Walter's Trichrome
for Elastic and Collagen

Solutions

Solution A
Ferric ammonium sulphate	2.5	g
Distilled water	100	mL

Solution B
Phosphotungstic acid	2	g
Distilled water	100	mL

Solution C
Eosin B	0.7	g
Acid fuchsin, sat. aqu	4	mL
Unna's elastic stain	35	mL
Ethanol, 50%	35	mL
Glycerol	40	mL

Tissue sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to 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 24 hours.
3. Rinse quickly with distilled water.
4. Place into solution B for 10 minutes.
5. Rinse quickly with distilled water.
6. Place into solution C for 15-20 minutes.
7. Rinse quickly with 95% ethanol until clouds of dye stop being extracted.
8. Dehydrate with 100% ethanol for 1 minute.
9. Clear with xylene and mount with a resinous medium.

Expected results

• Nuclei  –  purple
• Elastic fibres  –  purple
• Erythrocytes  –  orange
• Collagen  –  blue

Notes

1. This is a modification of Pasini's method.

 

Reference

Gray, Peter. (1954)
The Microtomist's Formulary and Guide.
Originally published by:– The Blakiston Co.
Republished by:– Robert E. Krieger Publishing Co.
  Citing:–
    Walter, (1930)
    Zeitschrift für wissenschaftliche Mikroskopie und für mikroskipische Technik,
    v. 46, pp. 458