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Rhodanine Staining Protocol
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Rhodanine Staining Protocol


Excess copper is found in the cytoplasm of cells bound to copper-associated protein. DMAB-rhodanine is a bidentate chelating agent, which has a strong affinity for proteinaceous copper deposits in tissue sections. Increased amounts of copper are found in the liver in several disease states including Wilson’s disease (an inherited condition), the excess copper has a toxic effect causing liver damage ranging from acute hepatitis and advanced cirrhosis. Wilson’s disease may also cause an excess of copper in the brain and the cornea of the eye. Other disease states which cause an excess of copper in the liver are chronic biliary obstruction and chronic hepatitis.

The reagent is not considered by the textile industry as having any commercial value, and as a result, the compound has not been allocated a Colour Index number.

Technical Points

1.   A known positive control section must be used to ensure correct demonstration has been achieved.

2.   (step 2) - Sections stained at 60°C may occasionally show some background staining.

3.   Rhodanine is leached from tissue sections by xylene in Depex mountant, obscuring copper staining.

4.   The slides should not be left sitting in xylene for long periods of time before coverslipping otherwise there may be a decrease in staining intensity.


1.      Bring sections to distilled water

2.      Stain in DMAB-rhodanine stain at 60°C 3 hrs (Check microscopically after 1 hour).

3.      Rinse well in distilled water

4.      Lightly counterstain with alum haematoxylin 5 secs

5.      Differentiate in acid alcohol

6.      Wash in water

7.      Blue in Scotts

8.      Wash in water 5 mins

9.      Dehydrate, clear and mount


        copper deposits..............................brick red

        nuclei..........................................pale blue

Reagent Formulae

1.   DMAB-rhodanine, Stock solution
                  dimethylamino benzylidene Rhodanine
                  absolute ethanol
Make a saturated solution of the DMAB-rhodanine in the alcohol.  Store the stock solution at 4°C.
Note - dimethylamino benzylidene Rhodanine has no CI number.

2.   DMAB-rhodanine, Working solution (shake well)
                DMAB-rhodanine, Stock solution    3.0 ml
                distilled water                              47.0 ml
Prepare fresh before use.


1.   Okamoto K, and Utamura M,(1938),Acta Scholae Medicinalis Universitatis Imperialis in Kioto,32,99

2.   Howell JS,(1959), ‘Histochemical demonstration of copper in copper fed rats and in the hepatolenticular degeneration’. J.Path.Bact.,77,423

3.   Pauline Hall , Histopathology, Flinders Medical Centre.

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