Deamination by the diazotization-deazotization method

Synopsis The diazotization-deazotization method for deaminating proteins in fixed tissue section, as originally described by Stoward (1963), has been investigated further with the following results. Blocking of arginine was confirmed by the Sakaguchi method and with anionic dyes. What appeared to be staining of lysine by anionic dyes was blocked but the dinitrofluorobenzene reaction was not. Blocking of histidine could not be demonstrated. Tyrosine was blocked to Millon's reagent but not to the coupled tetrazonium reaction. Staining of sulphydryl groups by Barrnett & Seligman's dihydroxydinaphthyldisulphide method was reduced but not obliterated. The colour of the reaction product between tryptophan residues and dimethylaminobenzaldehydenitrite was altered. Explanations for these findings are suggested.     

The histochemical specificity of High Iron Diamine—Alcian Blue

Summary The specificity of the High Iron Diamine—Alcian Blue pH2.5 (HID—AB 2.5) procedure was examined in tissue sites containing sialogycoproteins alone or differing proportions of sialo- and sulphosialoglycoproteins. Studies with HID in differing final concentrations of hydrochloric acid or sodium chloride confirmed that staining is dependent upon both the pH and the ionic strength of the dye bath and demonstrated a marked heterogeneity in the pKa of the anionic groups of sialosulphoglycoproteins. Use of the sequence High Iron Diamine—Alcian Blue pH 1.0 demonstrated that complete or almost complete staining ofO-sulphate esters occurred when HID was prepared in water (final pH 1.3). However, under these conditions HID—AB 2.5 was shown to be non-specific because only black HID staining was observed in sites containing large quantities of sialic acids. This non-specificity was due either to the masking of Alcian Blue staining by HID and/or the black HID staining of anionic groups other than sulphate. These results may account for some of the conflicting data obtained by different groups of investigators who have studied transitional mucosa in human colonic diseases. Caution should be used in drawing conclusions from the use of HID—AB 2.5 without confirmatory evidence from other more specific procedures.     

Observations on the use of diazo-I-H-tetrazole and phenylglyoxal in protein histochemistry as applied to human gingiva

Synopsis A solution of diazo-1-H-tetrazole, freshly prepared by the diazotization of 5-amino-1-H-tetrazole under conditions to avoid explosion, was adjusted to pH 8.8, diluted (11 or 19) with 0.67 M bicarbonate buffer, pH 8.8, and used immediately as a histochemical reagent for demonstrating histidine, tryptophan, and tyrosine residues in deparaffinized sections of frozen-dried human gingiva, rat abdominal skin, and mouse larynx fixed in modified Newcomer's solution. Diazo-1-H-tetrazole reacted histochemically like other diazonium coupling reagents in common use, except that in sections pretreated with bromoacetic acid at pH 7, diazo-1-H-tetrazole staining was increased, rather than decreased as expected. Pretreatment with bromoacetic acid also increased staining in gingival sections exposed to an acetic anhydride-pyridine mixture and then reacted with diazo-1-H-tetrazole. Similarly, pretreatment with bromoacetic acid increased the intensity of Millon's reaction in gingival sections. Sections of human gingiva or mouse larynx pretreated with diazo-1-H-tetrazole stained less intensely with Biebrich Scarlet used respectively at pH 2.62 and 6.50.In test-tube experiments to check the specificity of diazo-1-H-tetrazole for amino acids, only histidine, tryptophan, and tyrosine gave solutions with colours visually distinguishable from the buffer blank. In similar tests a solution of ribonuclease A gave a colour like that given by histidine and tyrosine. Whereas pyridine failed to yield a colour with undiluted diazo-1-H-tetrazole reagent in test-tube experiments, gingival sections exposed to pyridine for 24 hr stained more intensely with diazo-1-H-tetrazole, but diazo-1-H-tetrazole staining of abdominal skin sections was not altered by prior treatment with pyridine.Phenylglyoxal, used as a 1.5% w/v solution inN-ethylmorpholine-acetate buffer (0.2 M acetate) pH 8, blocked the Sakaguchi reaction in human gingival sections. Pretreatment with phenylglyoxal also led to a reduction in their staining by Biebrich Scarlet at pH 2.62, dinitrofluorobenzene, or diazo-1-H-tetrazole. In addition the dimethylaminobenzaldehyde nitrite reaction for tryptophan was reduced. Phenylglyoxal blockade of arginine residues in gingival sections was labile to 1% acetic acid containing 0.05 M choline chloride after 60 min; but in test-tube experiments extending over 320 min, di(phenylglyoxal)-l-arginine hydrochloride was more stable in this acetic acid-choline solution than in water. It is suggested that after treatment of gingival sections with bromoacetic acid at pH 7.0, additional tyrosine residues become available for reaction with diazo-1-H-tetrazole. Pyridine is thought to remove bound lipid from gingival epithelium, thereby exposing protein residues reactive with diazo-1-H-tetrazole. The use of diazo-1-H-tetrazole and phenylglyoxal for characterizing amino acid residues of gingival proteins responsible for anionic dye binding is discussed.     

