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

• Nuclei  –  black
• Erythrocytes  –  yellow
• Cytoplasm  –  red
• Muscle  –  red
• Collagen  –  blue

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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