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Which staining method for copper is best?
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Which staining method for copper is best?


Which histochemical staining method is best for copper in human or animal tissues? The choice seems to be between rubeanic acid (not in catalogs) and some impossibly long name that ends in "rhodanine."


This question to the HistoNet listserver elicited replies that generally favored the "rhodanine" reagent over "rubeanic acid." Nomenclature can be confusing! Don't confuse rhodaNine with rhodaMine, and note that in any chemical catalog, p-dimethyl- is indexed under the letter D, not P. A few general references for copper histochemistry are added at the end of this FAQ item.

J.A. Kiernan.

Answer 1.

Rubeanic acid is H2NCSCSNH2 and is listed in catalogs as Dithiooxamide (by Aldrich, Sigma and other vendors).

I prefer the "rhodanine" method for the demonstration of Copper:

  Fixation:   10% neutral buffered formalin.

  Embedding: Paraffin sections cut at 6 microns


  Distilled water, preferably deionized, should be used in all solutions and rinses.

    Rhodanine saturated solution (stock) -

       p-Dimethylaminobenzylinene-rhodanine   0.2 g
       Absolute ethanol                                  100 ml

    Rhodanine solution (working) -

       Rhodanine saturated solution (stock)     6 ml
       Distilled water                                    94 ml

    Diluted Mayer's hematoxylin

       Mayer's hematoxylin              50 ml
       Distilled water                      50 ml

   0.5% aqueous sodium borate (borax)

Note: The use of chemically clean glassware is necessary. Shake stock solution before measuring and mixing solutions and shake the working solution before pouring it onto the slides.


     1. Hydrate slides to distilled water.
     2. Incubate slides in rhodanine working solution at 37 degree C for 18 hours.
     3. Wash slides well in several changes of distilled water.
     4. Stain slides in diluted Mayer's hematoxylin for 10 minutes.
     5. Rinse slides with distilled water.
     6. Quickly rinse slides in 0.5% sodium borate.
     7. Rinse slides with distilled water.
     8. Dehydrate slides through 95% alcohol to absolute ethanol,
         clear, and coverslip with a synthetic mountant.


       Copper - orange/red.
       Tissue elements - light blue.

Eric C. Kellar
University of Pittsburgh Medical Center

Answer 2.

A few references for copper histochemistry.

Irons,RD; Schenk,EA; Lee,CK (1977): Cytochemical methods for copper. Archives of Pathology and Laboratory Medicine 101, 298-301. Cytochem methods for copper. Comparison of dithiooxamide, diaminobenzylidene-rhodanine, diethylthiocarbamate.

Pearse, AGE (1985) Histochemistry, Theoretical and Applied, 4th ed. Vol. 2. Metal histochemistry is extensively reviewed in Chapter 20.

Szerdahelyi,P; Kasa,P (1986): A highly sensitive method for the histochemical demonstration of copper in normal rat tissues. Histochemistry 85, 349-352. Highly sensitive histoch method for Cu histochemistry. Magnesium-dithizone, followed by silver intensification.

Szerdahelyi,P; Kasa,P (1986): Histochemical demonstration of copper in normal rat brain and spinal cord. Histochemistry 85, 341-347. Histochemical demonstration of Cu in normal brain, spinal cord.

John A. Kiernan
London, Canada

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