Masson's trichrome protocol

V6

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
tissue)

3
1
1
1

x
x
x
x

3
3
3
5

Min
Min
Min
Min

100% ethanol
95% ethanol
80% ethanol
DDW

2. Rinse in Bouin's Solution at room temperature overnight in a

hood.
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
sections.
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

xylene)

3 x 5 Min

Xylene

* 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
powder.
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
#HT1079).
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
powder.
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
period.

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