Copper Stain Kit Procedure
*Modified Uzman's Microwave
*Modified Uzman's Microwave
APPLICATION
Mammalian Tissue
DESIGNED TO SHOW
Excessive pathologic Ionic Copper deposits in tissue.
SPECIMEN REQUIREMENTS
Fixation: Tissue: Any well fixed tissue , however, it is suggested to use formalin fixed tissue. Do not use metal based primary fixatives.
Section : Cut paraffin sections at 5 microns.
COMPONENTS AND REAGENTS
1. Rhodanine Solution - 4oz / 120 ml
2. Concentrated Sodium Acetate - 4oz / 120 ml
3. Nuclear One - 4oz / 120 ml
4. 1 stained and 3 unstained control slides
5. Pap Jars
*Rhodanine Working Solution
Add 10ml of Concentrated Sodium Acetate per every 100ml of Distilled Water. Mix . Add 5ml of Rhodanine Solution. Mix. Solution should be made fresh before use.
STAINING PROCEDURE
1. Deparaffinize sections and hydrate to distilled water.
2.Treat sections in * Rhodanine Working Solution,
3. incubating in a Laboratory Microwave at low power for 5 minutes.
4.Allow sections to stand in hot solution for 15 minutes.
5. Wash sections through several changes of distilled water.
6. Stain section in Nuclear One for 10 minutes.
7. Rinse excess stain using distilled water and blue in distilled water for 3-4 minutes.
8. Dehydrate sections through three changes of Reagent Alcohol and clear through two changes of Xylene or three changes of Xylene Substitute.
9. Mount section using Clearslip.
RESULTS
Copper-Green-Black precipitate
Nuclei-Blue
NOTES
1. If a Laboratory Microwave is not accessible, a conventional room temperature procedure can be incorporated as follows; 18 hours in the * Rhodanine Working Solution. Follow the treatment with section washing through seven changes of deionized or distilled water.
2. Low levels of copper in tissue will demonstrate slight fading after coverslipping . Use tested or non-reducing mounting mediums.
3. Trace heavy metals in the primary fixative will cause non-specific staining.
REFERENCES
1. Uzman L. L. : Histochemical localization of Copper with rubeanic acid. Lab Invest. 5:299-305; 1958
2. Mallory F.B. and Parker, F., Jr.: Fixing and staining methods for lead and copper in tissues, Am J. Pathol. 15: 517-522, 1939
3. Luna, L.G. (1968): In: Manual of Histologic Staining Methods of the AFIP, pp. 187-188. McGraw-Hill, New York.
Dev. 09/03/97
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