Quarterly Journal of Microscopical Science, Vol s3-103, 457-475, Copyright © 1962 by Company of Biologists

Observations on a Highly Specific Method for the Histochemical Detection of Sulphated Mucopolysaccharides, and its Possible Mechanisms

¹ Department of Anatomy, University of St. Andrews, Queen's College, Dundee. Present address: General Hospital, Nottingham

Whereas basic dyes in aqueous solutions stain chromatin, all mucins, mast cells, the ground substance of cartilage, and epidermis, it has been shown that a 0.03% solution of basic dye in 5% aluminium sulphate produces a highly specific staining reaction for sulphated mucopolysaccharides. The best dyes are nuclear fast red (Herzberg) and methylene blue. Acid dyes in solutions of aluminium salts are induced to stain the ground substance of cartilage. These observations have been confirmed in a number of species.

Other metallic ions have similar properties and the use of green and purple chromic salts indicate that co-ordination plays a part in the reaction.

Methylation, saponification, and sulphation experiments show that the sulphate group is essential. This has been confirmed by using pure chemical substances in gelatin models. Oxidation of keratin with performic acid, which produces sulphonic groups, causes hair (previously negative) to react. From this it is suggested that sulphonic groups may also react, and that the reactive groups need not be attached to mucopolysaccharides. It is further suggested that the specificity of sulphated mucopolysaccharides is due to the fact that they are the only substances present in the tissues with a sufficient concentration of sulphate groups.

Experiments with solochrome azurine show that the aluminium is attached to all tissue elements irrespective of their nature. It is suggested by analogy with the work of others on the action of mordants that this attachment is by co-ordinate linkage.

Acid dyes of strong and weak type buffered to a variety of pH levels show that ionization plays little if any part in the staining of the sulphated elements, but may be of considerable importance in the staining reactions of the other tissue elements. By analogy with solochrome azurine, which is known to co-ordinate with aluminium, it is suggested that the dye is attached, by means of a further co-ordinate bond, to the metal. It is suggested that the sulphate group may owe its specificity to its strongly acid character, and the fact that such groups are capable of forming extremely stable complexes with metallic ions.

It is concluded that this is a highly specific method of staining sulphated mucopolysaccharides, depending upon the formation of a link by a metallic ion between the tissues and the dye, in the fashion of a true mordant. The ability of the dye to attach itself to the metallic ion depends finally upon the resultant of the electrostatic charges on the tissues and the metallic ion.