McFarlane's Trichrome
3rd Variant for Muscle and Collagen

Solutions

Regaud's iron hematoxylin or equivalent
Solution A
Picric acid 1 g
Orange G 0.25 g
Ethanol, 95% 80 mL
Distilled water 20 mL
Solution B
Acid fuchsin 0.25 g
Ponceau 2R 0.25 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Solution C
Acetic acid, glacial 2 mL
Distilled water 98 mL
Solution D
Picric acid 1 g
Phosphotungstic acid 10 g
Ethanol, 95% 80 mL
Distilled water 20 mL
Solution E
Aniline blue 2.5 g
Acetic acid, glacial 2.5 mL
Distilled water 97.5 mL
Solution F
Picric acid 0.5 g
Phosphotungstic acid 5 g
Ethanol, 95% 20 mL
Distilled water 801 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 Régaud's iron hematoxylin, but do not differentiate with iron alum.
  3. Place into solution A until nuclei are differentiated.
  4. Wash with water until erythrocytes alone remain yellow.
  5. Place into solution B for 5-10 minutes.
  6. Rinse with solution C.
  7. Place into solution D for 5 minutes until differentiated.
  8. Rinse with solution C.
  9. Place into solution E for 10 minutes.
  10. Rinse with solution C.
  11. Place into solution F until differentiated.
  12. Wash with solution C.
  13. Dehydrate with ethanol.
  14. 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, D and F require dry weights of picric acid. These could be provided from a saturated ethanolic (1 g in 12 mL) solution. Alternate formulas are:–
     
    Solution A   Solution D   Solution F
    Picric acid, sat. alc. 12 mL Picric acid, sat. alc. 12 mL Picric acid, sat. alc. 6 mL
    Ethanol, 95% 68 mL Ethanol, 95% 68 mL Ethanol, 95% 14 mL
    Distilled water 20 mL Distilled water 20 mL Distilled water 80 mL
    Orange G 0.25 g Phosphotungstic acid 10 g Phosphotungstic acid 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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