Polyacrylamide films as a tool for investigating qualitative and quantitative aspects of the staining of glycosaminoglycans with basic dyes

Synopsis With the introduction of model films of polyacrylamide gel into which purified glycosaminoglycans (GAGs) have been incorporated, the direct recording of metachromatic spectra with virtually no interference of the corresponding orthochromatic peaks has become possible. Because this model system yields situations comparable to those of stained sections under the microscope, it is well suited for investigating qualitative and quantitative aspects of histochemical staining procedures. Previous model experiments have shown that under aqueous conditions only minor differences can be observed between the metachromatic peaks of different GAGs complexed with a suitable dye (e.g. Toluidine Blue O, Thionin, Safranin O, Cresyl Violet, Crystal Violet). In non-aqueous media, such as glycerol and ethylene glycol, the complexes with Toluidine Blue O revealed a special pattern for heparin, having a metachromatic peak (517 nm) about 30 nm lower than that of all other GAGs. This observation has formed the basis of a method for the qualitative microspectro-photometric detection of heparinin situ which was worked out by combining model film experiments with microspectrophotometric data obtained from rat mast cells. Since only a limited number of cells is necessary for obtaining reliable data with this method, the presence of heparin in the cytoplasmic granules of normal human mast cells and basophilic granulocytes could thus be proved directly.Alcian Blue 8GX, another basic dye frequently used in GAG histochemistry, has also been investigated with polyacrylamide films. In contrast to the metachromatic dyes, the rate of staining with Alcian Blue depends to a large extent on the rate of penetration of the dye into the model films. The rate of penetration is also a phenomenon of great importance for dye bindingin situ, where complex basic protein molecules may form a barrier for the Alcian Blue molecules. The model film studies performed so far have yielded conditions that provide maximal staining (up to an optimal level) and a linear relationship between the concentration of GAG and the AB binding. The presence of basic protein, electrostatically bound to the GAG, was not found to influence either the rate of staining or the maximal amount of dye binding.Paper presented at a symposium The Changing directions of carbohydrate histochemistry, at the fifth International Congress of Cytochemistry and Histochemistry in Bucharest, Romania on 1 September 1976.     

The role of phosphotungstic and phosphomolybdic acids in connective tissue staining I. Histochemical studies

