Masson’s Trichrome Stain
Masson’s Trichrome Stain
Nuclei, Cytoplasm, Erythrocytes, Keratin, Muscle Fibers, Collagen, Mucus
Stock Solutions
Bouin’s Solution – Add glacial acetic acid (to 5%) just before use.
Alcoholic Hematoxylin Stock (Solution A)
Component Cat. No. Amount
Hematoxylin Sigma H9627 5 g
95% Ethanol 500 mL
Dissolve over gentle heat – do not boil. Label with date and initials. Store at room temperature up to 1 year.
10% Ferric Chloride Stock (Solution B)
Component Cat. No. Amount
Ferric chloride (Iron III chloride) Sigma F7134 5.8 g
Milli-Pure water 495 ml
Glacial Acetic Acid 5 ml
Dissolve ferric chloride in 50 ml water, then add remaining water to 990 ml. Add acetic acid last.
Store at room temperature for up to 1 year.
Acid Fuchsin, 1% Aqueous Stock Solution
Dissolve 1 g acid fuchsin (Sigma #857408) in 100 mL Milli-Pure water.
Label and store at room temperature.
Biebrich Scarlet, 1% Aqueous Stock Solution
Dissolve 10 g biebrich scarlet (aka Ponceau BS, Sigma #B6008) in 1000 mL Milli-Pure water.
Label and store at room temperature.
Aniline Blue Stock Solution
Component To make 50 mL To make 75 mL To make 250 mL
Aniline Blue (CAS #66687-07-8) 1.25 g 1.875 g 6.25 g
Glacial acetic acid 1.0 mL 1.5 mL 5.0 mL
DI water 50 mL 75 mL 250 mL
Store at room temperature for up to 1 year.
Phosphomolybdic-Phosphotungstic Acid Solution
Component To make 50 mL To make 75 mL To make 250 mL
Phosphomolybdic acid (Sigma #221856) 0.625 g 0.938 g 3.125 g
Phosphotungstic acid (Sigma #P4006) 0.625 g 0.938 g 3.125 g
DI Water 50 mL 75 mL 250 mL
Store at room temperature for up to 1 year.
Working Solutions
1% Acetic Acid Solution
Component To make 50 mL To make 75 mL To make 250 mL
Glacial acetic acid 0.5 mL 0.75 mL 2.5 mL
Distilled Water 49.5 mL 74.25 mL 247.5 mL
Make fresh just before use.
Weigert’s Hematoxylin – Working Solution Masson’s Trichrome Stain
Nuclei, Cytoplasm, Erythrocytes, Keratin, Muscle Fibers, Collagen, Mucus
Component To make 50 mL To make 75 mL To make 250 mL
Solution A – Alcoholic Hematoxylin Stock 25 mL 37.5 mL 125 mL
Solution B – 10% Ferric Chloride Stock 25 mL 37.5 mL 125 mL
Prepare just before use. Discard in “Hematoxylin Waste” container.
Biebrich Scarlet-Acid Fuchsin Solution – Working Solution
Component to make 50 mL To make 75 mL To make 250 mL
Biebrich scarlet, 1% stock 44.5 mL 66.75 mL 222.5 ml
Acid fuchsin, 1% stock 5.0 mL 7.5 mL 25 ml
Glacial Acetic Acid 0.5 mL 0.75 mL 2.5ml
Prepare just before use. Can be used for a maximum of two runs if within 24 hours.
Aniline Blue Working Solution
Component
Aniline Blue Stock Solution 50 ml 75 ml 250 ml
Glacial acetic acid 1.0 ml 1.5 ml 5.0 ml
Add glacial acetic acid just before using.
Masson’s Trichrome Stain
Nuclei, Cytoplasm, Erythrocytes, Keratin, Muscle Fibers, Collagen, Mucus
Procedure:
**Always run a control slide with every stain!**
1. Deparaffinize and re-hydrate tissues to PBS*
2. Distilled water* 1 min.
3. Mordant in Bouin’s – in fume hood, covered Overnight
4. Running tap water rinse – until yellow color clears 1 min.
5. Distilled water rinse 1 min.
6. Weigert’s Hematoxylin working solution* 12 min.
a. Discard after use.
7. Running tap water rinse 10 min.
8. Distilled water rinse 1 min.
9. Biebrich scarlet-acid fuchsin solution 1 min.
a. Solution may be used twice only, then discarded.
10. Distilled water rinse 1 quick dip only
11. Phosphomolybdic-phosphotungstic acid solution 30 min.
a. Discard after use.
12. Aniline blue solution* 10 min.
a. Solution may be used twice only, then discarded.
13. Distilled water rinse 1 quick dip only
14. 1% Acetic acid solution 4 min.
a. Discard after use.
15. 95% Ethanol 2 x 1 min.
16. 100% Ethanol 2 x 1 min.
17. Formula 83 3 x 1 min.
18. Mount with Permount
*Modifications For Cryo-Sections:
- Fix sections in 10%formalin (not Zinc) x 30 minutes.
- Replace steps 1, 2 with 3 x 3 min. rinses in distilled water.
- Reduce time in hematoxylin and aniline blue to 7 minutes each.
Results:
- Cytoplasm, keratin, muscle fibers, Erythrocytes – red
- Nuclei – black
- Collagen and mucus – blue