Hematoxylin

Hematoxylin is extracted from the heartwood of the logwood tree, Haematoxylum campechianum. It is not itself the dye: it must be oxidized to hematein, either slowly in air or quickly with an oxidant such as sodium iodate. Hematein then needs a mordant, usually aluminum, to form a positively charged dye-metal complex that binds the phosphate groups of nucleic acids, which is why chromatin stains strongly.

Freshly applied, the color is reddish purple. A brief alkaline rinse, the bluing step, turns it the familiar blue. Formulations are progressive, applied until the right depth is reached (Mayer's, for example), or regressive, overstained and then partly removed with dilute acid (Harris's, for example).

The final color depends on the formulation, the staining time, the bluing solution and its time, how old and how oxidized the solution is, and section thickness. The same tissue stained in two laboratories can differ visibly, and so can two runs in one laboratory weeks apart.

For image analysis that matters because color deconvolutionLoading... needs a stain vector, the exact color of pure hematoxylin. A published default may not fit your slides. Estimate it from a hematoxylin-only control section stained in the same run, and re-estimate when the staining protocol or reagent lot changes.

In brightfield IHCLoading..., hematoxylin is the counterstain: it marks every nucleus so the DABLoading... signal can be read against the tissue. For nuclear markers, DAB and hematoxylin sit in the same compartment, and a heavy counterstain overlaps and partly masks weak nuclear DAB, both to the eye and to deconvolution.

A lighter counterstain makes weak nuclear positivity easier to detect and separate, at the cost of fainter nuclei for segmentation. Settle the balance on control slides at the start of a study and keep it fixed, because a change in counterstain depth changes which weakly positive nuclei are called positive.