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Malay - Aim of special stain:
- Differential demonstration of specific connective tissues such as muscle, collagen fibres, fibrin and erythrocytes
- Diagnostic applications:
- Muscular pathologies, cardiac conditions, cirrhosis of the liver, glomerular fibrosis of the kidney
- Material supplied for surveys:
- Composite block containing appendix, lung and liver cut at 3µm
- Recommended control:
- Composite block containing a combination of lung, liver, small bowel, kidney or skin for better demonstration of muscle, collagen, fibrin and red blood cells
1. Masson trichrome staining protocol
Reagents – commercial source unknown, unless specified:
| 1. Ponceau Acid Fuchsin | |
| a) Ponceau de xylidine | 0.5 g |
| b) Acid fuchsin | 0.5 g |
| c) Distilled water | 100 mL |
| d) Glacial Acetic Acid | 1 mL |
| 2. 1% Phosphomolybdic acid solution | |
| a) Phosphomolybdic Acid | 1 g |
| b) Distilled Water | 100 mL |
| c) Solution A: | Commercially purchased |
| d) Solution B: | Commercially purchased |
For Use: Mix equal parts 1 and 2.
| 3. Aniline Blue 0.3% in 1% Acetic Acid | |
| a) Aniline Blue | 0.3 g |
| b) Acetic Acid | 1 mL |
| c) Distilled Water | 100 mL |
Method:
- Take section to distilled water.
- Mordant in Bouins solution (in a container) at 56°C in a water bath for 1 hour.
- Wash well in tap water until all the yellow disappears.
- Stain nuclei with Weigert’s Iron Haematoxylin for 10 minutes.
- Wash well in tap water and then rinse with distilled water.
- Ponceau Acid Fuschin on slide for 10 minutes.
- Wash with distilled water.
- Differentiate with 1% Phosphomolybdic acid solution (until connective tissue is clear of red stain) for 5 minutes.
- Rinse in distilled water.
- Place Aniline blue on the slide for 5 minutes.
- Dehydrate, clear and mount the section.
Results:
Nuclei – Blue/Black
Cytoplasm, muscle, erythrocytes – Red
Collagen – Blue
2. Picro Mallory staining protocol
Reagents – commercial source unknown, unless specified:
| 1. Weigert’s Iron Haematoxylin | |
| a) Solution A | |
| Haematoxylin | 1 g |
| Absolute Alcohol | 100 mL |
| b) Solution B | |
| 30 % Aqueous Ferric Chloride | 4 mL |
| Distilled water | 100 mL |
| Concentrated HCL | 1 mL |
For Use: Mix equal parts of Solution A and Solution B immediately before use. Make up a total of 10 mL (5 mL of Solution A and 5 mL of Solution B).
| 2. Masson’s Stain A | |
| a) Aqueous Biebrich Scarlet – 1 % aqueous | 90 mL (C.I. 26905) |
| b) Aqueous Acid Fuschin – 1% aqueous | 10 mL (C.I. 42685) |
| c) Glacial Acetic Acid | 1 mL |
| 3. Aqueous Phosphotungstic Acid – 5% |
| 4. Masson’s Stain B | |
| a) Aniline Blue | 2.5 g (C.I 42755) |
| b) Glacial Acetic Acid | 2.5 mL |
| c) Distilled water | 100 mL |
| 5. Acetic Acid – 1% |
| 6. Picro-Orange Solution | |
| a) 80% alcohol saturated with Picric Acid | 100 mL |
| b) Orange G | 0.2 g (C.I. 16230) |
Please use the fume hood or equivalent when making up solutions.
Method:
- Deparaffinize and bring sections to water.
- Stain in Weigert’s Iron Haematoxylin for 10 minutes.
- Wash in running water.
- Stain section in Picro-orange solution for 1 minute.
- Rinse rapidly in water.
- Stain in Masson’s stain A for 1 minute.
- Rinse sections in water.
- Rinse sections in 1% Acetic Acid.
- Rinse sections in water.
- Treat sections with 5% Phosphotungstic Acid for 5 minutes.
- Rinse sections in water.
- Rinse sections in 1% Acetic Acid.
- Stain sections in Masson’s Stain B for 3-4 minutes.
- Rinse in distilled water.
- Rinse in 1% Acetic Acid (Check microscopically to ascertain correct staining of collagen) for approximately 1 minute.
- Rinse in water.
- Dehydrate, clear and mount.
Results:
Nuclei – Black
Cytoplasm and muscle – Red
Collagen – Blue
Red blood cells – Yellow
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
2018 RCPAQAP Technical General Survey 1.
Speranza. Vinne Della in Richard W. Brown. ’Trichrome stains’: Histologic preparations Common Problems and Their Solutions. College of American Pathologists; 2009:95-101.
M Lamar Johns, John D Bancroft and Marilyn Gamble, Theory and practice of Histological Techniques. John D Bancroft; Marilyn Gamble. 6th New York, NY: Churchill Livingstone; 2008:146-150.