Hematoxylin and Eosin Staining Protocol

The oxidation product of hematoxylin is haemitin, and is the active ingredient in the staining solution. Hematoxylin is not classified as a dye since the molecule possesses no chromophore. The in-situ oxidation of hematoxylin is affected by the addition of a strong oxidant to the stain, in this case sodium iodate.
In acidic conditions, haemitin binds to lysine residues of nuclear histones by linkage via a metallic ion mordant, in this case aluminum. This undesirable coloration is selectively removed by controlled leaching in an alcoholic acidic solution, the process being termed 'differentiation'. Differentiation is arrested by returning to an alkaline environment, whereupon the haemitin takes on a blue hue, the process of 'bluing-up'. The haemitin demonstrates cell nuclei. Full cellular detail is obtained by counterstaining with the eosin mixture.

The length of time necessary to over-stain the tissues will depend upon fixation and the type of alum hematoxylin employed.
Differentiation with acid alcohol requires some practical experience to ascertain the correct end-point, since the acid solution alters the color of the tissue to red. The correct end-point is when, after bluing up, the background is almost colorless.
If Scott's tap water substitute is employed, bluing up is achieved in a much shorter time.
Eosin is highly soluble in water. Over-staining is removed by washing in running water.

For research use only. Not for any other purpose.

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