Hollande's Trichrome
for Mitoses, Keratin and Collagen
Materials
- An alum hematoxylin
- Solution A
Material Amount Basic fuchsin 1 g Ethanol, 70% 100 mL - Solution B
Material Amount Hydrochloric acid, conc. 0.1 mL Ethanol, 70% 100 mL - Solution C
Material Amount Phosphomolybdic acid 1 g Distilled water 100 mL - Solution D
Material Amount Orange G to saturation Distilled water 100 mL - Solution E
Material Amount Light green SF yellowish 0.2 g Distilled water 100 mL
Tissue Sample
Fixation requirements are unspecified, but likely many fixatives are satisfactory. 5µ paraffin sections of neutral buffered formalin fixed tissue are likely suitable. Many trichrome stains benefit from Bouin or mercuric chloride fixation of tissue or sections.
Protocol
- Bring sections to water via xylene and ethanol.
- Stain nuclei with an alum hematoxylin nuclear stain and blue.
- Place into solution A for 6-12 hours.
- Wash with water for 5 minutes.
- Place into solution B, a few seconds, until clouds of dye stop being extracted.
- Wash well with water.
- Place into solution C for 5 minutes.
- Rinse with water.
- Place into solution D for 5 minutes.
- Place into fresh solution E, 30-60 seconds, until differentiated.
- Dehydrate with amyl alcohol.
- Clear with xylene and mount with a resinous medium.
Expected Results
- Nuclei – blue
- Mitoses – red
- Cartilage – purple
- Erythrocytes – orange
- Keratin – orange
- Collagen – green
Notes
- Amyl alcohol was recommended for dehydration. If this is not available rapid dehydration with ethanol may suffice. Often t-butanol dehydrates satisfactorily without extracting excess dye.
- The method specified clearing with benzene. For safety reasons this should be avoided. Xylene or toluene should be quite satisfactory.
- Magenta was specified for solution A. This is an obsolete synonym for basic fuchsin.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Hollande, (1912)
Archives de zoologie expérimentale et générale, v.10, pp.62