Synopsis An investigation of the role of phosphotungstic and phosphomolybdic acids in Mallory-like trichrome methods showed unexpectedly that, rather than acting as mordants to anionic dyes, these polyacids selectively blocked staining of all tissue components other than connective tissue fibres to Aniline Blue and other similar fibrereactive dyes. Connective tissue components were found to contain residues resembling histidine that are easily accessible to anionic dyes. Blocking towards typical anionic dyes for demonstrating plasma proteins, such as Biebrich Scarlet, was also demonstrated but was less complete. The blockade of both types of dye was labile if the staining times were extended; plasma dyes were more sensitive than fibre dyes in this respect. Histochemical reactions for tyrosine residues were blocked. In connective tissue, phosphotungstic acid did not block histidine residues demonstrable by the coupled tetrazonium reaction with previous iodination. Thus it is postulated that differential trichrome staining occurs by binding of Aniline Blue to basic residues in the connective tissue not blocked by phosphotungstic acid and subsequent replacement of the blocking agent by an anionic dye. The binding of phosphotungstic acid to both epithelium and connective tissue was demonstrated by the quenching of autofluorescence in these regions and by the reduction of the bound PTA to blue coloured products with titanium trichloride.     

Histochemistry of protein-bound disulphide groups in the duct secretory granules of the rat submandibular gland

Synopsis The existence of disulphide groups in the granules of the secretory portion of the ducts of rat submandibular glands has been demonstrated with methods that reveal thiol groups formed after reducing the disulphide groups first. Disulphide groups were also demonstrated with cationic dyes by staining the cysteic acid residues obtained after oxidation with permanganate. Alcian Blue at pH 3.0 was used for this purpose. Two kinds of granules, characterized by their reactions with Alcian Blue at different pH levels, were apparent in differing stages of the same secretion.     

An alternative Alcian Blue dye variant for the evaluation of fetal cartilage

BACKGROUND: The most comprehensive evaluation of vertebrate skeletal development involves the use of Alizarin Red S dye to stain ossified bone and various other dyes to stain cartilage. The dye used most widely to stain fetal cartilage in rodents and rabbits is Alcian Blue 8GX. However, the global supply of this specific dye has been exhausted. Several forms of the dye marketed as Alcian Blue 8GX are now available, although they are not synthesized via the original 8GX manufacturing process. METHODS: One new Alcian Blue 8GX form and two Alcian Blue dye variants were evaluated in rats and rabbits using standard staining procedures. The staining quality of these dyes were evaluated relative to the original form of Alcian Blue 8GX based on cartilage uptake of the dye, clarity of the cartilaginous components, staining intensity of the dye, and overall readability of the specimens under stereomicroscopic evaluation. RESULTS: Staining with the newer form of Alcian Blue 8GX resulted in poor staining quality. The Alcian Blue-Pyridine variant performed well, although staining intensity was less than optimal. The Alcian Blue-Tetrakis variant provided staining characteristics that were most similar to the original form of Alcian Blue 8GX. CONCLUSIONS: Alcian Blue-Tetrakis was markedly better in its ability to stain fetal cartilage than the newer form of Alcian Blue 8GX.     

The Alcian Blue and combined Alcian Blue-Safranin O staining of glycosaminoglycans studied in a model system and in mast cells

Synopsis Polyacrylamide films containing different glycosaminoglycans have been applied to the study of the Alcian Blue and combined Alcian Blue-Safranin O staining procedures. It was found that the polyacrylamide matrix can be interpreted as some kind of barrier around the substrate molecules, a situation which can be compared to a certain extent with what occursin situ, where complex protein molecules can likewise form a barrier.The Alcian Blue staining of the model films was found to follow the Lambert-Beer law. The time to reach optimal dye binding depended on the concentration of the glycosaminoglycan enclosed in the model films and on the concentration of Alcian Blue in the dye solution. Lowering the pH of the dye solution appeared to increase the rate of staining. Optimal staining of model films in the presence of salt or urea was not possible, because under these conditions the pores of the polyacrylamide matrix became blocked. Alcian Blue was found to bind irreversibly to the glycosaminoglycan molecules enclosed in the polyacrylamide films.The results of the combined Alcian Blue-Safranin O staining applied to model films appeared to be highly dependent on the amount of Alcian Blue bound to the glycosaminoglycan in the first step of the double staining procedure. No specific differences were noticed between the behaviour of the different glycosaminoglycan-Alcian Blue complexes towards the Safranin O binding in the mext step. As the theoretical basis for the application of the combined Alcian Blue-Safranin O staining was also found not to be completely valid, the conclusion was reached that this double staining cannot be used for the histochemical identification of glycosaminoglycans. The colour retained by a certain glycosaminoglycan-containing part of the specimen only delivers information about the accesibility of that part for Alcian Blue.     

