Laboratory Protocols
Histological Staining Techniques
Miller's elastin stain (E.V.G.)
Miller, 1971.
- Dewax sections, rinse in alcohol, rinse in water.
- Acid potassium permanganate - 5 minutes (0.5% potassium permanganate in 3% sulphuric acid)
- Rinse in distilled.
- Decolourise in 1% oxalic acid - 2 minutes.
- Wash in several changes of distilled water.
- Rinse in 95% alcohol.
- Place in Miller's stain in a closed container for 1-3 hours. (Do the full 3 hours unless urgent - 1.5 hrs. may be enough but check under a microscope after the next two steps. Elastin should be virtually black and sharply stained.)
- Rinse in 95% alcohol.
- Rinse in water.
- Stain with Weigert's haematoxylin - 10 minutes.
- Wash in tap water - 1 minute.
- Differentiate in acid alcohol (1% hydrochloric acid in 70% alcohol) - 5 seconds.
- Wash in tap water - 5 minutes.
- Rinse in distilled water.
- Stain with van Gieson's stain - 2 minutes.
- Rinse quickly in distilled water.
- Blot dry.
- Rinse very quickly in alcohol, clear and mount.
Results:
Elastin and mast cell granules - black/purple.
Collagen - red.
Muscle and cytoplasm - yellow.
Miller's stain
Victoria blue - 2g
New fuchsin - 2g
Crystal violet - 2g
Dissolve the dyes in 400mls of distilled water.
Add in the following order:
Resorcin - 8g
Dextrin - 2g
Freshly prepared 30% aqueous ferric nitrate - 100mls
Boil for 5 minutes and filter whilst still hot.
Transfer the precipitate plus the filter paper to the original flask
and redissolve in 400mls of 95% alcohol.
Boil (using a hotplate NOT a naked flame) for 15-20 minutes.
Cool, filter and make up to 400mls with 95% alcohol.
Add 4mls of concentrated HCl.
This solution keeps very well and if stored in a sealed coplin jar
can be reused many times.