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Malay - Aim of special stain:
- Demonstrate the presence of copper deposits
- Diagnostic applications:
- Wilson’s and primary biliary cirrhosis disease
- Material supplied for surveys:
- Liver tissue with copper deposits, cut at 3µm
- Recommended control:
- Liver with deposits of copper
1. Okamoto and Utamura rhodanine copper stain
Reagents – commercial source unknown, unless specified:
| 1. 0.5% Celloidin Stock Solution | |
| a) Celloidin | 2.5 g |
| b) Absolute Alcohol | 250 mL |
| c) Diethyl Ether | 250 mL |
| Dissolve celloidin in absolute alcohol and ether. Store in closed stoppered bottles to prevent evaporation. |
|
| 2. Celloidin Working Solution | |
| a) 0.5% Celloidin, stock solution | 15 mL |
| b) Absolute Alcohol | 235 mL |
| Pour 0.5% celloidin stock solution into staining dish. Add absolute alcohol. |
|
| 3. Rhodanine (Stock Solution) | |
| a) Absolute Alcohol | 100 mL |
| b) 5-(4-dimethylaminobenzylidine) rhodanine | 0.5 g |
| Keep stock solution in amber bottle in refrigerator Solution stable for 1 month. |
|
| 4. Rhodanine (Working Solution) | |
| a) Sodium Acetate-trihydrate | 1 g |
| b) Distilled Water | 50 mL |
| c) 37% Formaldehyde, concentrated | 0.15 mL |
| d) Rhodanine stock solution | 3 mL |
| Note: Do not shake rhodanine stock solution when preparing the working solution. | |
| 5. Harris hematoxylin |
| 6. 0.05% Sodium Tetraborate (Borax) | |
| a) Sodium Tetraborate (Borax) | 0.5 g |
| b) Distilled Water | 1000 mL |
Method:
- Deparaffinise sections in xylene then rinse in 2 changes of absolute alcohol.
- Place slides in a film of 0.5% celloidin for 2 minutes.
- Place slides in 80°C oven until dry for 2 minutes.
- Place slides in 70% alcohol (to harden celloidin) for 1 minute.
- Wash slides in running water. Rinse in distilled water.
- Place slides in filtered rhodanine in plastic Coplin jar.
- Heat in microwave oven on high (level 5, please check microwave oven settings) for 20 seconds.
- Remove Coplin jar from microwave oven, agitate slides and place Coplin jar in 54°C convection oven for 30 minutes.
- Rinse well in distilled water.
- Stain in Harris Hematoxylin for 20 seconds.
- Rinse in distilled water.
- Rinse in 0.05% sodium tetraborate (borax) to blue for 20 seconds.
- Rinse well in distilled water.
- Blot section dry and take sections directly to xylene.
- Mount in coverslipper.
Results:
Copper deposits – orange/red
Nuclei – blue
Note: Rhodanine demonstrates the protein to which the copper binds rather than the copper itself.
2. Orcein staining method
Reagents – commercial source unknown, unless specified:
| 1. Working Solution | |
| a) Orcein (commercial preparation – Gurr’s Orcein biological stain, Synthetic) | 1 g |
| b) 70% alcohol | 100 mL |
| (adjust pH to 1-2 using conc. HCl 1.0 mL) | |
| 2. 1% Potassium Permanganate | |
| a) Potassium Permanganate | 1 g |
| b) Distilled Water | 100 mL |
| 3. 1 % Oxalic acid | |
| a) Oxalic acid | 1 g |
| b) Distilled Water | 100 mL |
Safety aspects:
Potassium permanganate – Severe mucous membrane irritant. Corrosive.
Oxalic acid – Severe eye and skin irritant.
Orcein – Other dyes in this group (oxazine) are hazards to health. Handle with care.
Method:
- Sections to water.
- Potassium permanganate for 5 minutes.
- Wash in water.
- Oxalic acid for 2 minutes.
- Wash in water.
- Rinse in 70% alcohol.
- Place slide in working Orcein solution mailer at 37oC in waterbath for 30 minutes.
- Rinse well in 70% alcohol.
- Dehydrate, clear and mount.
Results:
Copper-associated protein – purplish/brown
Cytoplasm – pale pink/light brown
Elastic fibres – brown
Disclaimer:
These methods are intended as a guide only. Laboratories that wish to implement these methods should perform internal validation prior to use. The RCPAQAP does not make any claim or warranty for the accuracy or performance of these methods.
References
2021 RCPAQAP Technical General Survey 3.
Bancroft JD, Gamble M. Theory and Practice of Histology Techniques. 5th Edition. Churchill Livingstone; 2002:339.
Bunton T.E. Comparison of three histochemical staining methods for the detection of copper in white perch (Morone americana) with abnormal hepatic copper storage: Journal of Comparative Pathology, Vol 102, Issue 1, 1990:25-31.