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Masson's Trichrome Stain

Solutions

Bouin's fluid ( Sigma HT 10-1-32 )

Saturated picric acid 150ml

Formalin 50ml

Glacial acetic acid 10ml

Weigert's iron hematoxylin

Stock solution A

Hematoxylin 1gm

95% alcohol 100ml

Stock solution B

29% aqueous ferric chloride 4ml

ddH₂O 95ml

Hydrochloric acid 1ml

Working solution

Use equal parts solution A and solution B

Biebrich scarlet-acid fuchsin

Working solution

1% aqueous stock Biebrich scarlet 90ml

1% aqueous stock acid fuchsin 10ml

Glacial acetic acid 1ml

Mix and filter.

Phosphomolybdic-phosphotungstic acid solution

Phomolybdic acid 5gm

Phosphotungstic acid 5gm

ddH₂O 200ml

Aniline blue solution

Aniline blue 2.5gm

Acetic acid 2ml

ddH₂O 100ml

Acetic water

Glacial acetic acid 1ml

ddH₂O 100ml

Procedure

1. Deparaffinize and rehydrate slides to ddH₂O.
2. Place slides in Bouin's fixative overnight at RT. Make sure dish or jar is covered.
3. Wash in running water until sections are colorless.
4. Rinse in ddH₂O.
5. Weigert's iron hematoxylin for 10 minutes. Wash in running water for 10minutes.
6. Rinse in ddH₂O.
7. Biebrich scarlet-acid fuchsin solution for 15min.
8. Rinse in ddH₂O.
9. Phosphomolybdic-phosphotungstic acid solution 10-15 minutes.
   ( discard solution)
10. Transfer directly to the aniline blue solution 10-20 minutes (Solution may be filtered back and reused).
11. Rinse in ddH₂O.
12. 1% acetic water for 3 -5 min (discard solution)
13. Dehydrate, clear in xylene and coverslip, using a synthetic mounting medium.

Results

Nuclei----Black

Cytoplasm, keratin, muscle fibers---red

Collagen, mucin---Blue

 

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