Lillie's Trichrome
for Muscle and Collagen

Solutions

Weigert's iron hematoxylin or equivalent

Solution A
Biebrich scarlet	0.5	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Solution B
Phosphomolybdic acid	2.5	g
Phosphotungstic acid	2.5	g
Distilled water	100	mL

Solution C
Fast green FCF	2.5	g
Acetic acid, glacial	2.5	mL
Distilled water	97.5	mL

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

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 Bouin's fluid, formal sublimate or B5 fixation. If formalin is used, secondary fixation of sections with Bouin's fluid at 56°C for an hour will improve staining.

Method

1. Bring sections to water via xylene and ethanol
   • If formalin fixed, refix in Bouins fluid for one hour at 56°C
   • Wash well in tap water to remove the yellow.
2. Stain nuclei with Weigert's iron hematoxylin or equivalent.
3. Wash well in tap water, rinse with distilled water.
4. Place into solution A for 2 minutes.
5. Rinse with distilled water.
6. Place into solution B for 1 minute.
7. Rinse with distilled water.
8. Place into solution C for 2 minutes.
9. Place into solution D for 1 minute.
10. Dehydrate with acetone.
11. Clear with xylene and mount with a resinous medium

Expected results

• Nuceli  –  black
• Cytoplasm  –  red
• Muscle  –  red
• Collagen  –  green

Notes

1. This method is frequently, and erroneously, called "Masson's" trichrome.
2. Refixing the sections in Bouin's fluid intensifies the colours and increases the contrast between the tissue components. Although often called mordanting, this step is simply secondary fixation of sections.
3. Acetone is specified for dehydration. Absolute ethanol can be substituted.
4. Longer times in solutions A and C (5 minutes each) may improve staining.

 

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

Kiernan. J.A., (1999)
Histological and histochemical methods: Theory and practice, Ed. 3
Butterworth Heinemann, Oxford, UK.