Massons Trichrome
Purpose
Used to differentiate between collagen and smooth muscle in tumors, and the increase of collagen in diseases such as cirrhosis. Routine stain for liver and kidney biopsies.
Principle
As the name implies, three dyes are employed selectively staining muscle, collagen fibers, fibrin, and erythrocytes. The general rule in trichrome staining is that the smallest dye molecule colors the less porous tissues; whenever a dye of large molecular size is able to penetrate, it will always do so at the expense of the smaller molecule.
Others suggest that the tissue is stained first with the acid dye, Biebrich Scarlet, which binds with the acidophilic tissue components. Then when treated with the phospho acids, the less permeable components retain the red, while the red is pulled out of the collagen. At the same time causing a link with the collagen to bind with the aniline blue.
Solutions
Bouin’s Fixative
Saturated picric acid 75 ml
37%-40% Formaldehyde 25ml
Glacial acetic acid 10ml
Mix well, label, date and initial. Stable for 2 years.
Caution: Carcinogenic, irritant.
Can be Purchased
SAFETY: Picric acid can become explosive when dry
Biebrich Scarlet-Acid Fuchsin Solution: Make 1000 mls
1% Aqueous Biebrich Scarlet (CI 26905) 90 ml
Expiry: 1 month
1% Aqueous Acid fuchsin 10 ml
Expiry: 6 months
Glacial acetic acid 1 ml
Caution: Avoid contact and inhalation.
Expiry of Biebrich Scarlet-Acid Fuchsin Solution: 2 months
Weigert’s Iron Hematoxylin (acid resistant)
Stock Solution A
Hematoxylin 5 gm
95% alcohol 500 ml
Mix well, label with initial and date. Stable for 4 months.
Caution: Flammable, avoid contact and inhalation.
Stock Solution B: (make in fume hood)
30% ferric chloride 20.0 ml (Must be made up)
Distilled water 475.0 ml
Hydrochloric acid 5.0 ml
Mix well, label with initial and date. Stable for 1 year.
Caution: Corrosive, avoid contact and inhalation.
Weigert’s Hematoxylin Working Solution (make just before using)
Solution A 25 ml
Solution B 25 ml
Add solution B to Solution A immediately before use and filter.
Solution should be violet-black color and must be discarded if brown (over oxidized). Working solution should be discarded at end of day.
Phosphotungstic/Phosphomolybdic Acid Solution
Phosphotungstic acid 25 gm
Phosphomolybdic acid 25 gm
Distilled water 1000 ml
Mix well, label with initial and date. Solution is stable for 6 months. Store in fridge
Caution: Corrosive. Avoid contact and inhalation. Strong skin and eye irritants
Aniline Blue (CI 42755)
Aniline blue 25 gm
Distilled water 1000 ml
Glacial acetic acid 20 ml
Mix well, label with initial and date. Solution is stable for 2 months.
Caution: Avoid contact and inhalation.
1% Acetic Acid
Glacial acetic acid 10 ml
Distilled water 990 ml
Mix well, label with initial and date. Solution is stable for 6 months.
Caution: Avoid contact and inhalation.
Safety
- Wear gloves, goggles and lab coat. Avoid contact and inhalation of dyes and chemicals.
- Bouin’s contains: formaldehyde, a known carcinogen, picric acid can become explosive when dry. Toxic by skin absorption. Keep hot uncapped Bouin’s under the hood.
- Phosphomolybdic, phosphotungstic acid powders, and acetic acid solutions are skin and eye irritants, and strong corrosives.
PROCEDURE
- Take sections to water.
- Mordant in Bouin’s (postfix) solution for 35 seconds in the microwave, by placing the coplin jar (with cap loosely on top to prevent overflow) on absorbent towels, as picric acid is explosive when dry. It is important not to let it spill in the oven and be allowed to evaporate. Allow to stand 15 minutes in the fume hood. Always carefully open the lid of the coplin jar in the fume hood. Extreme caution must be used not to inhale the fumes.
- Wash in running tap water until the yellow color disappears. (At least 5 min.)
- Place sections in Weigert’s working hematoxylin for 10 minutes.
- Wash in running tap water for 5 minutes
- Rinse in distilled water.
- Place sections in Biebrich Scarlet-Acid Fuchsin solution for 3 minutes.
- Rinse in distilled water.
- Phosphotungstic/phosphomolybdic acid for 5 minutes (discard solution).
- Transfer directly into Aniline blue for 3 minutes.
- Rinse in distilled water.
- Place slides in 1% Acetic acid for 3 minutes (discard this solution).
- Transfer directly to 100% alcohol to dehydrate (limits differentiation of dyes)
- Dehydrate, clear, and mount.
Results
- Nuclei black
- Cytoplasm, muscle, erythrocytes red
- Collagen blue
Notes
- Light green may be substituted for Aniline blue but 5% aqueous phosphotungstic acid for 5 minutes must be substituted with Phosphotungstic/phosphomolybdic acid
- When staining liver biopsies, the collagen is better light blue than dark blue.
Control
Small Bowel (rich in collagen and muscle)
References
IWK. (2007). IWK Laboratory Histology Staining Manual, Special Stains.
Carson, F. L., & Cappellano, C. H. (2015). Histotechnology: A Self-Instructional Text (4th ed.). American Society of Clinical Pathologists Press.
