Masson's Trichrome
An Original Variant

Solutions

Solution A
Hematoxylin	1	g
Distilled water	100	mL

Solution B
Ferric ammonium sulphate	4	g
Distilled water	100	mL

Solution C
Ferric ammonium sulphate	2	g
Distilled water	100	mL

Solution D
Acid fuchsin	0.1	g
Distilled water	100	mL

Solution E
Phosphomolybdic acid	1	g
Distilled water	100	mL

Solution F
Aniline blue	0.5	g
Phosphomolybdic acid	0.5	g
Distilled water	100	mL
Dissolve separately and combine.

Tissue sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Trichrome stains often benefit from initial Bouin or formal sublimate fixation, or from secondary fixation in Bouin's fluid for an hour at 56°C

Method

1. Bring sections to water via xylene and ethanol.
2. Place into solution A for 5 minutes at 50°C.
3. Rinse rapidly with water at 50°C.
4. Place into solution B for 10-15 minutes at 50°C.
5. Differentiate in solution C until nuclei alone are stained.
6. Wash in running tap water for 15 minutes.
7. Place into solution D for 10 minutes.
8. If overstained, remove excess with tap water.
9. Place in solution E for 5-10 minutes.
10. Place in solution F for 20-60 minutes.
11. Rinse rapidly with water.
12. Dehydrate with 95% and absolute ethanols.
13. Clear with xylene and mount with a resinous medium.

Expected results

• Muscle  –  red
• Collagen  –  blue
• Nuclei  –  black

Notes

1. This method was published at the same time as his more commonly used technique.
2. Steps 1-6 are an acid resistant nuclear stain. Another could be substituted.

Reference
Gray, Peter. (1954)
The Microtomist's Formulary and Guide.
Originally published by:– The Blakiston Co.
Republished by:– Robert E. Krieger Publishing Co.