You are on page 1of 4

Masson's trichrome protocol


I've used Masson's trichrome kit (Sigma HT15-1KT) and a modified
Hematoxylin solution to get better results.
1. Deparaffinize and rehydrate slides:
3 x 3 Min - Xylene (blot excess xylene with a kimwipe without touching the






100% ethanol
95% ethanol
80% ethanol

2. Rinse in Bouin's Solution at room temperature overnight in a
Be careful, Bouin's solution is hazardous (read the MSDS).
** Bouin's Solution intensifies the final coloration of the tissue.
3. Wash slides in running tap water to remove yellow color from
4. Rinse in DDW for 2 minutes.
5. Stain in Working Iron Hematoxylin Solution for 10 minutes.
See appendix A for Hematoxylin preparation options.
** Hematoxylin stains nuclei blue-black.
6. Wash in running tap water for 10 minutes.
7. Rinse in DDW for 2 minutes.
8. Stain in Biebrich Scarlet-Acid Fuchsin solution for 5 minutes.
Decreased red staining usually indicates that the staining solution
has aged or been overused and should be discarded.
** Beibrich scarlet-acid fuchsin stains cytoplasm and muscle red.
9. Rinse in DDW for 2 minutes.
10. Place the slides in Phosphomolybdic/Phosphotungstic Acid
Solution for 5-10 minutes.
Freshly prepare Working Phosphotungstic/Phosphomolybdic Acid
Solution by mixing 1 volume of Phosphotungstic Acid Solution and 1
volume of Phosphomolybdic Acid Solution with 2 volumes of DDW.
Discard after one use.
* Formation of a precipitate in Phosphomolybdic Acid Solution does
not affect performance.
** This allows for uptake of the aniline blue stain.
11. Stain sections in Aniline Blue Solution for 5 minutes.

**Aniline blue stains collagen blue.
12. Rinse slides briefly in distilled water.
13. Place slides in 1% acetic acid solution for 3-5 minutes. Discard
this solution.
*Rinsing in acetic acid after staining renders the shades of color
more delicate and transparent.
** If blue staining of connective tissue appears faded, the section
has probably been overdifferentiated in the acetic acid solution.
14. Dehydrate slides.
2 x 3 Min

95% ethanol

2 x 3 Min

100% ethanol (blot excess ethanol before going into

3 x 5 Min


* Option: You can leave the slides in xylene overnight to get good
clearing of the ethanol.
15. Cover the slides using Permount or Polymount (xylene based).
• Place a drop of Permount on the slide using a glass rod.
• Angle the coverslip and let fall gently onto the slide.
• Allow the Permount to spread beneath the coverslip, covering all
the tissue.
• Dry overnight in a fume hood.

Appendix A – Iron Hematoxylin preparation.
There are 2 options to prepare an iron Hematoxylin solution.
A prepared Sigma kit, and a handmade solution from Hematoxylin
I've found that in the Extra-Strength solution the nuclei signal was
strong and clear and was better than the Sigma Hematoxylin kit.
Option 1) Weigert's Iron Hematoxylin Set kit (Sigma
Make Hematoxylin Solution fresh by adding equal volumes of
Solution A and Solution B. The working solution is good for
approximately 10 days.
Option 2) Extra-strength Weigert's Iron Hematoxylin from
Solution A: Hematoxylin powder (Sigma H3136) - 2.5gr
Ethanol 95% - 25ml.
Solution B: Ferric chloride ( Sigma 157740) - 0.292gr
DDW - 24.5ml
10%HCl - 0.25ml
Mix 5ml of A with 25ml of B with 20 ml Ethanol 100%.
Appendix B - Misc for the protocol:
 If the alinine blue appears faded, then you have over
differentiated the alinine blue dye. Stain longer in alinine blue,
and don't overuse the solutions, it outdate/become weak with
use over time.

1% Acetic acid = 0.5ml Acetic acid, glacial + 49.5ml DDW
DDW = Double-distilled water.
Running tap water – Put the slides inside a box under a weak
stream of regular running tap water and let the water to fill
the box from the corner and to spill for the specified time

Figure1: Masson's trichrome staining of a fibrotic MDX mouse diaphragm using this protocol
and the extra strength Hematoxylin option.
Written by: Itai