The Gomori trichrome technique is useful for the staining of muscle and connective proper tissues.
Procedure
Protocol modified by Elisa Inés Sánchez.
Samples are fixed with Bouin or 4% formaldehyde and embedded in paraffin. 8 µm thick sections are obtained and attached to gelatin coated slides.
1. 2x10 min in xylene
2. 2x10 min in 100º ethanol
3. 10 min in 96º ethanol
4. 10 min in 80º ethanol
5. 10 min in 50º ethanol
6. 5 min in distilled H2O2
7. 1 h in Bouin fixative at 56-60 ºC, or 24 h at room temperature
Bouin fixative works as mordant
8. Rinses in distilled H2O2 until the yellow color fades out.
9. 10 min in Weigert hematoxylin
If the Weighert hematoxylin solution is old, an increase of the staining time may be necessary.
10. 5-10 min in tap water
If sections lose the color or the staining is dull, the Weigert hematoxylin step can be extended for another 5 min. The staining intensity can be controlled under the microscope.
11. 15 min int trichromic solution
Trichromic solution:
0.6 g cromo2R (CI 16570).
0.3 g light green (CI 42095).
0.8 g phosphotungstic acid.
1 ml glacial acetic acid.
Add distilled H2O2 until the total volume is 100 ml.
12. 1 min in 1 % of glacial acetic acid.
This is a differentiation step. If the color is no strong enough or the staining is irregular, sections can go back to the trichome solution for a longer time.
13. 30 s in distilled H2O2
14. 1 min in 100º ethanol
15. 1 min in 100º ethanol
16. 2 min in xylene
16. 3 min in xylene
17. Mounting and coversliped
Results
Collagen: dark green / light blue.
Muscle: red.
Cytoplasm: pinky.
Nucley: black.
Notes
The Weigert hematoxylin working solution older than 10 days loses staining power. It is a good practice to filter the solution before the staining to prevent precipitates and artifacts.
Adjust the trichrome solution to a pH value of 2, for a better staining.
The differentiation with lower concentrations of glacial acetic acid slows the differentiation speed.
The final dehydration has to be quick to prevent color fading.
Products
Xylene
50º, 70º, 80º, 90º, 96º and 100º ethanol
Weigert hematoxylin
Bouin
Cromo2R (CI 16570)
Light green (CI 42095)
Phosphotungstic acid
Glacial acetic acid
Labware
Stainig dishes
Slides racks
Beakers
Magnetic stirrer
Bottles for solutions
Coverslips
