, mix together. Good for one day only.

ALUM HEMATOXYLIN
Use Mayer or Gill hematoxylin used in H&E set up.

DILUTE AMMONIA WATER
Use dilute ammonia water used in H&E set-up.

 
PROCEDURE - Rhodanine:

1.	Deparaffinize and hydrate sections through graded alcohol to
distilled water.

2.	Place slides in WORKING rhodanine solution in 60o C. oven	1
hour

(OR, heat in 750 watt microwave oven on HIGH for 30 seconds. DO NOT
LEAVE UNATTENDED. Allow to set on counter at room temperature for 5
minutes.)

3. 	Rinse in distilled water, 3-5 changes	5-10 seconds each

4. 	Stain lightly in alum hematoxylin	10 seconds

5.	Wash in running water	10 seconds

6.	Blue in dilute ammonia water	2-3 seconds

7.	Wash in running water	5 minutes

8.	Dehydrate through graded alcohols and clear in xylene.

9.	Coverslip using a synthetic mounting media.


RESULTS:

Copper deposits 	orange/red
Heavy metals (mercury, silver, cadmium)	orange/red
Bile		yellow
Red blood cells	yellow
Lipofuchsin	golden
Nuclei		blue

PROCEDURAL NOTES:

1. 	Avoid fixatives with heavy metals (mercury, zinc), as possible
false-negative staining have been reported.

2.	This is not a specific test for copper. This demonstrates the
protein to which heavy metals, such as copper, would bind. Therefore, it may
be demonstrating other metals, such as silver or cadmium.

3.	Time given for the microwave procedure is for a 750 watt microwave
oven. Adjust time accordingly for microwave ovens with other wattage.
Solution should be heated to about 80o C.

4.	As the Working Rhodanine solution is alcoholic, it heats up faster
than an aqueous solution heated in a microwave oven. As a result, the
working rhodanine solution boils very quickly when heated in the microwave
oven. Do not leave unattended.

5.	Fading of staining after coverslipping is common.

6.	Stain may also be done at 37o C. Stain for 18 hours.

REFERENCES:
Bancroft JD, Stevens A: Theory and Practice of Histological Techniques, 3rd
ed. New York, NY, Churchill Livingstone, 1990.
Carson FL: Histotechnology: A Self-Instructional Text, Chicago, IL, ASCP
Press, 1990.
Sheehan DC, Hrapchak BB: Theory and Practice of Histotechnology, 2nd
edition. Columbus, Ohio, Battelle Press, 1980.
Vacca L: laboratory Manual of Histochemistry. New York, NY, Raven Press,
1985.
* Source of procedure is unknown.

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
JMaslanka <@t> stpetes.org
Sent: Wednesday, May 28, 2008 1:27 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] ?Quicker Copper Stain

Hi, does anyone have a quicker procedure for staining copper in liver other
than the overnight method? If so would you please share.
Thanks

Joe Maslanka
Cyto/Histo Coord.
St Peter's Laboratory

" Not everything that can be counted counts.....
  Not everything that counts can be counted."
          Albert Einstein
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet