Progressive haemalum (alum-haematoxylin) stains the DNA, not nucleoprotein, in cell nuclei.

What is it about?

Haemalum (the H of the H&E staining method) exists as a positively charged dye-metal (aluminium-haematein) complex in solutions at pH2.0-2.5, which progressively stain nuclear chromatin and very little else. Earlier investigators of the staining mechanism have suggested that: (a) in analogy to mordant textile dyeing, aluminium ions (+ve) form a bridge, involving coordinate bonds, to the phosphate groups of DNA and to haematein. (b) the organic (haematoxylin or haematein) moiety of the dye-metal complex binds to the basic nucleoprotein (histones) that lie alongside the DNA molecules in nuclear chromatin. The work presented in this paper shows that haemalum has demonstrable affinity for DNA but not for histone. Sequential staining experiments with an aluminium salt and haematein, both at pH2.0-2.5, do not support the mordant dyeing hypothesis. Nuclear staining by hemalum at pH ≤ 2 (and also by other acid dye-metal complexes such as chromium-gallocyanine and iron-celestine blue) is attributed to electrostatic attraction to the protonated phosphate groups of DNA (pKa~1.0). The bulky, non-planar red aluminium-haematein complex is unlikely to to be held in place by hydrogen bonds or van der Waals forces. The stability of the stain is due to conversion to an insoluble blue product when stained preparations are "blued" by exposure to water at pH>5.5,

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Photo by Luke Chesser on Unsplash

Photo by Luke Chesser on Unsplash

Why is it important?

Haemalum and eosin (H&E) staining of human and animal tissues has been routinely used in the teaching of normal histology and in diagnostic histopathology for more than 140 years. Results vary among different laboratories and with different tissues. Different pathologists have different ideas concerning the ideal colours in an H&E-stained section. Understanding the mechanisms of all preparative techniques for microscopy is essential if the methods are to be intelligently used and modified to suit different tissues and different users.