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Electron Microscopy Sciences

Technical Data Sheets

Goldner's Trichrome Method

EMS Catalog #26386

Fixation

10% Buffered Neutral Formalin or Bouin's Solution

Sections

Paraffin, frozen or celloidin

Microtomy

Cut paraffin sections at 6 micrometers.

Procedure

  1. Decerate slides in Xylene, 2 changes, 2 minutes each. Place in absolute alcohol, 2 changes, 2 minutes each; 95% alcohol, 2 changes, 2 minutes each; then rinse in distilled water.
  2. Mordant in Bouin's Fluid solution for 1 hour at 56°C, or overnight at room temperature, if formalin fixed.
  3. Cool and wash in running tap water until yellow color disappears.
  4. Rinse in distilled water.
  5. Place in Weigert's Hematoxylin for 10 minutes.

    ***To prepare Weigert's Solution, mix equal parts of:
    Weigert's Iron Hematoxylin A
    Weigert's Iron Hematoxylin B
    *** stir vigorously.
  6. Wash in running tap water for 10 minutes.
  7. Rinse in distilled water.
  8. Stain in Ponceau Acid Fuchsin for 5 minutes.
  9. Wash in Acetic Acid, 1%.
  10. Place in Phosphomolybdic Acid-Orange G solution until collagen is decolorized.
  11. Rinse in Acetic Acid, 1% for 30 seconds.
  12. Stain in Light Green Stock Solution for 5 minutes.
  13. Rinse in Acetic Acid, 1%, for 5 minutes.
  14. Blot sections but do not allow to dry.
  15. Dip quickly in absolute alcohol.
  16. Dehydrate slides in 95% alcohol and absolute alcohol 3 changes each. Clear in Xylene, 3 changes.
  17. Mount coverglass with appropriate medium (refractive index 1.48-1.56).

Stain Results

Nuclear chromatin Brownish black
Cytoplasm Bright red
Erythrocytes Orange
Muscle Red
Collagen Green

 

References

Luna, Lee G. Histopathologic Methods and Color Atlas of Special Stains and Tissue Artifacts
Johnson Printers, Downers Grove , IL p 151-152, c 1992.

Product Information

Bouin’s Fluid
Weigert’s Iron Hematoxylin A
Weigert’s Iron Hematoxylin B
Ponceau Acid Fuchsin
Acetic Acid 1%
Phosphomolybdic Acid Orange G Solution
Light Green Stock 0.2%
Goldner's Trichrome Method