Verhoeff's Iron Hematoxylin
for Elastic
Materials
Stock Verhoeff A
| Material | Amount | |
|---|---|---|
| Hematoxylin | 1 | g |
| Ethanol, absolute | 20 | mL |
Stock Verhoeff B
| Material | Amount | |
|---|---|---|
| Ferric chloride | 10 | g |
| Distilled water | 100 | mL |
Stock Verhoeff C
| Material | Amount | |
|---|---|---|
| Potassium iodide | 4 | g |
| Iodine | 2 | g |
| Distilled water | 100 | mL |
Working solution
| Material | Amount | |
|---|---|---|
| Stock solution A | 20 | mL |
| Stock solution B | 8 | mL |
| Stock solution C | 8 | mL |
Differentiator
| Material | Amount | |
|---|---|---|
| Stock solution B | 10 | mL |
| Distilled water | 40 | mL |
Tissue Sample
A 10% Formalin variant is suitable. Paraffin sections at 5µ are preferred.
Protocol
- Bring sections to water via xylene and ethanol.
- Place in working Verhoeff’s solution for 15 minutes.
- Wash well with tap water to remove all excess hematoxylin.
- Differentiate until the elastic fibres are satisfactory. This should be controlled microscopically based on the appearance of fibres in the area of interest.
- Rinse well with tap water.
- Place into 95% ethanol for 5 minutes to remove iodine discolouration.
- Wash well with tap water to remove all residual chemicals.
- Counterstain with Van Gieson.
- Dehydrate with absolute ethanol.
- Clear with xylene and mount with a resinous medium
Expected Results
- Nuclei – black
- Elastic fibres – black
- Muscle – yellow
- Collagen – red
Notes
- The alcoholic hematoxylin solution should be freshly made. It does not need to have been ripened as ferric chloride is an oxidising agent. Some technologists keep pre-weighed aliquots of 1 gram hematoxylin and dissolve it in 20 mL ethanol as needed.
- The working solution should be made just before use and allowed to stand for five minutes to ripen.
- While this is a popular elastic stain, it is difficult to stain both coarse fibres and fine fibres optimally in a single section. If coarse fibres are well stained, the finest fibres may be completely decolourised, and if fine fibres are optimally stained, the coarse fibres are underdifferentiated. Ensure differentiation is optimised for the fibres of interest.
- If Van Gieson is used as a counterstain, the elastic fibres should be very slightly underdifferentiated as picric acid will also remove some hematoxylin staining.
- This method likely stains by two mechanisms: ionic attachment of iron mordanted hematoxylin to most structures and by van der Waal’s forces to elastic fibres. The differentiation step is accomplished either by oxidative bleaching of hematoxylin by ferric chloride, or by displacement of the mordanted hematoxylin by free mordant in solution. The dye attached to elastic fibres by van der Waal’s forces is not extracted easily. It is, however, still susceptible to bleaching, which likely explains why fine fibres may be pale. That bleaching takes place is shown by the differentiating fluid remaining clear during differentiation, extracted dye being seen to bleach as it dissolves out.
- Note that nuclei are only adequately, not well, stained.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Histological demonstration techniques, (1974)
Cook, H C.
Butterworths, London, England - Culling, C.F.A., (1963)
Handbook of histopathological techniques, 2nd ed.
Butterworths, London.
