On the mechanism of Mallory's phosphotungstic acid-haematoxylin stain.

The mechanism of Mallory's (1900) phosphotungstic acid-haematoxylin (PTAH) stain is not yet fully understood; staining properties vary with the age of the dye solution. In this study, a 1 year-old solution was employed as described previously. Paper chromatograms revealed blue, red and yellow components of phosphotungstic acid-haematoxylin. Modification of reactive groups as well as consecutive treatment of sections with haematoxylin and phosphotungstic acid indicated hydrogen bond formation by the blue component. Substitution of brazilin for haematoxylin proved that two free phenolic --OH groups per dye residue are essential for binding of the blue phosphotungstic acid-haematoxylin chelate. Uptake of the red fraction was determined by the polyacid moiety of the dye. Van der Waals forces presumably contributed to dye binding, but did not govern the selectivity of the red and blue components of PTAH for various tissue structures.