Figure 1. Cytoplasmic granules of copper in dog liver, left 400X, right 1000X.
Rubeanic Acid Staining Procedure for Copper
Purpose:
This standard operating procedure (SOP) establishes the standard for staining
copper using the Rubeanic Acid staining procedure.
Reagents and Materials:
Reagents
10% Sodium Acetate
Sodium Acetate 10gm
Distilled water 100ml
Rubeanic Acid, Stock
Rubeanic Acid, 0.05gm
Absolute Alcohol 50ml
Rubeanic Acid, Working
Rubeanic Acid, Stock 2.5ml
10% Sodium Acetate 50ml70% Alcohol
95% Alcohol 70ml
Distilled water 25mlAbsolute Alcohol
Eosin
Commercially prepared by Surgipath Co.
Expiration date provided by manufacturer.
Store at room temperature.
Materials
Tissue slides and control slides for special stain requested.
Microwave to bake slides.
Staining rack, 20 slide capacity.
Coverslips and synthetic mounting media.
Hazards
and Precautions:
See Material Safety Data Sheets.
Sample
Preparations:
Paraffin tissue section slides cut at 4 microns.
Calibration
and Standardization:
A tissue section slide containing copper is stained with each procedure.
Procedure:
1. Deparaffinize
and hydrate to distilled water.
2. Place in working Rubeanic Acid in a plastic coplin jar with lid loosely applied.
Microwave at 70% power for 1 minute.
3. Remove control slide, rinse in 70% alcohol and check with microscope. If
result is not dark enough, replace in warm Rubeanic Acid Solution - 1minute.
4. 70% alcohol - 5 minutes.
5. 100% alcohol - 5 minutes.
6. Eosin - 15 seconds.
7. Dehydrate , clear and coverslip.
Positive
control: dog liver with copper accumulation
Results:
Copper - greenish – black granules
Background – pinkish - orange
Figure 1. Cytoplasmic granules of copper in dog liver, left
400X, right 1000X.
Effective:
July 1, 2004
Cherie Chapman
James R. Turk
References:
Sheehan DC, Hrapchak BR: Theory and Practice of Histotechnology, 2nd Ed, CV Mosby, St. Louis, 1980, pp. 229-230.