20 to 30 Sec)

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 recommended 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 pancreas 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. Orange G, a strongly acid dye containing the sulphonate group, and the weakly acidic dyes eosin B and solochrome azurine which possess carboxyls as their acidic groups, were buffered at pH values from 1 to 9, at unit intervals, in aqueous and aluminium solutions, and sections stained in these solutions. Results The full results are given in the tables (see Appendix, p. 472), but some points require further elaboration. The fixative was of little importance except in the case of a Zenker's fluid,

FIG. 1 (plate). A, dog's ear (formaldehyde dichromate, NFRAL), to show staining of mast cells and ground substance of cartilage. B, thyroid of sheep (formaldehyde calcium, toluidine blue in 5% aluminium sulphate). Note lack of reaction in colloid. c, skin of rat (formaldehyde saline, solochrome azurine in aluminium sulphate followed by aqueous methylene yellow). Photographed with a minus yellow filter. Note the 'clear' nucleus. D, posterior pituitary of ox (Helly's fluid, PFA, NFRAL 3h). Neurosecretory substance reacts. E, Cheek pouch of hamster (formaldehyde calcium, Nile blue sulphate in 5% aluminium sulphate). F, intestine of sheep (Huber, pararosaniline in ferric). Mast cells and the elastic tissue in the arteriole take the stain. G, base of a peptic ulcer (mercuric formaldehyde, NFRAL). H, skin of rat (formaldehyde calcium, PFA, NFRAL). Strong reaction in the hair shafts and fainter reaction in the keratinized layer surrounding the hair follicle and on the surface of the epidermis. I, intestine of ox (formaldehyde dichromate, aqueous methylene blue). j, salivary glands of ox (formaldehyde saline, NFR in unboiled purple chromic sulphate). No reaction in the gland tissue or nuclei. Compare fig. 3, E. K, intestine of ox, adjacent section to I (methylene blue in aluminium sulphate). Note that only mast cells and some of the musus reacts. B

To, 20

D A

JlL H G .

50)1

FIG. I I. D. HEATH 40/x Heath—-Method for sulphated mucopolysaccharides 461 which completely suppressed the staining reaction by basic dyes in aluminium or aqueous solutions, and also the staining of the ground substance of cartilage with acid dyes in aluminium solution. In general, the fixatives with pH values between pH 4 and pH 7 were best, 'analar' formaldehyde buffered at pH 6 and formaldehyde-calcium being the fixatives of choice. Some difficulty was experienced with sheep material fixed in formaldehyde-saline, but this appeared to vary from animal to animal Alcoholic fixatives were of no particular value, and fixed and unfixed frozen sections reacted similarly to paraffin sections. When basic dyes are dissolved in aluminium sulphate solution they give highly specific reactions for the granules of mast cells, the ground substance of cartilage, certain mucins, and epidermis (fig. 1, A, K). AS these have all been reported to contain sulphated mucopolysaccharides, for the sake of brevity they will be referred to as the 'sulphated elements'. The resulting colour was orthochromatic in some cases and metachromatic in others. It was found that where specificity is poor, other mucins and chromatin staining, it may be improved by increasing the concentration of aluminium present. A critical concentration of the metallic ion appears to be necessary, since 5% chromic alum gives low specificity whereas 10% chromic alum gives absolute specificity. If account is taken of the large amount of water of crystallization in chrome alum it will be realized that 10% is roughly equivalent to 5% chromic sulphate. Further examples of this phenomenon are demonstrated in the varying concentrations of cupric, cobaltous, and nickel salts necessary for specificity (see table 3). The dyes which gave the most specific results were those of the basic formula shown here.

—X—{ Ni=> X = O, S, or N Kl = C or N h2 = CH3, C2H5, etc. Of these the azines (XX1 = N, N) (neutral red (Conn, 1953), nuclear fast red (Herzberg), (Gurr, i960)) and thiazins (XX1 = S, N) (toluidine blue and the various members of the methylene series) give the highest degree of specificity, and are certainly dyes of choice for this method of staining. The oxazines (XX1 = O, N) (e.g. cresyl fast violet and celestin blue) give good specificity, although gallamine blue and gallocyanine stain poorly. Xanthines (XX1 = O, C), particularly the acridines, give good specificity, and these may prove useful in ultra-violet microscopy: rhodamines again are fairly good, but in addition tend to stain red blood-corpuscles very deeply (fig. 2, E). This may be useful as a method of detecting red blood-corpuscles, since sulphated elements differentiate out more rapidly. The phenylmethanes (basic fuchsin, crystal violet, malachite green, &c.) differentiate out very rapidly owing to their high solubility in alcohol (Conn, 1953), and for this reason are of little value in this method. Ferric salts improve this to a certain extent, and in addition, with the magentas (particularly pararosaniline) elastic tissue takes the stain, 462 Heath—Method for sulphated mucopolysaccharides (fig. 1, F). It is of interest to note that this and related dyes dissolved with ferric salts are used in Weigert's elastic tissue stain. Azo dyes (Janus green) give low intensity of staining, but specificity is high. This was also noted with the phthalocyanins (alcian green) (Lubs, 1955) and various miscellaneous dyes mentioned in the table. In simple aqueous solutions the basic dyes behave in a normal fashion, staining the sulphated elements and in addition chromatin, all mucins, and several other elements (fig. 1,1). Acid dyes dissolved with aluminium sulphate stain all tissue elements a deep even colour, including cartilage ground substance (fig. 2, B) which is not stained in simple aqueous solution (fig. 2, A). The tissues from various other species all react similarly to those of ox and rat (figs. 1, B, E, G; 2, c; 3, c). Other aluminium salts gave exactly similar results to aluminium sulphate, both as simple salts and alums. Mordanting of the sections in aluminium sulphate solution and subsequent staining in aqueous NFR again gives specificity of action. Other metallic salts give similar results to aluminium in greater or lesser degree, the best being cobalt, cadmium, and nickel. In general, those salts which form co-ordination complexes with ease give the best results. Purple chromic salts when prepared without boiling give specificity, whereas green chromic and boiled purple chromic salts do not (figs. 1, J; 3, E). Salts of non- metals do not confer specificity. Potassium and sodium give no staining at all. Solochrome azurine in aqueous solution stains all tissue elements except sulphated elements, a faint red-orange. When mixed with aluminium salts the dye solution changes to a deep red-purple which stains all tissue elements dark blue. Sections mordanted with aluminium and stained in aqueous solochrome azurine solution give similar results to those stained in aluminium solochrome azurine solution. Sections mordanted in aluminium, stained in aqueous solochrome azurine, and subsequently stained in aqueous methylene yellow or thioflavine T show the sulphated elements stained yellow presumably

