Notre Dame Integrated Imaging Facility

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:

1. Fix sections in 10%formalin (not Zinc) x 30 minutes.

2. Replace steps 1, 2 with 3 x 3 min. rinses in distilled water.

3. Reduce time in hematoxylin and aniline blue to 7 minutes each.

 

RESULTS: Cytoplasm, keratin, muscle fibers, Erythrocytes – red

Nuclei – black

Collagen and mucus – blue