McFarlane's Trichrome
2nd Variant for Muscle and Collagen

Solutions

Weigert's iron hematoxylin or equivalent
Solution A
Picric acid 0.2 g
Acid fuchsin 0.8 g
Acetic acid, glacial 2 mL
Distilled water 98 mL
Solution B
Acetic acid, glacial 2 mL
Distilled water 98 mL
Solution C
Picric acid 1 g
Phosphotungstic acid 10 g
Ethanol, 95% 40 mL
Distilled water 60 mL
Solution D
Aniline blue 2.5 mL
Acetic acid, glacial 2.5 g
Distilled water 97.5 mL
Solution E
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.

Method

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with Weigert's iron hematoxylin or equivalent.
  3. Place into solution A for 5 minute.
  4. Rinse with solution B.
  5. Place into solution C for 5 minutes.
  6. Rinse with distilled water.
  7. Place into solution D for 5-10 minutes.
  8. Rinse with solution B.
  9. Place into solution E for 5 minutes.
  10. Wash with solution B.
  11. Dehydrate with ethanol.
  12. Clear with xylene and mount with a resinous medium.

Expected results

Notes

  1. McFarlane also has another multi-step method and a one step method.
  2. 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   Solution C   Solution E
    Picric acid, sat. aqu. 16 mL Picric acid, sat. alc. 12 mL Picric acid, sat. aqu. 20 mL
    Distilled water 82 mL Distilled water 60 mL Distilled water 70 mL
    Acetic acid, glacial 2 mL Ethanol, 95% 28 mL Ethanol, 95% 10 mL
    Acid fuchsin 0.8 g Phosphotungstic acid 10 g Phosphotungstic acid 2.5 g

 

Reference
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. 23

 


 

 

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