Humberstone's Stain
for Elastic Fibres

Solutions

Weigert's iron hematoxylin or equivalent

Van Gieson's picro-fuchsin

Potassium permanganate, 0.25% aqu., for the Mallory bleach

Humberstone's solution
Preparing the precipitate
Victoria blue 4R	1	g		In an oversized flask, add the dyes to the water, and boil to dissolve. Add the resorcin, dextrin and ferric chloride. Boil for 3 minutes. Cool and filter.
Ethyl violet	1	g
Resorcin	4	g
Dextrin	0.5	g
Ferric chloride, 30% aqu.	25	mL
Staining solution
Ethanol, 95%	100	mL		Place the precipitate and filter paper back into the original flask. Add the ethanol and simmer gently for 15 minutes. Cool and filter. Make up the volume to 350 mL with 95% ethanol. Add the phenol and hydrochloric acid.
Phenol	10	g
Hydrochloric acid, conc.	4	mL
Distilled water	200	mL

Tissue sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Method

1. Bring sections to water via xylene and ethanol.
2. Do a Mallory bleach with 0.25% potassium permanganate for ten minutes.
3. Rinse with 95% ethanol.
4. Place into Humberstone's solution overnight.
5. Wash with 95% ethanol to remove excess solution.
6. Wash in water.
7. Counterstain with iron hematoxylin and van Gieson.
8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected results

• Elastic fibres  –  blue-black
• Nuclei  –  black
• Cytoplasm  –  yellow
• Collagen  –  red

Notes

1. The authors state that buffered formalin is an unsuitable fixative. I have not found this so and have used this method regularly with such tissue. It may be that the modern practice of minimalist fixation has an effect.
2. As the staining solution ages, the demonstration of elastic fibres improves in both quality and speed. When the solution is a few years old elastic is demonstrated within about 4 hours, although overnight is generally preferable.
3. The solution is stable for more than ten years. Due to the improvement in staining as the solution ages, it is suggested that a litre, or more, be made at a time.
4. The authors suggest as suitable counterstains, iron hematoxylin and van Gieson, Masson's trichrome or MSB (with green collagen).

 

Reference
Humberstone, G. C. W., and Humberstone, F. D., (1969)
An Elastic Tissue Stain
Journal of Medical Laboratory Technology, V. 26, No 2, pp. 99.

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