Masson’s Trichrome Blue Protocol

Masson’ Trichrome Blue
Safety equipment: Work under a hood with lab coat, gloves, and glasses.
1. De-paraffin slides in xylene (1)———————- 2 minutes (re-use)
2. De-paraffin slides in xylene (2)———————- 2 minutes (re-use)
3. Clear slides in 100% alcohol————————– 2 minutes
4. Clear slides in 100% alcohol————————– 2 minutes
5. Hydrate slides in 95% alcohol————————- 2 minutes
6. Hydrate slides in running water———————– 5 minutes
7. Bouins at 60 degrees Celsius————————– 1 hour (re-use)
8. Wash in running water ——————————– 10 minutes
9. Weigert’s Hematoxylin (make fresh) —————— 10 minutes (dump)
10. Wash in running water ——————————– 10 minutes
11. Biebrich Scarlet Acid Fuchsin————————- 10 minutes (re-use)
12. Rinse in running water——————————– 2 minutes
13. Phosphomolybdic / phosphotungstic acid—————- 10 minutes (re-use)
14. No rinse, goes directly into blue
15. Analine Blue—————————————— 10 minutes (re-use)
16. Rinse in running water——————————– 1-2 minutes
17. 1% Glacial Acetic Acid——————————– 5 minutes (re-use)
18. Dehydrate in 95% alcohol—————————— 1 minute
19. Dehydrate in 95% alcohol—————————— 1 minute
20. Dehydrate in 100% alcohol—————————– 1 minute
21. Dehydrate in 100% alcohol—————————– 2 minutes
22. Dehydrate in 100% alcohol—————————– 2 minutes
23. Clear in xylene (3)———————————– 2 minutes (re-use)
24. Clear in xylene (4)———————————– 5 minutes (re-use)

Results:
Nuclei- Black
Cytoplasm, keratin, muscle fiber and intercellular fibers- Red
Collagen- Blue


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