Masson's Trichrome
Yellow Collagen Variant

Solutions

Régaud's iron hematoxylin or equivalent

Solution A
Acid fuchsin	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Solution B
Phosphomolybdic acid	1	g
Distilled water	100	mL

Solution C
Metanil yellow	to	saturation
Distilled water	100	mL

Solution D
Acetic acid, glacial	1	mL
Distilled water	99	mL

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. Stain nuclei with Régaud's iron hematoxylin or equivalent.
3. Wash well with water.
4. Place into solution A for 5 minutes.
5. Rinse rapidly with water.
6. Place into solution B for 5 minutes.
7. Drain.
8. Pour on solution C and leave 5 minutes.
9. Place in solution D for 5 minutes.
10. Rinse rapidly with water.
11. Dehydrate with absolute ethanol.
12. Clear with salicylic xylene and mount with salicylic balsam.

Expected results

• Nuclei  –  black
• Muscle  –  red
• Collagen  –  yellow

Notes

1. This method was published at the same time as his more commonly used technique.
2. It was specified that solution C should be poured onto the slide while on a staining rack, probably to ensure the solution was not re-used.
3. It was originally considered important to mount sections stained with acid dyes in an acidified mounting medium, in the belief that it preserved brightness and clarity.
4. Salicylic xylene is made by saturating xylene with salicylic acid.
5. Salicylic balsam is made by adding salicylic acid to Canada balsam, allowing any excess to clear by settling out. A useful alternative is to dip the coverslip into salicylic xylene prior to mounting. The dissolved salicylic acid will then acidify the mounting medium.

 

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