FIG. 2 (plate). A, trachea of ox (aqueous ponceau 2R). Note lack of reaction in cartilage. B, trachea of ox, adjacent section to A (ponceau 2R in aluminium sulphate solution). Note intense reaction in cartilage. C, tendon and synovial membrane of sheep (NFRAL). Note reaction in tendon and mast cell in synovial membrane. D, intestine of sheep (fresh-frozen, NFRAL). Mast cell only reacts. E, skin of rat (rhodamine B in 5% aluminium sulphate), to show red blood-cells in a capil- lary reacting. FIG. 3 (plate). A, umbilical cord (NFRAL). Note that only mast cells react. B, umbilical cord (N.F.R., aqueous). Note nuclear staining in addition. C, intestine of guinea pig (NFRAL). Mucus in Brunner's glands reacts. D, umbilical cord (after sulphation stained NFRAL). Note intense reaction of all elements. E, intestine of sheep (NFR in green chromic sulphate). Note nuclear staining. Compare fig- 1, J. F, test-slide of chemical substances from mast cells (NFRAL). hep, heparin; hist, histamine; j = serotonin (5-hydroxytryptamine); 5 hp, 5-hydroxytryptophane; con, control blank. G, test-slide of chemical substances (NFRAL). spg = sulphated polyglucose. /-t

