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Malay - Aim of special stain:
- Demonstrating pathological changes in elastic fibres
- Diagnostic applications:
- Emphysema, arteriosclerosis and other vascular diseases, identification of venous invasion in tumours of colorectal carcinomas
- Material supplied for surveys:
- Liver, skin and lung composite tissue block cut at 3µm
- Recommended control:
- Tissues that demonstrate both fine and coarse fibres. For example, skin tissue staining should demonstrate both fine and coarse elastic fibres, the lung should demonstrate the internal and external elastic fibres in the alveoli pleura, and vascular tissue and the liver tissue should demonstrate the elastic fibres in the vessel walls.
1. Aldehyde fuchsin Gomori method
Reagent – commercial source unknown, unless specified:
| 1. Dubosq-Brazil Stock Solution | |
| a) Absolute alcohol | 220 mL |
| b) 40% Formaldehyde | 120 mL |
| c) Distilled water | 50 mL |
| d) Saturated Picric acid in 80% alcohol | 25 mL |
| e) Glacial Acetic Acid | 30 mL |
| 2. Aldehyde fuchsin | |
| a) Para-rosaniline | 1.25 g |
| b) 70% ethanol | 225 mL |
| c) Conc. Hydrochloric acid | 2.5 mL |
| d) Paraldehyde | 2.5 mL |
| Dissolve para-rosaniline in the 70% Alcohol and add the Hydrochloric acid and the Paraldehyde. Shake well and stand at room temperature until the colour is a deep purple (24-48 hours). | |
Store in the refrigerator. Solutions have an expiry of 3 months.
| 3. Iron hematoxylin | |
| a) Solution A: | |
| Dissolve 3 g Hematoxylin in 50 mL of Ethanol. Add 250 mL of Ethanol then add 200 mL of Distilled water. |
|
| b) Solution B: | |
| Ammonium ferric sulphate | 20 g |
| Distilled water | 440 mL |
| Conc. sulphuric acid | 5 mL |
| For use: Two parts of solution A to one part of solution B | |
| 4. 0.25% Orange G | |
| a) Orange G | 0.5 g |
| b) Saturated aqueous Picric Acid | 200 mL |
| 5. Trichrome solution | |
| a) Chromotrope 2R | 3 g |
| b) Light green | 1.5 g |
| c) Glacial acetic acid | 5 mL |
| d) Phosphotungstic Acid | 4 g |
| e) Distilled water | 500 mL |
| 6. 85% Ethanol and 0.2% Acetic acid |
Method:
- Bring sections to water.
- Treat sections in Duboscq-Brazil for 10minutes.
- Rinse well for 5 minutes.
- Stain in Aldehyde fuchsin for 4 minutes.
- Wash off with 85% Ethanol (brief, differentiation step).
- Rinse in water.
- Stain in freshly prepared Iron hematoxylin for 2 minutes.
- Wash in running tap water until the section is blue.
- Stain in Orange G solution for 30 seconds.
- Wash in running water until the orange stain remains only in the red cells.
- Stain in Trichrome solution for 4 minutes.
- Rinse briefly in 0.2% Acetic acid.
- Wash in water.
- Dehydrate.
Results:
Elastic tissue – Purple
Collagen – Green
Red cells – Orange
Muscle and cytoplasmic tissue – Reddish
2. Orcein with Van Gieson counter stain
Reagent – commercial source unknown, unless specified:
| 1. 1% Orcein in 70% Ethanol with 1 % HCL (Corrosive solution) | |
| a) Orcein (synthetic) | 4.0 g |
| b) 70% ethanol | 400 mL |
| c) Hydrochloric Acid | 4.0 mL |
| Add Orcein, 70% Ethanol and the stirring bar into a conical flask. Carefully add Hydrochloric Acid and stir until dissolved. Filter the solution into a stock bottle. |
|
| 2. 70% Ethanol | |
| a) Absolute Alcohol | 70 mL |
| b) Distilled water | 30 mL |
| 3. Van Gieson’s Stain (Explosive solution) | |
| a) Saturated aqueous picric acid (commercially available) | 100 mL |
| b) 1 % Acid Fuchsin (commercially available) | 10 mL |
| i) Acid Fuchsin | 1 g |
| ii) Distilled water | 100 mL |
| Add water and Acid Fuchsin into a conical flask. Stir on the magnetic stirrer to dissolve. Filer into a stock bottle. | |
Pour Picric Acid into a conical flask and add Acid Fuchsin, stir until dissolved.
