Howell's Rubeanic Acid
for Copper Deposits

Solutions

Eosin Y, 0.1% in absolute ethanol.

Sodium acetate, 10% aqueous.

Stock rubeanic acid
Rubeanic acid	50	mg
Ethanol, absolute	50	mL

Working rubeanic acid
Stock rubeanic acid	2.5	mL
Sodium acetate, 10% aqueous.	50	mL

Tissue sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are satisfactory.

Method

1. Bring sections to water via xylene and ethanol.
2. Place in the working rubeanic acid solution overnight at 37°C.
3. Place into 70% ethanol for 15 minutes.
4. Place into absolute ethanol for 6 hours.
5. Lightly counterstain with alcoholic eosin.
6. Rinse well with absolute ethanol.
7. Clear with xylene and mount with a resinous medium.

Expected results

• Copper  –  green-black granules
• Cytoplasm  –  pink

Notes

1. Use both negative and positive controls, as occasional failures in staining occur. A suitable positive control is a liver section from a known case of Wilson's disease. Failing that, copper may be found in liver from chronic active hepatitis.
2. Copper forms a chelate with rubeanic acid, as do some other metals. Of these, cobalt and nickel may be confused with copper, so sodium acetate is added to the solution to inhibit them.
3. Rubeanic acid is also known as ethanedithioamide, dithiooxamide or dithiooxalic diamide, with the formulae below. It would appear that the copper is chelated between the sulphur and nitrogen (see Kiernan).

   H2NCSCSNH2

 

Reference
Bancroft, J.D. and Stevens A. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.

Susan Budavari, Editor, (1996)
The Merck Index, Ed. 12
Merck & Co., Inc., Whitehouse Station, NJ, USA

Kiernan. J.A., (1999)
Histological and histochemical methods: Theory and practice, Ed. 3
Butterworth Heinemann, Oxford, UK.

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