F G — fucoidin heporin hep spg h/st ogor s dextrin control 5hp con

Fi<;. 3 I. D. HEATH Heath—Method for sulphated mucopolysaccharides 463 by displacement of the solochrome azurine from them; the other tissue elements retain the even, deep blue colour (fig. 1, c). Sections stained in aluminium solution of methylene yellow, differentiated in alcohol, rehydrated, and stained in aqueous solochrome azurine, again show the sulphated elements stained yellow with the surrounding tissue the deep blue colour of the solochrome azurine aluminium reaction. When tissues are stained in a mixture of light green SF and NFR in aluminium, sulphated elements stain red and the remainder of the tissues stain green, including chromatin. In aqueous solution the sulphated elements stain red as does chromatin, while the cytoplasm of cartilage cells stains green as do all the other tissue elements. The heparins, fucoidin, and the sulphated polyglucose give a positive staining reaction, but none of the other substances react, (fig. 3, F, G). Methylation blocks staining with NFRAL and both methylene blue solutions. Subsequent saponification does not restore staining with NFRAL, or methylene blue at pH 1-5, but does so at pH 5. Sulphation restores staining with NFRAL and methylene blue at pH 1-5, but in addition induces staining of most other tissue elements, including the general ground substance, although the sulphated elements stain more deeply in the case of NFRAL and are faintly metachromatic with methylene blue. Umbilical cord stains a deep, even red colour with NFRAL after sulphation, whereas only mast cells stained previously (fig. 3, A, B, D). With aqueous solochrome azurine the ground substance of cartilage stains red-brown after methylation, in contradistinction to the fact that, without methylation, it does not stain. In aluminium solution it stains blue, similarly to unmethylated sections in aluminium solution. Saponification greatly increases the intensity of staining in aluminium solution and suppresses to a large extent the staining in aqueous solution. Methylation has no effect upon the staining of elastic tissue with pararosaniline in ferric salts. Periodic acid has no effect upon the reaction. Potassium permanganate gives a more definite staining of the sulphated elements where this reaction is weak. No other elements react. After oxidation by performic acid hair shafts and keratinized epidermis react (fig. 1, H), as does neurosecretory substance (after staining for 3 h) (fig. 1, D), but no reaction is seen in the islets of Langerhans. Methylation for 16 h at 60° C blocks the reaction with neurosecretory substance and that of hair shafts and keratinized epithelium. Alcian blue reacts similarly. Sulphation restores the reaction in hair after its loss by methylation. Buffering NFR at various pH levels had no effect on the reaction. At no pH level does the aqueous solution give specificity of action for sulphated elements. With gallamin blue and gallocyanin in aqueous solution variable results are obtained. Staining does not occur at pH 4, but does occur above and below, with fair specificity for sulphated elements at low pH. Chromatin and connective-tissue elements stain more prominently above pH 4. In aluminium solution the dyes gave a fair degree of specificity for sulphated elements at all pH levels, including pH 4, but the staining tends to be of low 464 Heath—Method for sulphated mucopolysaccharides intensity. With orange G in aqueous solution an intense staining reaction is seen in all tissues, except the sulphated elements, at pH 1; there is a gradual fall-off in intensity until at pH 9 only eosinophils stain. In aluminium solution all tissue elements stain at all pH values. Eosin B in aqueous solution reacts differently, staining acidophil tissues at pH 3, 4, and 5 with a marked fall-off in intensity on each side of this peak. In aluminium solution there is a gradual increase in the intensity of staining of all elements in the tissues as the pH level rises, but at pH 1 to 3 the sulphated elements do stain faintly, the rest of the tissues being colourless. The results with solochrome azurine were similar to, but not so clear-cut as those with eosin B. There tends to be more background staining in aqueous solution at low pH levels. Discussion It is apparent from the above observations that many basic dyes when dissolved with aluminium salts stain the granules of mast cells, the ground- substance of cartilage, epidermis, and some mucins in a highly specific manner. Further, acid dyes are induced to stain highly acid elements. These phenomena are common to a large number of other metallic ions, most of which form co-ordination complexes readily. Other tissue elements known to contain sulphated mucopolysaccharides react in a similar fashion to the 'sulphated elements', as do the sulphated polysaccharide test-substances when stained with a basic dye in the presence of aluminium sulphate. The metallic ion is, therefore, necessary, since in aqueous solution many more tissue elements stain, as they do in solutions of non-metallic salts. Purple chromic salts are already in loose co-ordination with an ion or molecule, usually water, whereas green chromic salts are co-ordinated in an extremely stable fashion with a very low exchange rate (Martell and Calvin, 1952). If purple chromic salts are boiled, the co-ordination becomes much more stable and the salts turn green. The fact that green chromic and boiled purple chromic salts do not give specificity of action suggests that co-ordination plays some part in the mechanism. Sodium and potassium do not have any great tendency to form complex ions, although they do form some (Moeller, 1952), and in common with other salts they tend to reduce the effects of stains in solution by causing flocculation with consequent reduction of diffusibility (Baker 1958). This might possibly explain the suppression of staining with these particular salts. It may also explain the variable and poor results obtained with gallamin blue and gallocyanin in the buffered solution in which potassium ions were present. Further these dyes have their iso-electric point at about pH 4-1 (Baker, 1958) and would tend to be in an unchanged state, which would encourage flocculation. It is necessary to use buffers, as the pH of aluminium sulphate solution is difficult to adjust by the addition of acid or alkali alone (Baker, 1958). Methylation with absolute methanol in the presence of dilute mineral acid was performed by Charles and Todd (1940) in their investigation of the barium salt of heparin. They noted that this procedure reduced the amount Heath—Method for sulphated mucopolysaccharides 465 of sulphur present to a very low level. This action on tissue sections has been confirmed by Spicer, Swarm, and Burtner (1961), who methylated sections labelled with 35S sulphate, and found that the resulting radioautographs were almost devoid of activity. Fraenkel-Conrat and Olcott (1945) used a similar method for the esterification of carboxyl groups on peptide chains, and Wigglesworth (1952) and Fisher and Lillie (1954) have applied this technique to tissue sections. The latter authors are of the opinion that loss of metachromasia and basiphilia may be taken to indicate the loss of sulphate groups and the esterification of the carboxyl groups. Spicer and Lillie (1959) found that basiphilia returned at pH 3 to 4 and above, after saponification of the sections in 1% potassium hydroxide in 70% alcohol; and this, in their opinion, is indicative of the reversal of the esterification of the carboxyl groups. The Joss of reaction with NFRAL after methylation and its failure to return after saponification (its pH is 3-5) suggests that action depends upon the sulphate group. This is confirmed by the return of the reaction after sulphation and the appearance of a reaction after sulphation, in the ground substance of umbilical •cord, which, according to Moore and Schoenberg (1957), consists mainly of the unsulphated mucopolysaccharide, hyaluronic acid. The test-slides coated with pure chemical substances further confirm this opinion. However, the results with solochrome azurine suggest that, in the case of acid dyes at least, some other groups may be able to participate in the reaction. Saponification increases the intensity of staining with this dye, and