Apr 28, 2026
Standard Operating Procedure: Masson Trichrome Staining on Rat Femur Sections
- 1University of Southern Denmark
- Molecular Bone Histology, University of Southern Denmark
Protocol Citation: Birgitte Villadsen 2026. Standard Operating Procedure: Masson Trichrome Staining on Rat Femur Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov11ebovr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 12, 2025
Last Modified: April 28, 2026
Protocol Integer ID: 234869
Keywords: rat femur sections for differentiation, rat femur section, masson trichrome, mineralized bone, demineralized bone, collagen
Abstract
To perform Masson Trichrome staining on rat femur sections for differentiation of mineralized bone, demineralized bone, collagen, muscle fibers, and nuclei.
Materials
Bouin’s solution components:
- Picric acid (84512.260, VWR Chemicals; 12 g/L aqueous solution, 750 mL)
- Formaldehyde solution (15512-1L-R, Sigma-Aldrich; 250 mL)
- Acetic acid (2234.1000, Chem Solute; 50 mL)
Weigert-Lillie’s Iron Hematoxylin:
- FeCl3·6H2O (1.03943.0250, Merck; 15 g)
- FeSO4·7H2O (1.03965.0500, Merck; 27 g)
- Hydrochloric acid (20252.290, VWR Chemicals; 12 mL)
- Hematoxylin monohydrate (1.15938.0100, Merck; 10 g)
- Ethanol 99% (1000 mL)
Biebrich Scarlet/Fuchsin Acid:
- Biebrich Scarlet (1A-398, Waldeck; 4.5 g)
- Fuchsin Acid (1.05231.0025, Merck; 0.5 g)
- Acetic acid (2234.1000, Chem Solute; 5 mL)
Molybdotungstophosphoric Acid:
- Molybdotungstophosphoric acid hydrate (1.00532.0100, Merck; 25 g)
- Tungstophosphoric acid hydrate (1.00583.0250, Merck; 25 g)
2% Aniline Blue:
- Aniline Blue (21999.183, VWR; 20 g)
- Acetic acid (2234.1000, Chem Solute; 20 mL)
Safety warnings
Contains picric acid and formaldehyde. Toxic if inhaled, ingested, or in contact with skin. Irritates eyes, respiratory tract, and skin. Use protective clothing, gloves, and work under ventilation. In case of eye contact, rinse immediately and seek medical attention.
Ethics statement
This protocol requires prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Before start
Preparation of Solutions
Bouin’s solution: Mix 750 mL picric acid solution, 250 mL formaldehyde solution, and 50 mL acetic acid.
Weigert-Lillie’s Iron Hematoxylin: Prepare Solution A by dissolving 15 g FeCl3·6H2O, 27 g FeSO4·7H2O, in 1788 mL distilled water and 12 mL HCl. Prepare Solution B by dissolving hematoxylin monohydrate (10 g) in 1000 mL ethanol. For staining, mix 150 mL of Solution A with 50 mL of Solution B, filter, and use within 5 days.
Biebrich Scarlet/Fuchsin Acid: Dissolve 4.5 g Biebrich Scarlet and 0.5 g Fuchsin Acid in 500 mL distilled water with 5 mL acetic acid, stir and filter.
Molybdotungstophosphoric/Tungstophosphoric Acid: Dissolve 25 g of Molybdotungstophosphoric acid hydrate and 25 g of Tungstophosphoric acid hydrate.
2% Aniline Blue: Dissolve Aniline Blue (20 g) in 1000 mL distilled water with 20 mL acetic acid, stir with heat and filter.
Staining Procedure
2h 35m
Melt sections for 30 min 60 °C
Deparaffinize: Histoclear
10m
Deparaffinize: 99% ethanol
5m
Deparaffinize: 96% ethanol
5m
Deparaffinize: 70% ethanol
3m
Deparaffinize: Water
1m
Bouin’s solution: Preheat and incubate at 60 °C in fume hood.
1h
Rinse in running water.
15m
Stain with Weigert-Lillie’s Iron Hematoxylin
10m
Rinse in running water
5m
Stain with Biebrich Scarlet/Fuchsin Acid
7m
Dip in water twice.
Treat with molybdotungstophosphoric/tungstophosphoric acid
4m
Stain with 2% Aniline Blue
20m
Rinse in water 5–10 dips.
Dehydrate: 96% ethanol 5 dips
Dehydrate: 99% ethanol 5 dips
Dehydrate: Histoclear
10m
Mount with Dako Mounting Media.