Miller's Stain
for Elastic Fibres
Materials
- Weigert’s iron hematoxylin or equivalent
- Van Gieson’s picro-fuchsin
- Potassium permanganate, 0.25% aqu., for the Mallory bleach
- Miller’s solution
Material Amount Victoria blue 4R 1 g New fuchsin 1 g Crystal violet 1 g Resorcin 4 g Dextrin 1 g Ferric chloride, 30% aqu. 50 mL Distilled water 200 mL Ethanol, 95% 200 mL Hydrochloric acid, conc. 2 mL
Preparation
- In an oversized flask heat the water and add the three dyes.
- When dissolved, add the resorcin, dextrin and ferric chloride in the order given.
- Boil for 5 minutes, then filter while hot.
- Place the precipitate and filter paper back into the original flask, add the 95% ethanol and simmer gently for 20 minutes.
- Cool and filter.
- Restore the volume to 200 mL with 95% ethanol, then add 2 mL of concentrated hydrochloric acid.
Tissue Sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.
Protocol
- Bring sections to water via xylene and ethanol.
- Do a Mallory bleach with 0.25% potassium permanganate for ten minutes.
- Rinse with 95% ethanol.
- Place into Miller’s solution up to three hours.
- Wash with 95% ethanol to remove excess solution.
- Wash in water.
- Counterstain with iron hematoxylin and van Gieson.
- Dehydrate with ethanol, clear with xylene and mount with a resinous medium.
Expected Results
- Elastic fibres – black
- Nuclei – black
- Cytoplasm – yellow
- Collagen – red
Notes
- Three hours is recommended, although staining may be adequate after 90 minutes.
- Dilute with an equal quantity of 95% ethanol for overnight staining.
- The staining solution is stable for years.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Miller, P.J., (1971)
Journal of Medical Laboratory Technology, V. 28 - Ellis, R.,
Miller’s Elastic Staining Protocol
IHC World