On the alcianophilia of the drug suramin used as a tool for inducing experimental mucopolysaccharidosis

The trypanocidal drug suramin was previously reported to induce mucopolysaccharidosis in rats; apart from the biochemical demonstration of increased tissue concentrations of sulfated glycosaminoglycans (GAGs), a strongly positive staining reaction with the cationic dye Alcian Blue was taken as indicating GAG-storage (Constantopoulos et al. 1983). The purpose of the present report is to point out a methodical pitfall. In model experiments it was found that suramin itself, being a polysulfated compound, gives a strongly positive reaction with Alcian Blue at pH 1. It is known that suramin is accumulated in the lysosomes and that high drug concentrations are retained in the tissues for weeks. Therefore a positive staining reaction with Alcian Blue observed in a given cell cannot be conclusively attributed to the storage of sulfated GAGs as has been done in the past. The present report may be a warning that, in the case of the suramin-induced animal model of mucopolysaccharidosis, the usual histochemical strategy, i.e. staining with cationic dyes, is not suitable for analysing the cellular distribution pattern of GAG-storage, since the inducing drug by itself reacts with the indicator dye.     

Nuclear stains with soluble metachrome metal mordant dye lakes

Summary Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe²⁺ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO₄ sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control.Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe²⁺ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results.Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes.The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes¹. Benzil at pH 13, which prevents the -naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.Assisted by Contract Nol-CB-43912 National Cancer Institute     

The effect of pH on Alcian Blue staining of epithelial acid glycoproteins. I. Sialomucins and sulphomucins (singly or in simple combinations)

Synopsis This study is concerned with the staining of epithelial acid glycoproteins by Alcian Blue at various pH levels. A detailed analysis of the effect of pH on Alcian Blue staining of epithelial tissues at selected sites was made. Alcian Blue was combined with the periodic acid-Schiff technique, the Alcian Blue being used at pH levels between 2.6 and 0.5.Animal salivary glands, human foetal tracheal gland and epithelial goblet cells of the adult bronchial mucosa were chosen for study because the nature of their acid glycoprotein is known and is relatively simple.In sites containing sialomucin alone, no Alcian Blue staining took place below pH 1.5. A difference was demonstrated between sialidase-sensitive sialomucin which stained only between pH 2.6 and 1.7, and sialidase-resistant sialomucin which stained between pH 2.6 and 1.5. Two types of sulphomucin were identified: the usual one stained with Alcian Blue at all the pH levels studied, and the other, in the canine gland, stained only at the most acid pH levels, that is, between pH 1.5 and 0.5.     

DIAZOPHTHALOCYANINS AS REAGENTS FOR FINE STRUCTURAL CYTOCHEMISTRY

This paper reports the synthesis of 14 diazophthalocyanins containing Mg, Cu, or Pb as the chelated metal. To assess the usefulness of these compounds for fine structural cytochemistry, the relative coupling rates with naphthols were tested as well as the solubility of the resulting azo dyes. Three of the diazotates were reacted with tissue proteins in aldehyde-fixed material, and the density increases thus produced were compared in the electron microscope with those produced by staining similarly fixed material with the phthalocyanin dye, Alcian Blue. Finally, one of the diazotates was used as a capture reagent for the demonstration of the sites of acid phosphatase activity with the electron microscope.     

