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457 Observations on a highly specific method for the histochemical detection of sulphated mucopolysaccharides, and its possible mechanisms By I. D. HEATH (From the Department of Anatomy, University of St. Andrews, Queen's College, Dundee. Present address: General Hospital, Nottingham) With 3 plates (figs, i to 3) Summary 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. Introduction IN the course of a previous investigation (Coupland and Heath, 1961) it was noted that when nuclear fast red (Herzberg) (NFR) (E. Gurr) is dissolved in [Quart. J. micr. Sci., Vol. 103, pt. 4, pp. 457-75, 1962.] 458 Heath—Method for sulphated mucopolysaccharides aluminium sulphate solution (NFRAL), it stains the granules of mast cells, the ground substance of cartilage, and some mucins with a high degree of specificity, all of which have variously been reported to contain sulphated mucopolysaccharides (Stacey, 1946; Meyer, 1955-6). Since the methods in common use for the detection of mucopolysaccharides in tissues, namely Hale's (1946) method, PAS, alcian blue (Steedman, 1950; Mowrey, 1956), and the use of basic dyes at various pH levels are not sufficiently specific to distinguish between simple acid mucopolysaccharides, sulphated mucopolysaccharides, and in some cases nucleic acids, it was felt that a more specific method would be of value. A preliminary report (Heath, 1961) has been published. Materials and methods The experiments are divided into two main sections. The first concerns the effects of a large number of dyes, dissolved in 5% aluminium sulphate solution, on ox trachea and rat skin. These tissues contain sulphated mucopolysaccharides in several forms and are also rich in SH and SS groups. Other salt solutions commonly used as mordants in the dye industry (chromic, ferric, &c), were also employed in some cases. The second is a group of experiments intended to elucidate the possible mechanism of action. Preparation of tissue Pieces of fresh ox trachea and rat skin were fixed in formaldehyde dichromate (buffered to pH 6 with sodium acetate buffer) and in formaldehyde calcium (Pearse, i960). In addition tissues from a variety of species were fixed in the fluids mentioned in table 1. After fixation for 48 h the tissues were dehydrated, embedded in paraffin wax in the routine manner, sectioned at 5 fx, and mounted on glass slides, egg albumen being used as adhesive. Tissues fixed with dichromate were washed for 12 h in running water before embedding. Fresh and fixed frozen sections were also used. Preparation of staining solutions o-i% stock solutions of the various dyes were prepared by dissolving the requisite amount of the dye in boiling 5% aluminium sulphate, filtering, and cooling. This stock solution was further diluted to 1 in 3 with cool 5% aluminium sulphate solution. Aqueous solutions of the dyes were also prepared in a similar fashion and diluted with water before use. In some cases dyes were prepared in ferric sulphate or chloride, or purple chromic sulphate, all at 5%. Staining procedure 1. Dewax and hydrate sections. 2. Remove mercury crystals where necessary. 3. Stain in above diluted solutions, 5 to 30 min. 4. Rinse in water. Heath—Method for sulphated mucopolysaccharides 459 5. Differentiate in 70% alcohol till tissue background is colourless (usually 20 to 30 sec). 6. Dehydrate in graded alcohols. 7. Clear in xylene. 8. Mount in polystyrene or Canada balsam. The tissues may be counterstained after step 3 in aqueous solutions of acid dyes, but the procedure is simplified and a better result obtained if the requisite quantity of acid dyes of suitable solubility and colour is added to the staining solution, to make a final concentration of 0-03% of the acid dye. Subsidiary experiments Mordanting experiments. Aluminium chloride and solutions of a number of salts of other metals and non-metals were used as solvents and diluents for NFR and for other dyes detailed in the tables, allowance being made where necessary for water of crystallization in the salt. Dewaxed hydrated sections were mordanted for 1 h in 5% aluminium sulphate, and then stained with aqueous NFR or o-i% solochrome azurine (I.C.I.). This is a dye recom- mended by Pearse (1957) for the detection of aluminium in tissue sections. Sections mordanted in aluminium sulphate and stained in aqueous solochrome azurine were subsequently stained in aqueous methylene yellow or thioflavine T. Other sections stained in aluminium solutions of one of the two latter dyes, were differentiated in alcohol, rehydrated, and stained in aqueous solochrome azurine. Control sections were stained in aqueous solochrome azurine, and also in an aluminium solution of solochrome azurine. Further sections were stained in mixtures of light green SF and NFR, in aqueous and aluminium solutions. Pure chemical test slides. Pure chemical substances were dissolved in 5% gelatin to a final concentration of 0-2% w/v, smeared on slides, fixed in formaldehyde vapour overnight, and stained by the above method. The substances used were the known constituents of mast cells, namely heparin (Holmgren and Willander, 1937) from sheep, ox, and pig; histamine (Riley and West, 1956); 5-hydroxytryptamine (Benditt, Wong, Areas, and Roeper, 1955; Coup- land and Riley, i960), and in addition 5-hydroxytryptophane (Lagunoff and others, 1957). A variety of sulphated polysaccharides, heparin, fucoidin (Percival, 1949), and a sulphated polyglucose, also unsulphated polysaccharides, starch, dextran, and dextrin were used. Gelatin blanks were run as controls. Methylation, saponification, and sulphation. A batch of ox-trachea sections were methylated in o-i N HC1 in absolute methanol (Fisher and Lillie, 1954). Some of these sections were subsequently saponified in 1% potassium hydroxide in 70% alcohol (Spicer and Lillie, 1959). Slides from each of these groups were sulphated in a 1:1 mixture of glacial acetic acid and concentrated sulphuric acid (Grillo and Lewis, 1959), again with previously untreated control sections, and sections of umbilical cord. Slides from each of the resultant groups were stained in NFRAL, solochrome azurine in aluminium and 460 Heath—Method for sulphated mucopolysaccharides aqueous solutions, and in aqueous methylene blue solutions atpH 1 -5 and pH 5 (Spicer and Lillie 1959), again with control sections. Slides which had only been methylated were stained in a solution of pararosaniline in 5% ferric chloride. The reason for the latter experiment will be made apparent in the Discussion. Effect of oxidation. Sections of ox trachea and rat skin were treated with °'5% periodic acid or 1% potassium permanganate for 10 min, others with performic acid for 5 min (Pearse, i960). Sections of pituitary and pan- creas were similarly treated, and all the resultant sections stained in NFRAL. In addition the performic acid/alcian blue technique (Adams and Sloper 1955-6) was performed on pituitary sections as control. Sections oxidized by performic acid were subjected to methylation for periods up to 20 h at 6o° C; some of these were sulphated subsequently. Alteration ofpH. NFRAL solution was buffered at pH 1, 2, 3, 4, and 5. Above pH 5 precipitation occurred. Aqueous solutions were similarly buffered, and also at pH 3-5—the 'natural' pH of the NFRAL. Sections were stained in similarly buffered solutions of gallamine blue and gallocyanin in aqueous and aluminium solutions.

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