Figure 1. Canine small intestine. Fibrous tissue stains blue (arrows) and smooth muscle stains red.
Masson Trichrome Staining Procedure
Purpose:
This standard operating procedure (SOP) establishes the standard for staining
collagen, mucin, keratin and muscle fibers using the Masson Trichrome staining
procedure.
Reagents and Materials:
Reagents
Bouin’s Solution
Commercially prepared by Sigma Diagnostics.
Expiration date provided by manufacturer.
Store at room temperature.Weigert’s Iron Hematoxylin, Solution A:
Hematoxylin 5gm
95% ETOH 500mlWeigert’s Iron Hematoxylin, Solution B:
Ferric Chloride 5.8gm
Distilled water 495ml
Hydrochloric Acid 5mlMake fresh:
Solution A 20ml
Solution B 20ml
Biebrich Scarlet-Acid Fuchsin Solution
10% Phosphomolybdic Acid Solution
10% Phosphotungstic Acid SolutionAniline Blue Solution
Commercially prepared by Sigma Diagnostics.
Expiration date provided by manufacturer.
Store at room temperature.
1% Acetic Acid
Distilled water 99ml
Acetic Acid 1ml
Materials
Tissue slides and control slides for special stain requested.
Microwave to bake slides.
Staining rack, 20 slide capacity.
Coverslips and synthetic mounting media.
Hazards
and Precautions:
See Material Safety Data Sheets.
Sample
Preparations:
Paraffin tissue section slides cut at 4 microns.
Calibration
and Standardization:
A tissue section slide containing collagen, mucin, keratin and muscle fibers
is stained with each procedure.
Procedure:
1. Deparaffinize
and hydrate to distilled water.
2. Place in Bouin’s in a plastic coplin jar with the lid loosely applied.
Microwave at 70% power for 45 seconds.
3. Distilled water rinse – until sections are colorless.
4. Weigert’s Iron Hematoxylin – 2 minutes.
Make fresh:
Solution A 20ml
Solution B 20ml
Microwave at full power for 15 seconds.
5. Distilled water rinse.
6. Biebrich Scarlet-Acid Fuchsin Solution – 2 minutes.
Microwave at 70% power for 30 seconds.
7. Distilled water rinse.
8. Phosphomolybdic Acid- Phosphotungstic Acid Solution.
Make fresh:
Phosphotungstic Acid 10ml
Phosphomolybdic Acid 10ml
Distilled water 20ml
Microwave at full power for 15 seconds.
9. Aniline Blue Solution – 5 minutes.
Microwave at full power for 20 seconds.
10. Distilled water rinse.
11. 1% Acetic Acid Solution - 2 minutes.
12. Dehydrate, clear and coverslip.
Positive
control: small intestine (Figure 1)
Results:
Nuclei – dark blue
Cytoplasm, keratin, muscle fibers, intercellular fibers - red
Collagen, mucin - blue
Figure 1. Canine small intestine. Fibrous tissue stains blue
(arrows) and smooth muscle stains red.
Effective: July
1, 2004
Cherie Chapman
James R. Turk
References:
Armed Forces Institute of Pathology Laboratory Methods in Histotechnology, eds. Prophet EB, Mills B, Arrington JB, Sobin LH, American Registry of pathology, Washington D.C. , 1992, pp132..Sheehan DC, Hrapchak BR: Theory and Practice of Histotechnology, 2nd Ed, CV Mosby, St. Louis, 1980, pp. 190.