On the alcianophilia of the drug suramin used as a tool for inducing experimental mucopolysaccharidosis

Summary The trypanocidal drug suramin was previously reported to induce mucopolysaccharidosis in rats; apart from the biochemical demonstration of increased tissue concentrations of sulfated glycosaminoglycans (GAGs), a strongly positive staining reaction with the cationic dye Alcian Blue was taken as indicating GAG-storage (Constantopoulos et al. 1983). The purpose of the present report is to point out a methodical pitfall. In model experiments it was found that suramin itself, being a polysulfated compound, gives a strongly positive reaction with Alcian Blue pH 1. It is known that suramin is accumulated in the lysosomes and that high drug concentrations are retained in the tissues for weeks. Therefore a positive staining reaction with Alcian Blue observed in a given cell cannot be conclusively attributed to the storage of sulfated GAGs as has been done in the past. The present report may be a warning that, in the case of the suramin-induced animal model of mucopolysaccharidosis, the usual histochemical strategy, i.e. staining with cationic dyes, is not suitable for analysing the cellular distribution pattern of GAG-storage, since the inducing drug by itself reacts with the indicator dye.     

Investigations of dye binding in the sequential staining of mucosaccharides by Alcian Yellow-Alcian Blue. 1. Histochemical experiments on different animal and human mucosaccharides

Synopsis It may be assumed that, histochemically, carboxyl groups and sulphate half-ester groups of muco electrolyte concentration of the dye baths in the two steps of a sequential Alcian Yellow-Alcian Blue method. In the present study the specificity and reliability of this method has been investigated.When the staining conditions were the same in both steps, the second dye (Alcian Blue) was found to stain mucosubstances in spite of the prior staining with Alcian Yellow. Binding of Alcian Blue was observed in all but very dilute Alcian Blue solutions. The degree of Alcian Blue binding depended on the dye concentration and staining time of the second step (Alcian Blue), and it varied widely for different mucosubstances. Although an incomplete saturation of anionic groups with dye molecules in the first step cannot be completely excluded, it is thought that Alcian Blue displaces Alcian Yellow from the carboxyl and sulphate groups of mucosubstances in tissue sections.It seems that the sequential Alcian Yellow-Alcian Blue method, under the conditions investigated, does not provide a reliable means for differentiating carboxyl and sulphate groups of mucosbstances in tissue sections simultaneously, because the second dye obviously is capable of displacing the first dye from sulphate groups. However, it is possible to distinguish non-sulphated acid mucosaccharides from sulphated mucosaccharides.     

Histochemical evidence of a functional heterogeneity of the chondrocytes of adult canine articular cartilage

Synopsis Twenty humeral heads were collected from 10 adult English Pointer dogs, fixed in 15% formalin containing cetylpyridinium chloride, decalcified, processed for paraffin sections, and cut serially. The articular cartilage was studied by staining principally with Alcian Blue in the presence of 0.4 or 0.9m MgCl₂ with and without a van Gieson counterstain. The results of the differential staining procedures demonstrated the existence of two groups of chondrocytes with distinctly different staining affinities. One group reacted intensely for the presence of protein-polysaccharides within its cytoplasm while the other group completely lacked this property. An approximate proportionality of 31 of the protein polysaccharide-positive and-negative chondrocytes was observed in the tangential layer and upper intermediate zone. In the lower intermediate zone, radiate zone, and zone of calcified cartilage, the chondrocyte types were present in equal proportions. Staining with Alcian Blue in the presence of 0.9m MgCl₂ with and without a van Gieson counterstain indicated a further subdivision of the protein-polysaccharide positive group of chondrocytes. This blocking technique has been reported to distinguish between chondroitin sulphate and high mol. wt. keratosulphate. Thus, based upon a greatly decreased number of the blue-stained chondrocytes after staining with Alcian Blue in the presence of 0.9m MgCl₂ the hypothesis is put forward that some chondrocytes produce primarily chondroitin suphate and others produce both chondroitin sulphate and keratosulphate.     

