Pasini's Trichrome
for Elastic and Collagen

Solutions

Alum hematoxylin

Solution A
Phosphotungstic acid	2	g
Distilled water	100	mL

Solution B – Var I				Var II
Eosin B	0.7	g		Eosin B, 2% in 50% ethanol	30	mL
Acid fuchsin, sat. aqu.	4	mL		Acid fuchsin, sat. aqu.	4	mL
Unna's elastic stain	35	mL		Unna's elastic stain	30	mL
Glycerol	40	mL		Glycerol	15	mL
Ethanol, 50%	35	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 an alum hematoxylin nuclear stain.
3. Place into solution A for 10 minute.
4. Rinse quickly with distilled water.
5. Place into solution B for 15-20 minutes.
6. Rinse quickly with 70% ethanol.
7. Dehydrate and differentiate with 100% ethanol for a few seconds.
8. Clear with xylene and mount with a resinous medium.

Expected results

• Nuclei  –  blue
• Elastic fibres  –  purple
• Erythrocytes  –  orange
• Collagen  –  blue

Notes

1. Ehrlich's alum hematoxylin was recommended. An iron hematoxylin may be more suitable.
2. Solution B var I is as given by Gray, var II is as given by Gatenby & Beams.

 

Reference
Gray, Peter. (1954)
The Microtomist's Formulary and Guide.
Originally published by:– The Blakiston Co.
Republished by:– Robert E. Krieger Publishing Co.
  Citing:–
    Gatenby, J. B. & Cowdry, E. V., (1928)
    Bolles Lee's Microtomist's Vade Mecum, pp. 280
    Blakiston, Philadelphia

Bolles Lee, A.. Edited by Gatenby, J.B. and Beams, H.W., (1950)
The Microtomist's Vade-Mecum. 11 ed., pp. 485
Churchill, London, UK.