it would appear that the carboxyl group and other groups not esterified by methylation are responsible. Giles (1944) suggested that hydroxyl groups chelate chromium ions in the mordanting of wool. Treatment of tissues with iodine and potassium permanganate (Scott and Clayton, 1953) has been used in the histochemical detection of SS and SH groups. Pearse (i960) is of the opinion that should oxidation be stronger, e.g. by performic acid, sulphinic (SO2H) and sulphonic (SO3H) groups will be formed. These are detectable by basic dyes in strongly acid solution, and form the basis of the performic acid / alcian blue techniques for the detection of cystine (Adams and Sloper, 1955-6). After oxidation with performic acid, hair-shafts and neurosecretory substance give a positive reaction with NFRAL. This reaction is reversible by methylation, and this suggests that sulphonic groups may give a positive reaction or that sulphate groups have been formed during the oxidation, and also that they need not necessarily be attached to mucopolysaccharides. Harms (1957) is of the opinion that ester sulphates play a part in the staining of elastic tissue; a view with which Pearse (i960) disagrees, and which does seem unlikely, in view of the fact that it is only with iron salts that this reaction is observed and that methylation has no effect upon it. Solochrome azurine is an acid triphenylmethane dye (Neal, 1961), which on chelation with aluminium forms a deep red-purple lake. This colour change has been used for the histochemical detection of aluminium (Pearse, 1957). It can be deduced from the experiments with this dye that aluminium is 466 Heath—Method for sulphated mucopolysaccharid.es attached to all tissue elements in a fairly firm fashion (it resists washing in running water), and that the aluminium which is associated with sulphated elements has such a high afinity for basic dyes, that the dye is able to displace the chelated solochrome azurine from the aluminium in such positions. Solo- chrome azurine is not unique in that it changes colour when it chelates with aluminium. Anthroquinone dyes are also known to form dye lakes of different colours with such metals as calcium and chromium (Conn, 1953). However, it is known that many chelates are colourless or have absorption spectra similar to those of the constituents (Martell and Calvin, 1952). Consequently it may be that many dyes behave in the fashion of solochrome azurine, forming chelates with the metal although not changing colour. This would appear to be the case with light green SF and NFR in aluminium solution, in which a similar selectivity of staining occurs to that observed with solochrome azurine and methylene yellow. It would therefore appear that the aluminium is attached to the tissues, and that dyes may form chelates similar to solochrome azurine with the aluminium—acid dyes forming chelates in relation to all tissue elements, and basic dyes only in relation to sulphated elements. The factors involved in this reaction now appear to be (1) the tissues and the mode of attachment of the metallic ion to them, and (2) the final reaction of the dye with the tissue and attached metal. Tissues consist of a mixture of acidic and basic groups which, dependent upon position, relative numbers, and ionizing strength, cause any particular tissue to react as predominantly basic (acidophil) or acidic (basiphil). Generally speaking, according to Baker (1958), over the pH range of normal staining, tissues act in a fairly consistent fashion as either basic or acidic, irrespective of the pH of staining. He also lists as the more common acidic tissues, nucleoproteins, the ground substance of cartilage, cartilage, and some mucins, and as basic tissues, collagen, red blood-corpuscles, and eosinophil granules. The groups which give the tissues their characteristic nature are mainly carboxyl and amino in the proteins, and in addition phosphate and sulphate groups on certain other tissue constituents. Seki (1933) has found that the majority of dyes act either as basic or acid dyes over the usual pH range of staining, irrespective of the actual constituent groups. The degree of ionization of any particular dye will depend presumably upon the pH of the solution in which staining is performed and the nature and strength of the characteristic group. Thus we may assume that the tissues and dyes will behave in a predictable fashion regarding their relative states of ionization in any particular pH. To turn to the mode of attachment of the metal to the tissues, a great deal of information has been amassed from investigations of the action of mordants (mainly chromic, ferric, and aluminium salts) in the textile dye industry. Some of this is applicable to the staining of tissues in metallic salt solutions and will be discussed briefly along with work done on biological tissues. Wigglesworth (1952) has studied extensively the sites and modes of Heath—Method for sulphated mucopolysaccharides 467 attachment of iron to tissues in sections and is of the opinion that the iron is held by co-ordinate bonds formed between the hydroxyl of the carboxyl group of the peptide chains, which, depending uponpH of solution and position in the chain, will not be excessively ionized. When special care is taken to preserve the phospholipids in the tissues these substances also show uptake of iron. He suggests that the phosphate groups and the hydroxyls of the sugars of the nucleoproteins and phosphoproteins will form such links, particularly at low pH. Chromium and aluminium react in a slightly different fashion, being only lightly attached to cytoplasmic proteins and elastic tissue (Mollendorf and Tomita, 1926), which would give a more likely explanation of the staining reaction for elastic tissue noted with iron salts, rather than the possible presence of sulphate groups mentioned above. The affinity of iron for hydroxyl groups may also explain the staining of red blood-corpuscles with rhodamines, as these have a benzoic acid group attached in position X1 in the basic formula above and this may form a complex with the ferrous iron in the haemoglobin. Investigating the chrome mordanting of wool, Giles (1944) came to the conclusion that various groups on the wool protein molecule were capable of displacing the co-ordinated water or hydroxyl groups from round the chromium ion and forming chelates, thus attaching the metal to the wool. The groups involved were stated to be hydroxyl, amino, amido, and also S—S bonds which had become split to form SH groups. Those observations are confirmed by the solochrome azurine methylation experiments, which indicate that groups other than the sulphate group are capable of chelating aluminium and thus allowing further staining. Sandritter (1955), investigating biological tissues, was of the opinion that the acid groups of nucleic acids and certain mucosubstances form co-ordination complexes with chromium and aluminium more readily than cytoplasmic proteins, particularly at low pH. It, therefore, appears that aluminium is most probably attached to the tissues by means of co-ordinate linkages. Kantor and Schubert (1957), investigating the staining reaction of blocks of cartilage, noted that acid dyes could only stain the ground substance of cartilage if the charge upon the cartilage was removed—in their particular case by methylation. These observations have been confirmed, for aqueous solochrome azurine will stain the methylated ground substance of cartilage. This also suggests that the aluminium may either remove or neutralize the charge upon the cartilage ground substance in aluminium solution. As was noted, orange G even in strongly acid aqueous solution will stain in its normal fashion, whereas at high pH only eosinophils stain. If this is considered in the light of the present concept of tissue staining (Conn, 1953), one must assume that the dye is ionized, as the basic groups in the tissue undoubtedly will be. At high pH the dye will be more highly ionized, but ionization of the basic groups in the tissue will be depressed. Orange G owes its acid nature to the sulphonic group, which is strongly acid. Eosin B, on the contrary, owes its acid nature to the carboxyl group, which is a more weakly acid, and is evidently not ionized at pH 1 to 3 in aqueous solution. At pH 3 to 5 it will be more strongly 