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
Hi! Could you please let me know what are the modifications for Masson Trichrome staining on cryosections from soft tissues? Thank you in advance.
To stain frozen tissues with the Masson’s trichrome you must first put the tissues on positively charged slides. This will help the tissues adhere to the slides. The next step is to fix your tissues as soon as they come out of the cryostat (unless already fixed). Fixation times need only approximately 5 minutes in 10% NBF for every 5 microns. This means, if your tissues are 10 microns, soak them for 10 minutes and so on. After that the slides can be placed into running water for 5-10 minutes and proceed with the staining beginning with the 1 hour in bouins.
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