Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
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.
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) -
Absolute ethanol 100 ml
Rhodanine solution (working) -
Rhodanine saturated solution (stock) 6 ml
Diluted Mayer's hematoxylin
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
4. Stain slides in diluted Mayer's hematoxylin for 10
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
clear, and coverslip with a synthetic
Copper - orange/red.
Tissue elements - light blue.
Eric C. Kellar
University of Pittsburgh Medical Center
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
Pearse, AGE (1985) Histochemistry, Theoretical and Applied, 4th ed. Vol. 2. Metal histochemistry is extensively reviewed in
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
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