468 Heath—Method for sulphated mucopolysaccharides ionized and staining results, but at pH 5 to 9 ionization of the basic groups of the tissues will be suppressed and again little or no staining results. In aluminium solution orange G stains all elements at all pH values. However, with eosin B at low pH only sulphated elements stain weakly. With rising pH a gradual increase in intensity of staining of all elements occurs. The experiments with orange G suggest that the charge on the cartilage has been removed or neutralized and those with eosin B that with acid dyes at least, ionization of the dye has little effect on the staining of sulphated elements, but appears, to play a part in the staining of other tissue elements. Observations on the importance of electrostatic forces in staining were made by Neal (1947), who noted that such forces were active over distances of 100 A°, whereas the forces involved in electrovalent change are only active over distances of 5 A0. Presumably the electrostatic forces either prevent or encourage the approach of dye ions to the tissues, or in this case the tissue /metal complex. The orange G experiments also suggest that amino groups in the tissues- play little part in the reaction, for the tissues stain in aluminium solution irrespective of the state of ionization of the amino groups. The sulphonic group in the dye appears to be of no particular importance in the reaction, as solochrome azurine does not contain this group, nor do many of the basic dyes which react. Why the un-ionized eosin B should chelate with the aluminium in relation to sulphated elements only at low pH is not known. It may be that a more stable complex is formed when aluminium is in relation to these elements. Ease of formation and stability of the final complex may also explain the displacement of solochrome azurine by basic dyes, and the preferential uptake of basic dyes in mixture of acid and basic dyes. Moeller (1952) lists various groups in order of ease of formation and stability of final complex, and it is interesting to note that the amino group is placed before the carboxyl group, for this suggests that the amino group might at least in part be responsible. Thus far the following situation appears likely. 1. The metallic ion attaches itself to all the tissue elements, almost certainly by co-ordinate bonds, since this occurs irrespective of the nature of the tissue. This is consistent with the present theories of mordanting. 2. The dye then attaches itself to this tissue metal complex, once again, in all probability, by means of co-ordinate bonds. The ability of the dye tO' form such links is influenced by the electrostatic forces present locally. Let us now consider the various tissue elements as they would be affected by such a mechanism, in acid solution, i.e. the condition prevailing in aluminium sulphate solution. Consider first the strongly acid groups such as sulphates. These will almost certainly be ionized, and, as a consequence, be negatively charged. Such a charge will attract the metallic ion and may hold it there by electrostatic forces or by formation of a co-ordinate bond with the sulphate group, or any other suitable group, e.g. the hydroxyls of the mucopolysaccharides, as is suggested by the results obtained after methylation, Heath—Method for sulphated mucopolysaccharides 469 with solochrome azurine. By whatever method the metallic ion is held there will tend to be a neutralization of charge, and, dependent upon the relative amounts of each of the two reactive groups, the net charge will be positive, negative, or neutral. If the charge is positive, basic dyes, which will be ionized in this particular solution, will be repelled. This does not happen. Similarly, if it was negative, acid dyes would be repelled if ionized, although un-ionized acid dye molecules could still form co-ordinate links. The orange G experiments show that even at pH 1 orange G is ionized to a certain extent, and consequently it is unlikely that the charge is negative. It is most probable neutral. This supports the suggestion that the final mode of attachment to the metal is by chelation, since it is the only method by which both positive and negative ions may attach themselves to a single element, namely aluminium. In this particular case the removal of the sulphated groups by methylation would leave the tissues in a highly positive state after the attachment of the aluminium, which has been shown by the methylation and solochrome azurine experiments to occur, presumably by co-ordinate links with other groups as suggested above. These groups would not be of sufficient strength to neutralize the charge on the aluminium, and the tissue/metal complex would be positively charged, consequently repelling basic dye ions, and block- ing the reaction. Acid ions, however, would be attracted and could consequently stain in the fashion noted. Consider next the weaker acid groups such as phosphate and carboxyl. These will presumably be less highly ionized, and in addition they do not occur in such high concentrations as sulphate groups, e.g. there are 5 or 6 sulphate groups on each repeating unit of the heparin molecule (Foster and Huggard, 1955). However, the metallic ion will be attracted to them and may form co-ordination complexes with these groups, or as suggested above, with other groups in the molecules to which they are attached. In all probability the resultant electrostatic charge will be positive owing to the higher charge on the metallic ion and the relatively low concentration of negative tissue charges. This will repel basic dye ions, but attract acid dye ions or have no effect upon un-ionized acid dye molecules under the con- ditions of pH assumed above. That the forces linking the metal to the tissues are weaker in this particular case is suggested by the fact that if the dye concentration is raised, relatively to the metallic ion, to a sufficiently high level the chromatin will stain, presumably according to the law of mass action, the metal being displaced from the reactive groups in the tissue. This supports the view that at least in part the mode of attachment is governed by electrostatic forces. To turn to acidophil (basic) tissue elements, these will have a positive charge, presumably due to amino group, which will tend to repel the metallic ions. However, as has been shown by the experiments with orange G and eosin B, the amino groups appear to have very little influence upon the reaction even at low pH, presumably on account of their position and numbers. In addition the aluminium ion has definitely been shown by the solochrome azurine experiments to be present on all tissue elements and it appears therefore that these groups do not in fact have any significant influence on the 470 Heath—Method for sulphated mucopolysaccharides attachment of the metal to the tissues. In fact, Giles (1944) is of the opinion that un-ionized amino groups co-ordinate with chromium. Once again the resultant charge on the tissues is likely to be positive, and so will repel basic dye ions and attract acid dye ions, as is observed. It is interesting to note that acid dyes stain much more deeply in aluminium than in aqueous solution. Ideally, confirmation of this theory might be sought in the use of an amphoteric dye buffered at various pH levels. Unfortunately, when such dyes as gallamin blue and gallocyanin form lakes with metals, they behave as basic dyes (Baker, 1958). The results obtained with this particular set of experiments confirm this. On the basis of the above theory this reaction need not be specific for sulphate groups, since theoretically any other strongly acid group in an ionizable state in the tissue would give a positive reaction. It may be, therefore, that just as the sulphated mucopolysaccharides are the only naturally occurring substances with a sufficiently high concentration of sulphate groups, so the sulphate group may be the only acid group of sufficient strength present in the tissues. However, it is known that certain ion-exchange resins of sulphonic acid type are capable of co-ordinating metallic ions in an extremely stable fashion and may only be removed by concentrated acid of the same type. Consequently it may be that the specificity is due to this property of the sulphate group rather than its strength as an acid.

