Principle
The oxidation product of haematoxylin is haematin, and is the active ingredient in the staining solution. Haematoxylin is not classified as a dye since the molecule possesses no chromophore. The in situ oxidation of haematoxylin is effected by the addition of a strong oxidant to the stain, in this case sodium iodate. Lillie’s variant of Mayer’s haemalum is discussed in Lynch et al. (pp 1032)
(please refer to Lillie Mayer)
Haematin exhibits indicator-like properties, being blue and less soluble in aqueous alkaline conditions, and red and more soluble in alcoholic acidic conditions. In acidic conditions, haematin binds to lysine residues of nuclear histones by linkage via a metallic ion mordant, in this case aluminium. To ensure saturation of chemical binding sites, the stain is applied longer than necessary, resulting in the overstaining of the tissues with much non-specific background colouration. This undesirable colouration is selectively removed by controlled leaching in an alcoholic acidic solution, (acid alcohol), the process being termed "differentiation". Differentiation is arrested by returning to an alkaline environment, whereupon the haematin takes on a blue hue, the process of "blueing-up". The haematin demonstrates cell nuclei.
Full cellular detail is obtained by counterstaining with the eosin mixture. There are three commonly used forms of eosin - eosin Yellowish (tetrabromofluorescein, disodium salt CI 45380), eosin Bluish (the dinitro- dibromo-derivative CI 45400), and eosin Alcohol Soluble (the ethyl derivative CI 45386), the former is preferred. Colour enhancement is achieved by fortifying the stain with phloxine, a chemical member of the same family as eosin( halogenated fluorosceins). The mechanism of their staining is not fully understood, but is believed to be of an electrostatic nature. Visualisations most acceptable to the histologist are obtained by applying the dyes in acidic conditions, whereby more intense specific colourations are obtained, the more acidic tissue components taking up the dye to a greater intensity, hence the addition of acetic acid.