Goldner's Trichrome Method

EMS26386

Components

Bouin’s Fluid

Weigert's Iron Haematoxylin A

Weigert's Iron Haematoxylin B

Ponceau Acid Fuchsin

Acetic Acid, 1%

Phosphomolybdic Acid Orange G Solution

Light Green Stock, 0.2%

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 Haematoxylin for 10 minutes.
   
   ***To prepare Weigert's Solution, mix equal parts of:
   Weigert's Iron Haematoxylin A
   Weigert's Iron Haematoxylin 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

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.