McFarlane's Trichrome 2nd Variant
for Muscle and Collagen
Materials
- Weigert’s iron hematoxylin or equivalent
- Solution A
Material Amount Picric acid 0.2 g Acid fuchsin 0.8 g Acetic acid, glacial 2 mL Distilled water 98 mL - Solution B
Material Amount Acetic acid, glacial 2 mL Distilled water 98 mL - Solution C
Material Amount Picric acid 1 g Phosphotungstic acid 10 g Ethanol, 95% 40 mL Distilled water 60 mL - Solution D
Material Amount Aniline blue 2.5 mL Acetic acid, glacial 2.5 g Distilled water 97.5 mL - Solution E
Material Amount Picric acid 0.25 g Phosphotungstic acid 2.5 g Ethanol, 95% 10 mL Distilled water 90 mL
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
- Bring sections to water via xylene and ethanol.
- Stain nuclei with Weigert’s iron hematoxylin or equivalent.
- Place into solution A for 5 minute.
- Rinse with solution B.
- Place into solution C for 5 minutes.
- Rinse with distilled water.
- Place into solution D for 5-10 minutes.
- Rinse with solution B.
- Place into solution E for 5 minutes.
- Wash with solution B.
- Dehydrate with ethanol.
- Clear with xylene and mount with a resinous medium.
Expected Results
- Nuclei – black
- Erythrocytes – yellow
- Cytoplasm – red
- Muscle – red
- Collagen – blue
Notes
- McFarlane also has another multi-step method and a one step method.
- Solutions A, C and E require dry weights of picric acid. These could be provided from saturated aqueous (1 g in 78 mL) or ethanolic (1 g in 12 mL) solutions. Alternate formulas are:
Solution A
Material Amount Picric acid, sat. alc. 16 mL Distilled water 82 mL Acetic acid, glacial 2 mL Acid fuchsin 0.8 g Solution C
Material Amount Picric acid, sat. alc. 12 mL Distilled water 60 mL Ethanol, 95% 28 mL Phosphotungstic acid 10 g Solution E
Material Amount Picric acid, sat. alc. 20 mL Distilled water 70 mL Ethanol, 95% 10 mL Phosphotungstic acid 2.5 g
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:
McFarlane, (1944)
Stain Technology, v. 19, pp. 29
