How to De-Stain and Re-Stain Masson’s Trichrome


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.

Porcine Aorta Masson’s Trichrome Green

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
    1. Clear in xylene – 5 minutes

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

3 thoughts on “How to De-Stain and Re-Stain Masson’s Trichrome

  1. It is truly a nice and helpful piece of info. I am happy that you shared this helpful info with us. Please stay us informed like this. Thanks for sharing.

  2. Its August 4th 2022 and I just attempted the protocol with some rat myocardial Masson Trichrome slides that were faded. The destaining procedure worked quite well.

Leave a ReplyCancel reply