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Malay - Aim of special stain:
- Demonstration of ferric iron deposits
- Diagnostic applications:
- The presence of ferric iron in tissue – hemochromatosis
- Material supplied for surveys:
- Liver tissue cut at 3µm
- Recommended control:
- Liver
1. Perls Prussian Blue Method A
Reagent – commercial source unknown, unless specified:
| 1. 2% Hydrochloric acid | |
| a) Hydrochloric Acid | 2 mL |
| b) Distilled water | 98 mL |
| 2. 2% Potassium Ferrocyanide | |
| a) Potassium Ferrocyanide | 2 g |
| b) Distilled water | 100 mL |
| 3. Working solution (prepare fresh before each use) | |
| Mix equal parts of 2% hydrochloric acid and 2% potassium ferrocyanide |
| 4. 0.1% Nuclear Fast Red | |
| a) Nuclear fast red | 0.1 g |
| b) Aluminium Sulphate | 5 g |
| c) Distilled water | 100 mL |
| 5. Eosin staining solution |
Method:
- Deparaffainise section and take it to distilled water.
- Filter potassium ferrocyanide- hydrochloric acid working solution into Coplin jar and stain slides for 10 minutes.
- Rinse in distilled water.
- Wash well in water.
- Counterstain with nuclear fast red for 5 minutes.
- Wash well in water.
- Dehydrate, clear and mount the section.
Results:
Iron deposits – blue
Background – various shades of red
2. Perls Prussian Blue Method B
Reagent – commercial source unknown, unless specified:
| 1. Prussian blue solution: | |
| a) 2% aqueous potassium ferrocyanide | 25 mL |
| Store at 4 degrees. Stable for 3 months. | |
| b) 2% aqueous hydrochloric acid | 25 mL |
| Stable indefinitely at room temperature. | |
| Mix A and B together. Prepare fresh before use. Stable for 2 hours. | |
| 2. Pararosaniline solution | |
| a) Pararosaniline stock solution | |
| i) Pararosaniline | 2 g |
| ii) Methanol | 200 mL |
| Shelf life 3 months. Record the date on the bottle. Store at room temperature. | |
| b) Pararosaniline working solution | |
| i) Pararosaniline stock solution | 1 mL |
| ii) Distilled water | 50 mL |
| Prepare fresh. Stable for 2 hours. | |
| 3. 1% Aqueous Safranin or Alcoholic Eosin |
Method:
1. Deparaffainise section and take it to water.
2. Wash the section in distilled water.
3. Transfer sections to freshly prepared Prussian blue solution for 20 minutes at room temperature.
4. Rinse in distilled water.
5. Wash well in tap water.
6. Counterstain with eosin for 30 seconds.
7. Wash well in tap water.
8. Dehydrate, clear and mount the section.
Results:
Ferric iron – blue
Nuclei – Red
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
2019 RCPAQAP Technical General Survey 1.
Charles J, Churukian. Theory and practice of Histological Techniques. John D Bancroft; Marilyn Gamble. 6th edition. New York, NY: Churchill Livingstone; 2008:233-236.
Solarbio life sciences, “Prussian Blue Iron Stain Kit (with Eosin)”, 2005-2021 Solarbio, http://solarbio.net/goods-476.html [accessed February 2025].