Transfer into a stock bottle.
| 4. 1% Acid Alcohol (Corrosive solution) | |
| a) Hydrochloric Acid | 20 mL |
| b) Absolute Alcohol | 1400 mL |
| c) Distilled water | 600 mL |
| Mix alcohol and water in the stock bottle. Carefully add the hydrochloric acid and mix thoroughly. Transfer to stock bottle. |
|
Method:
- Take sections down to 70% Alcohol.
- Stain in Orcein Solution (in a sealed Coplin jar) for 1 hour.
- Wash in running water for 1 minute.
- Differentiate in acid alcohol until the excess stain is removed.
- Wash in running water for 5 minutes.
- Counterstain with Van Gieson for 3 minutes.
- Rinse in running water.
- Dehydrate in 100% alcohol only, avoid using 70% and 95% alcohol. Clear and mount.
Results:
Coarse elastic fibres – reddish brown
Smooth Muscle – red
Fine elastic fibres – dark brown
Red Blood Cells – yellow
Method Limitations
- Store Orcein solution in a sealed Coplin jar to avoid precipitation of stain.
- Gurr/BDH Orcein has given consistent results and should be used in this method.
- Discard solution after one month.
- Van Gieson’s Stain may be stored, but a freshly prepared solution will give a sharper result.
3. Millers Method
Reagent – commercial source unknown, unless specified:
| 1. New Fuchsin | 1 g |
| 2. Crystal Violet | 1 g |
| 3. Victoria Blue 4R |
1 g |
| Dissolve the dyes in 200mls of hot water, then add the following in order. | |
| Resorcin | 4 g |
| Dextrin | 1 g |
| Fresh 30% aqueous Ferric chloride | 50mL |
Boil the mixture on a hot plate in a fume cupboard for 5 minutes and then filter while still hot. Let cool then return the precipitate with the filter paper to the original vessel and re-dissolve in 200 mL of 95% alcohol. Boil for a further 15-20 minutes. Filter and discard the filter paper and let cool. Restore the volume to 200 mL with 95% alcohol. Finally, add 2.0 mL of concentrated Hydrochloric Acid. Mix well and store.
Miller’s elastic stain has excellent keeping properties and may be reused for many years.
| 4. Curtis’ Stain | |
| a) 1% aqueous Ponceau S | 10 mL |
| b) Sub Saturated aqueous Picric Acid Solution | 90 mL |
| c) Glacial Acetic Acid | 1 mL |
| Mix the solutions, the stain keeps well | |
| 5. 1% Potassium Permanganate | |
| a) Potassium Permanganate | 1 g |
| b) Distilled Water | 100 mL |
| 6. 1% Oxalic Acid | |
| a) Oxalic Acid | 1 g |
| b) Distilled Water | 100 mL |
Method:
- Bring sections to water.
- 1% Potassium Permanganate for 5 minutes.
- Rinse in water.
- 1% Oxalic Acid for 1 minute.
- Wash in water.
- Rinse in 95% alcohol.
- Stain in Miller’s stain for 3 hours.
- Wash in 95% alcohol.
- Rinse in distilled water.
- Counterstain with Curtis’ stain for 2 minutes.
- Rinse with Absolute Alcohol.
- Dehydrate, clear and mount.
Results:
Elastic fibres, mast cell granules – blue/black
Collagen – red
Muscle, cytoplasm, fibrin – yellow
Nuclei – brownish/green
Erythrocytes – yellow
Notes:
- Differentiation is not required. The stain may be diluted with an equal volume of 95% alcohol for overnight staining.
- Picric Acid is toxic and a percussive explosive when dry.
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
2010 RCPAQAP Technical General Survey 2.
2020 RCPAQAP Technical General Survey 1.
Gomori G. Aldehyde-fuchsin: a new stain for elastic tissue. American Journal of Clinical Pathology. 1950 Jul;20(7):665-6. PMID: 15432365.
Carson. Frieda L. Elastin Stains in W. Brown. Histologic preparations Common Problems and Their Solutions. College of American Pathologists press, 2009:109-115.
Bancroft, JD and Gamble, M. Theory and Practice of Histological Techniques. 6th Edition, Churchill Livingstone. London. 2008:148-155.