Tissue sample
A 10% Formalin variant is suitable.
Paraffin sections at 5µ are preferred.
Method
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.
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 very 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 decolorised, 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 probably depends on a dual staining mechanism: iron mordanted attachment to most structures and hydrogen bonding 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 hydrogen bonds is not extracted easily because the hydrogen bond is between the dye and the protein rather than between the mordant and the protein. 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.
Reference Histological demonstration techniques, (1974) Cook, H C.
Butterworths, London, England
