Masson's Trichrome for Connective Tissue

EMS26367

Components:

Bouin's Fixative

Weigert's Iron Haematoxylin A

Weigert's Iron Haematoxylin B

Biebrich Scarlet Solution 1%

Phosphomolybdic/Phosphotungstic Acid Solution

Aniline Blue Solution

Acetic Acid 1% Aqueous

Fixation:

Bouin’s or 10% Buffered Formalin

Sections:

Paraffin at 6µm

Staining Procedures:

1. Deparaffinise and hydrate to distilled water.
2. Mordant in Bouin’s Fixative for 1 hour at 56°C or overnight at room temperature.
3. Cool and wash in running water until yellow colour disappears.
4. Rinse in distilled water.
5. Stain in Weigert’s Iron Hematoxylin Working* solution for 5 minutes. Wash in running water for 10 minutes.

1. To prepare working solution, mix equal parts of Weigert’s Iron Haematoxylin A and Weigert’s Iron Haematoxylin B.

6. Rinse in distilled water.
7. Place in Biebrich Scarlet-Acid Fuchsin for 15 minutes.
8. Rinse in distilled water.
9. Place in Phosphomolybdic Acid-Phosphotungstic Acid for 15 minutes.
10. Stain in Aniline Blue Solution for 10 to 20 minutes.
11. Rinse in distilled water.
12. Differentiate in Acetic Acid, 1% for 3 to 5 minutes. Discarded solution.
13. Dehydrate in 95% alcohol and absolute alcohol, and clear in xylene, 2 changes each.
14. Mount.

Shorten staining times when using 2-4µm sections of renal biopsy specimens:

Biebrich Scarlet – Acid Fuchsin: 5 minutes
Phosphomolybdic Acid-Phosphotungstic Acid: 10-12 minutes
 Aniline Blue Solution: 3 minutes

Stain Results:

NOTE: To ensure proper mordanting Bouin’s Fixative should be preheated to 58 – 60°C

References:

Sheehan and Hrapchak. Theory and Practice of Histotechnology. St. Lous. The Mosby Comp. 1980, p. 190