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Walter’s Trichrome for Elastic and Collagen

Walter's Trichrome

for Elastic and Collagen

9
steps
8
materials

Materials

Solution A

MaterialAmount
Ferric ammonium sulphate2.5g
Distilled water100mL

Solution B

MaterialAmount
Phosphotungstic acid2g
Distilled water100mL

Solution C

MaterialAmount
Eosin B0.7g
Acid fuchsin, sat. aqu4mL
Unna’s elastic stain35mL
Ethanol, 50%35mL
Glycerol40mL

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.

Protocol

  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

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. 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