I wish to express my thanks to Dr. Golberg (Benger Ltd.), Mrs. S. M. Neal (I.C.I.), Dr. Riding (Evans Medical Ltd.), and Dr. D. M. Sheppherd for supplying dextran, solochrome azurine, the heparins, and the sulphated polyglucose respectively. I am also indebted to Professor R. E. Coupland and to Dr. R. Jameson for many helpful discussions, to the members of the technical staff for their generous help, and to Miss E. Trueland and Miss S. Allton for typing the manuscript. References ADAMS, C. W. M. and SLOPER, J. C, 1955-6. J. Endocrinol., 13, 221. BAKER, J. R., 1958. Principles of biological microtechnique. London (Methuen). BENDITT, E. P., WONG, R. L., ARASE, M., and ROEPER, E. 1955. Proc. Soc. exp. Biol. Med., 9O. 3°3- CHARLES, A. F., and TODD, A. R., 1940. Biochem. J., 34, 112. CONN, H. J., 1953. Biological stains, 6th ed. Geneva (N.Y.). (Biotech. Pub.) COUPLAND, R. E., and HEATH, I. D., 1961. J. Endocrinol. 32, 71. and RILEY, J. F., i960. Nature, 187, 1128. FISHER, E. R., and LILLIE, R. D., 1954. J. Histochem. Cytochem., 2, 81. FOSTER, A. B., and HUGGARD, A. J., 1955. Advances in carbohydrate chemistry, 10, 335. New York (Academic Press). FRAENKEL-CONRAT, H., and OLCOTT, H. S., 1945. J. biol. Chem., 161, 259. GILES, C. H., 1944. J. Soc. Dyers and Col., 60, 303. GILES, R. B., and CALKINS, E., 1955. J. clin. Invest., 34, 1476. GRILLO, T. A. I., and LEWIS, P. R., 1959. J. Anat., 93, 581. GURR, E., i960. Encyclopaedia of microscopic stains, London (Hill). HALE, C. W., 1946. Nature, 157, 802. Heath—Method for sulphated mucopolysaccharides 471 HARMS, H., 1957. Acta Histochem., 4, 314. HEATH, I. D., 1961. Nature, 191, 1370. HOLMGREN, H., and WILLANDER, O., 1937. Z. Zellforsch mikr. Anat., 42, 242. KANTOR, T. G., and SCHUBERT, M., 1957. J. Histochem. Cytochem., 5, 28. LAGUNOFF, D., LAM, K. B., ROEPER, E., and BENDITT, E. O., 1957. Fed. Proc, 16 (1552), 363. LUBS, H. A., 1955 (ed.). The chemistry of synthetic dyes and pigments. New York (Rhein- hold). MARTELL, A. E., and CALVIN, M., 1952. Chemistry of the metal chelate compounds. Englewood Cliffs (N.J.) (Prentice Hall). MEYER, K., 1955-6. Harvey lectures, 1935-56, 88. New York (Academic Press). MOELLER, T., 1952. Inorganic chemistry, an advanced textbook. New York (Wiley). MOLLENDORF, W., and TOMITA, T., 1926. Z. Zellforsch. mikr. Anat., 3, 1. MOORE, R. D., and SCHOENBERG, M. G., 1957. A.M.A. Arch. Path., 64, 39. MOWRY, R. W., 1956. J. Histochem. Cytochem., 4, 30.7 NEAL, S. M., 1947. J. Soc. Dyers & Col., 63, 368. 1961. Personal communication. PEARSE, A. G. E., 1957. Acta Histochem., 4, 95. i960. Histochemistry, theoretical and applied, 2nd ed. London (Churchill). PERCIVAL, E. G., 1949. Quart. Rev. chem. Soc, 3, 369. QUINTARELLI, G., TSUIKI, S., and HASHIMOTO, Y., 1961. J. Histochem. Cytochem., 9, 176. RILEY, J. F., and WEST, G. B., 10.56. A.M.A. Arch. Dermatol., 74, 471. SANDRITTER, W., 1955. Zeit. wiss. Mikr., 62, 283. SCOTT, H. R., and CLAYTON, B. P., 1953. J. Histochem. Cytochem., 1, 336. SEKI, M., 1933. Z. Zellforsch. mikr. Anat., 18, 1. SPICER, S. S., SWARM, R. L., and BURTNER, H. J., 1961. Lab. Invest. 10, 256. and LILLIE, R. D., 1959. J. Histochem. Cytochem., 7, 123. STEEDMAN, H. F., 1950. Quart. J. micr. Sci., 91, 377. STACEY, M., 1946. Recent advances in carbohydrate chemistry, 2, 161. New York (Academic Press). WIGGLESWORTH, V. B., 1952. Quart. J. micr. Sci., 93, 105.

