Miller'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

Miller' solution
Preparing the precipitate
Victoria blue 4R	1	g		In an oversized flask heat the water and add the three dyes. When dissolved, add the resorcin, dextrin and ferric chloride solution in the order given. Boil for 5 minutes, then filter while hot.
New fuchsin	1	g
Crystal violet	1	g
Resorcin	4	g
Dextrin	1	g
Ferric chloride, 30% aqu.	50	mL
Distilled water	200	mL
Staining solution
Ethanol, 95%	200	mL		Place the precipitate and filter paper back into the original flask. Add the ethanol and simmer gently for 20 minutes. Cool and filter. Restore the volume to 200 mL with 95% ethanol. Add the hydrochloric acid.
Hydrochloric acid, conc.	2	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 Miller's solution up to three hours.
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  –  black
• Nuclei  –  black
• Cytoplasm  –  yellow
• Collagen  –  red

Notes

1. Three hours is recommended, although staining may be adequate after 90 minutes.
2. Dilute with an equal quantity of 95% ethanol for overnight staining.
3. The staining solution is stable for years.

 

Reference
Miller, P.J., (1971)
Journal of Medical Laboratory Technology, V. 28

Ellis, R.,
Miller's Elastic Staining Protocol
IHC World

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