Gladstone Institutes Histology and Light Microscopy Core
Modified Verhoeff’s – van Giesen staining Protocol
This technique is for demonstrating elastin tissue in arteries and lungs. Such as in emphysema
and thinning and loss of elastic fibers in arteriosclerosis and other vascular diseases.
The tissue is stained with haematoxylin and a ferric chloride and iodine mordant.
Differentiation of the stain is completed using ferric chloride to break the tissue-mordant dye
complex. The elastic tissue has the strongest affinity for the dye and will keep the dye longer
than other tissue constituents
Tissue:
Formalin fixed paraffin embedded tissue or cryosections (with less resolution and
quality), sectioned at 2-10m
Note: Bake slides prior to staining, the stain can make the sections float off – be gentle
Controls:
Artery or skin
Solutions:
Verhoeff’s hx stock solutions:
Alcoholic haematoxylin
Stable for 1 year
Haematoxylin (Am Mastertech AHHEM25)
100% alcohol
Dissolve using gentle heat
10% Ferric chloride
Ferric Chloride
diH2O
1g
22ml
Stable for 1 year
Lugols iodine
Iodine
Potassium iodine
diH2O
(or purchase from Sigma L6146-1L)
20g
200ml
2g
4g
100ml
Verhoeff’s working solution
Make fresh
Alcoholic haematoxylin
22ml
10% Ferric chloride
8ml
Lugols iodine
8ml
Add solutions in order given, mixing between solutions, make fresh and discard
Van Giesen counterstain
01 Jan 2013
Oil Red O
Page 1 of 2
1% aqueous acid fuschsin
saturated (1.3%) aqueous picric acid
Differentiating solution
2% Ferric chloride
10% ferric chloride
diH2O
5ml
195ml
Make fresh
10ml
40ml
Fixing solution
5% hypo
Sodium thiosulphate
diH2O
25g
500ml
Procedure:
1. Dewax and rehydrate to water
2. Stain in Verhoff’s working solution
30min
3. Wash in running water
5 min
4. Differentiate in 2% ferric chloride
Determine microscopically
The elastin should be seen as black fibers on a grey background
5. Wash in running tap water
5 min
6. Counterstain in van Giesen
5 min
7. 95% alcohol
5 dips
8. 100% alcohol
2 x 1 min
9. Xylene
3 x 1 min
10.Mount in DPX (Gurr)
Results:
Elastic fibers and nuclei
Collagen
Other tissue elements
- Black
- Red
- Yellow
References:
Bancroft J, Stevens A, Theory and Practice of Histological Techniques, 2nd
Ed, 1982,pp 125, 139, Churchill Livingstone, NY
http://www.ihcworld.com/_protocols/special_stains/vvg.htm
library.med.utah.edu/WebPath/HISTHTML/.../EVG.PDF
01 Jan 2013
Oil Red O
Page 2 of 2