Kk 472 Heath—Method for sulphated mucopolysaccharides Appendix The following symbols are used in the tables: Fixatives 1. Formaldehyde saline (pH 3-8). 7. Zenher's fluid (pH 2-3). 2. Formaldehyde dichromate (pH 6). 8. Acetic formaldehyde alcohol (pH 3). 3. Formaldehyde calcium (pH 5-8). 9. Huber's fluid (pH 1-3). 4. Formaldehyde mercuric (pH 2 ••£). 10. Bouin's fluid (pH 1-9). 5. Helly's fluid (pH 4-1). 11. Lead subacetate 3% in 10% formalin 6. Formaldehyde at pH 1 to 9. (pH 5-8). A = acid. M = metachromasia (presence indicated Am = amphoteric. by+). B = basic. S = specificity for sulphated elements. SMP = sulphated mucopolysaccharides.

TABLE I Experiments on fixation and differences between species

Reference for Species and tissue Fixatives SMP Results with NFRAL Man skin 1, 2, 3 4. Meyer, 1955-6 mast cells +, epidermis + peptic ulcer 4 — mast cells +, mucus 0 colon 4 — appendix 4 — jejunum 4 — uterus, body I. 4, 8 9 — Imast cells +, mucus in cervix-f- but I varies to some extent uterus cervix I, 4. 8, 9 uterine tube 4 — mast cells + prostate 4 — mast cells + pituitary i. 5 — mast cells in posterior lobe + aorta 3. i — medial coat + secondary amyloid 4 Giles and Calkins nil (no metachromasia with toluidine of liver 1955 blue) adrenal I, 2, 3 — mast cells + umbilical cord 4 Moore and mast cells +, also some of blood-vessels Schoenberg, and occasional faint background stain- 1957 ing. tongue 10 — mast cells + Horse lip *| S — mast cells + liver capsule i, S — mast cells + Ox duodenum , 2, 3 mast cells + pancreas i 2, 3 — mast cells 4- liver capsule i 2, 3 4. S, — mast cells + 7, 8, 9, i°.

adrenal , " 3 mast cells •+• trachea , 2, 3 — mast cells + tendon . 2, 3 Meyer, 1955-6 tendon reacts synovial membrane , 2, 3 — mast cells only react pituitary i 2. 3 — mast cells in post pituitary + only salivary gland , 2, 3 Quintarelli and mast cells only + others, 1961 Dog skin I. 2 — mast cells + cartilage I, 2 Meyer, 1955-6 ground substance of cartilage + liver , 2 — mast cells only react Heath—Method for sulphated mucopolysaccharides 473 TABLE I (cont.)

