Masson’s Trichrome Green Protocol
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. Bouin’s Fixative (60 Celsius)———————- 30 minutes (re-use)
8. Wash in running water—————————— 5 minutes
9. Weigerts——————————————- 10 minutes (dump)
10. Wash in running water—————————— 5 minutes
11. Biebrich scarlet acid fuchsin———————- 5 minutes (re-use)
12. Rinse quickly————————————– 30-60 seconds
13. Phostung-Phosmoly acid (PTA-PMA)——————- 5 minutes (re-use)
14. No rinse
15. 2.5% Fast green———————————— 5 minutes (re-use)
16. Rinse quickly ————————————- 30 seconds
17. 1% Glacial acetic acid—————————– 2 minutes (re-use)
18. Dehydrate in 95% alcohol————————— 30 seconds
19. Dehydrate in 100% alcohol————————– 1 minute
20. Dehydrate in 100% alcohol————————– 2 minutes
21. Dehydrate in 100% alcohol————————– 2 minutes
22. Clear in xylene (3)——————————– 2 minutes (re-use)
23. Clear in xylene (4)——————————– 5 minutes (re-use)
Results:
Nuclei——————— Black to purple
Cytoplasm- —————- Shades of pink, red
Erythrocytes and myelin—- Scarlet
Collagen fibers———— Strong bluish-green
Mucus———————- Lighter green