Milligan's trichrome
for Muscle and Collagen

Solutions

Solution A
Potassium dichromate	2.25	g
Hydrochloric acid, conc	2.25	mL
Ethanol, 95%	25	mL
Distilled water	75	mL

Solution B
Acid fuchsin	0.1	g
Distilled water	100	mL

Solution C
Phosphomolybdic acid	1	g
Distilled water	100	mL

Solution D
Orange G	2	g
Phosphomolybdic acid	1	g
Distilled water	100	mL

Solution E
Acetic acid, glacial	1	mL
Distilled water	100	mL
Directions

Solution F
Fast green FCF	0.1	g
Acetic acid, glacial	0.2	mL
Distilled water	100	mL

Tissue sample
5µ paraffin sections of neutral buffered formalin fixed were specified. Other fixatives may be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin's fluid.

Method

1. Bring sections to water via xylene and ethanol.
2. Place into solution A for 5 minute.
3. Rinse with distilled water.
4. Place into solution B for 5 minutes.
5. Rinse quickly with distilled water.
6. Place into solution C for 1-5 minutes.
7. Rinse with distilled water.
8. Place into solution D for 5-10 minutes.
9. Rinse with distilled water.
10. Place into solution E for 2 minutes.
11. Place into solution F for 5-10 minutes.
12. Place into solution E for 3 minutes.
13. Dehydrate with ethanol.
14. Clear with xylene and mount with a resinous medium.

Expected results

• Nuclei  –  red
• Erythrocytes  –  orange
• Muscle  –  red
• Collagen  –  green

Notes

1. Aniline blue may be substituted for fast green FCF at the same concentration. Collagen would then be blue.

 

Reference

Gray, Peter. (1954)
The Microtomist's Formulary and Guide.
Originally published by:– The Blakiston Co.
Republished by:– Robert E. Krieger Publishing Co.
  Citing:–
    Milligan, (1946)
    Technical Bulletin, v. 7, pp.57