RE: Copper Stain

From:"Morken, Tim" <>

Here is one I've used with good results.

Tim Morken, BA, EMT(MSA), HTL(ASCP)
Infectious Disease Pathology Activity
Centers for Disease Control and Prevention
1600 Clifton Road
Atlanta, GA 30333

PH: 404-639-3964
FAX: 404-639-3043


Copper:  Rhodanine Method
This method appears to be more sensitive than the rubeanic acid method,
however it has been suggested that rhodianine demonstrates the protein to
which the copper binds rather than the copper itself. Therefore, although
considered more sensitive, it may be less specific than the rubeanic acid
methods and false positive results may be obtained.
Tissue fixed with neutral buffered10% Formalin.  Acid fixatives may remove
copper. Cut paraffin sections at 6 to 10 microns.  (Thicker sections may
stain better).
Caution:  The following chemicals used in this procedure are hazardous:
Rhodanine	Poison
For more information on each chemical, refer to it's Material Safety Data

1.	Rhodanine Saturated Stock Solution
Rhodanine (D# 90) *	0.2 gm
Absolute ethanol	100.0 mL
Note:  Make sure jar has crystals on bottom.
Saturate until blood red.

*Rhodanine is listed in catalogs alternatively as
"(p-Dimethylaminobenzalrhodanine)"   or

2.	Rhodanine Working solution
Rhodanine saturated stock solution	1.5 mL
Distilled water	50.0 mL

Note:  Shake stock solution before measuring and mixing solutions.
Shake working solution when pouring it on slides.
Discard after use.
3.	Gill's Hematoxylin (See H&E Staining Procedure)

Liver or spleen known to have large amounts of copper
1.	Bring sections to distilled water.
2.	Incubate in Rhodanine working solution in a coplin jar overnight at
37oC or 2 hours at 56oC.
3.	Rinse well in distilled water.
4.	Stain nuclei in Gill's Hematoxylin for 1 minute.
5.	Blue in tap water.  DO NOT USE AMMONIA WATER (NH40H).
6.	Rinse well with distilled water.
7.	Dehydrate, clear and mount using permanent mounting medium.
Copper granules	bright red to red-yellow.
Nuclei	light blue.
With low copper concentrations in tissue, slight fading may occur after
coverslipping and the predcipitate may be difficult to distinguish from
Sheehan, D.C., and Hrapchak, B.B.: Theory and Practice of Histotechnology,
Second Edition, 1980, C.V. Mosby Co., St. Louis, p. 230. 


Stock Rhodanine solution	5 mL
Distilled water	45 mL

1.	Deparaffinize and hydrate slides to distilled water.
2.	Place slides in 50 mL of Working Rhodanine solution.
Place probe into solution, set microwave TEMP/COOK/HOLD to 135oF, power
level 4.  Press START.  
3.	When temperature is reached, stir solution, and set timer for 10
4.	Rinse well in distilled water. 
5.	Place slides in Harris's Hematoxylin for 1 minute.
6.	Rinse slides in running tap water.
7.	Blue sections in running tap water.
8.	Rinse slides in distilled water.
9.	Dehydrate, clear, and coverslip.

Copper	Bright red to red-yellow
Nuclei	Light blue

-----Original Message-----
From: George, Cheryl []
Sent: Wednesday, September 20, 2000 11:01 AM
To: ''
Subject: Copper Stain

Hi out there,

Does anyone know of a reliable copper stain for formalin fixed paraffin
embedded tissue?


Cheryl George, HT (ASCP)
Anatomic Pathology Supervisor

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