Today there was an issue with faded sections stained with Masson’s Trichrome.  The researcher wanted to have vibrant red and blue colors on his tissues like he is use to without cutting new sections.  He said the sections on the stained slide was exactly where he wanted to be and was afraid to cut deeper for more sections.  I understood and set off to ask the histology forum exactly how to do this, there was no answer.  I can make this work and set to first get the coverslips off just a couple of his slides.

After the coverslips are off, I used the same de-staining procedure for the H&E.  Run the slides into 100% alcohol, 95% alcohol and then running water for 10 minutes.  For an H&E stained section, normally most of the stain is washed out by the running water.  For the Masson’s trichrome this did not happen.  After an hour of washing in cold, hot and distilled running water, the blue was mostly gone but the red and purple nuclei were still present. So I thought about how the biebrich scarlet acid fuchsin stain works, it is an acid stain.  This means that maybe I could use a very weak base to help the stain loosen from the tissue.  The left over blue and purple nuclei could maybe taken out by an alcoholic acid like the one used in the H&E stain.

The staining was a success and looked as good as if it was never re-done.  To celebrate this new de-stain and re-stain protocol, I am posting it here just in case anyone else needs an unusual de-staining protocol for Masson’s trichrome.  The real procedure is as follows:

1. De-coverslip slides in xylene  – Until coverslips come off very easily
2. Soak in new-used xylene – 10 minutes after coverslips are off.
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 – 10 minutes
7. De-stain in 1% acid alcohol – 5 minutes
8. Dip in water – 5 quick dips
9. De-stain in ammonia water – 10 minutes
10. Wash in running cold water – 10 minutes
11. Weigert’s Hematoxylin (make fresh)  – 10 minutes (good 24hrs)
12. Wash in running water – 10 minutes
13. Biebrich Scarlet Acid Fuchsin – 10 minutes
14. Rinse in running water – 2 minutes
15. Phosphomolybdic / phosphotungstic acid – 10 minutes
16. No rinse, goes directly into blue
17. Analine Blue – 10 minutes
18. Rinse in running water – 1-2 minutes
19. 1% Glacial Acetic Acid  – 5 minutes
20. Dehydrate in 95% alcohol – 1 minute
21. Dehydrate in 100% alcohol – 1 minute
22. Dehydrate in 100% alcohol – 2 minutes
23. Dehydrate in 100% alcohol – 2 minutes
24. Clear in xylene – 2 minutes
25. 1. Clear in xylene – 5 minutes

This protocol has also been posted under the Other protocols page for printing.

http://atomic-temporary-32783013.wpcomstaging.com/histology/other-protocols-sops-equipment-manuals/