Reference for Species and tissue Fixatives SMP Results with NFRAL Pig duodenum I) 2 Meyer, 1955-6 mast cells only react Sheep duodenum I-II mast cells only -f- pancreas I, 2, 3 — mast cells +, some mucins faint + stomach I. 2, 3 no mast cells seen, mucus 0 trachea I. 2, 3 ground substance of cartilage, mast cells and some mucins + thyroid I, 2, 3 mast cells + uterine tube I. 2, 3 — mast cells 4- tendon I. 2, 3 tendon + synovial membrane I. 2, 3 ,— nil + pituitary i, 2, 3 — nil + salivary gland I, 2. 3 mast cells only + adrenal I, 2, 3 — mast cells + Rat jejunum I — no mast cells seen spleen I, 2 — nil + brain I — nil + skin all except 6 — mast cells and epidermis -4- lung all except 6 — mast cells + liver all except 6 — mast cells + omentum all except 6 — mast cells + bone and cartilage I, 2, 3 — faint reaction in bone, definite + in cartilage testis i. 2» 3 mast cells + adrenal 1,2, 3 — mast cells + Guinea-pig duodenum I, 2, 3 — /only very stomach i> 2, 3 occasion al mucus of Brunner's gland + 1 colon », 2, 3 _ nil else mast cells jejunum I, 2, 3 — I seen pancreas I, 2, 3 — (only skin I, 2, 3 — epidermiv s + occasional adrenal I, 2, 3 I mast cells Ueen Hamster cheek pouch x> z — mast cells + Rabbit skin I, 2, 3 nO mast groune d substance + ( ., cartilage I. 2, 3 \ cells seen Bat tongue 4 — mast cells + Mouse skin I, 2 — epidermis faint +, mast cells + cartilage I, z — ground substance + Chick skin I, 2, 3 — mast cells + bone I, 2, 3 — faint + cartilage I, 2, 3 — ground substance + c.n.s. I, 2, 3 — — Toad skin 4 — mast cells + Frog skin I, 2, 3 mast cells + 474 Heath—Method for sulphated mucopolysaccharides

TABLE 2 Various dyes in 5% aluminium sulphate and in simple aqueous solution

Dye M' Aluminium Aqueous Remarks Azo 1. Bismark brown B1 _ 5 nuclei and s cell mucins 2. chrysoidin T B — s 3. Janus green B _ s 4. Janus black B _ 5. orange G A - all elements s SMPs8 do not see section on pH experiments stain stain 2 6. ponceau 2R A — M 7. ponceau S A - „ ,, Quinone imine 8. thionin B — precipitate nuclei and dyes no. 10-14 and 18-22 are all mucins recommended along with other members of the methylene series (see Conn 1953) 9. azure C B — „ 10. azure A B + S 11. azure B B S 12. methylene blue B _ s (( nos. 20 and 21 being best for general use 13. toluidine blue B + tt 14. brilliant cresyl s blue B f> 3 + s 15. gallocyanin AM + s )( see pH experiments 16. gallamin blue AM + s tl 17. celestin blue B + s )} dyes 14-19 gave specificity in S% Cu SO, 18. Nile blue B _ s 19. cresyl fast violet B _ s "t 20. neutral red B _ s si. nuclear fast red B _ s "t 22. safranin O B + s 23. azocarmine G A _ all elements SMPs'do stain not stain Triphenyl methanes 24. basic fuchsin B — S nuclei and all differentiate out very rapidly all mucins alcohol; Fe3+ and Als+ salts improve this to a certain extent. 25. brilliant green B _ S 26. crystal violet B S " 27. Hoffmann's violet B _ S — 3+ 28. magenta II B — S () stains elastic tissue in Fe salts 29. malachite green B _ S (l 30. methyl green B + S tt 31. methyl violet B — s lt 32. night blue B — s 33. pararosanilinc B — s stains elastic tissue in ferric salts 34. rosaniline B — s |( 35. Victoria blue B B — s 36. Victoria blue 4R B _ s 37. acid fuchsin A _ all elements SMP do not stain stain 38. aniline blue A — 39. fast green A — )( 40. light green S.F. A — 41. methyl blue A - „ „ Xanthines 42. acridine orange B — S nuclei and all mucins Heath—Method for sulphated mucopolysaccharides 475 TABLE Z (cont.)

Dye Aluminium Aqueous Remarks 43. acridine red B + S nuclei and all mucins 44. acriflavine B - S 45. pyronin B. B — S 46. pyronin G (Y) B — s 47. rhodamine B. B stains red blood-cells in addion — s J 48- rhodamine 3^-*O B — s similar results to those in A1 + were obtained in 10% Fes+ with 47-50 40. rhodamine 69X B — s „ 50. rhodamine S B + s 51. eosin B A - all elements SMPs do not see pH experiments stain stain S2. eosin Y A - precipitate SMPs do not stain 53. fast acid blue A - all elements ,, stain Miscellaneous dyes 54. alican blue B — S nuclei and all staining much improved with mucins Fe3+ 55. alcian green B + S 56. thioflavine T B — S () 57. alizarin red S A — all elements SMP do not forms bright red lake with Al3+ stain stain

TABLE 3 Staining with NFR in various salt solutions

Salt Specificity Cone, tv/v Salt Specificity Cone, ic/v aluminium chloride + 5 ferric chloride + - aluminium sulphate + 5 ferric sulphate + 10 ammonium alum + 10 ferrous sulphate + 10 ammonium sulphate — 10 lithium carbonate precipitate 5 chrome alum + 10 magnesium sulphate + 5 ferric alum -1- 10 manganese sulphate + 10 potassium alum + S mercuric chloride + si. 5 precipitate barium chloride only faint 5 mercurous sulphate + 5 staining cadmium chloride + 5 nickel sulphate + 10 calcium chloride + 5 lead nitrate + 5 calcium nitrate + 5 potassium chloride no staining — chromic sulphate (green) - 10 silver nitrate + sl. 5 precipitate chromic sulphate + 10 sodium chloride no staining — (purple) 5 cobaltous sulphate + 10 stannic chloride + cobaltous nitrate + 10 stannous chloride + 5 cuprous chloride precipitate 5 zinc chloride + 5 cupric sulphate + 10 zinc sulphate +