Histochemical staining of lymphatic anchoring filaments

Summary Lymphatic anchoring filaments are stained by means of histochemical methods that demonstrate disulfide-groups. Thiosulfation of sections either followed by aldehyde-fuchsin staining or by Alcian Blue +0.8 M MgCl₂ staining has been used. Lymphatic anchoring filaments display striking fine structural similarities to elastic fiber microfibrils and both kinds of fibers are characterized by disulfide content.     

Impurities and staining characteristics of Alcian Blue samples

Synopsis The composition of various samples of Alcian Blue^* and related dyes was studied, using t.l.c. (with cellulose as adsorbent andn-butanol: acetic acid: water as developing solvent), solvent extraction and precipitation, i.r. spectroscopy and classical semimicro analysis. All the Alcian Blue samples appeared to contain the same coloured components. The Alcian Green samples were mixtures of these blue components and Alcian Yellow. All the Astra Blue samples examined were composed of the same blue constituents. Colourless components identified were boricacid, dextrin and sulphate and sometimes amounted to nearly three-quarters by weight of the crude dyes. Impurities had only a slight effect on staining with Alcian Blue in aqueous acetic acid but appreciably affected staining by the critical electrolyte concentration (C.E.C.) procedure. Dextrin as impurity raised C.E.C. limits while the inorganic salt impurities raised the C.E.C. values of some substrates and lowered those of others.     

Some effects of salts on staining: Use of the Donnan Equilibrium to describe staining of tissue sections with acid and basic dyes

Summary Using a wide variety of acid and basic dyes, and dyebaths of various pH’s and salt contents, it was shown that various effects of neutral inorganic salts on the staining of tissue sections could be explained by the Donnan Membrane Equilibrium. Thus the simultaneous increases in staining of some tissue components and decreases in staining of others, which occur on adding salt to the dyebaths, are often explicable in terms of the Donnan Equilibrium. The variation in staining intensity with dye charge, seen with a number of acid dyes, also agreed with the predictions of the Donnan Equilibrium. The Donnan Equilibrium was applied to understanding the mode of action of various biological staining methods requiring salt in the dyebaths; namely the Best’s Carmine glycogen stain, the Alcian Blue and Congo Red methods for amyloid, and the salty periodic acid-Schiff procedure for anionic mucosubstances.     

The effect of pH on Alcian Blue staining of epithelial acid glycoproteins. II. Human bronchial submucosal gland

Synopsis The effect of pH on Alcian Blue staining of sialomucins and sulphomucins in human bronchial submucosal glands has been analysed. Using Alcian Blue combined with periodic acid-Schiff, lowering the pH was associated with a decrease in the area staining with Alcian Blue and an increase in that staining with periodic acid-Schiff, save in one bronchus with a large sulphomucin content, in which an increase in the area staining with Alcian Blue was found at pH1.0. In all bronchi, an increase in the intensity of Alcian Blue staining was found at this pH. Sialomucin sensitive to sialidase was found to lose Alcian Blue staining at a higher pH than sialomucin resistant to the enzyme. Some sulphomucins stained with Alcian Blue throughout the pH range studied and some only at the more acid pH levels. At pH1.0 Alcian Blue stained only sulphomucins, thus distinguishing them from sialomucins. Alcian Blue staining combined with the high iron diamine technique has enabled three sulphate groups to be identified: one stained with high iron diamine, the other two did not, and, of the latter, one stained with Alcian Blue at pH 2.6 and1.0, and the other only at pH1.0.     

Application of selected cationic dyes for the semiquantitative estimation of glycosaminoglycans in histological sections of articular cartilage by microspectrophotometry

Summary Selected commonly used cationic dyes, viz. Thionin, Safranin O, Toluidine Blue O, Dimethylmethylene Blue, Cuprolinic Blue, Cupromeronic Blue,N, N-Diethylpseudoisocyanine, and a modified PAS-method, and staining method, with a variety of alternative procedures, e.g., variation of pH, use of the critical electrolyte concentration method, and blocking reactions (methylation-saponification, carboxymethylation), were tested to select optimal staining procedures for the semiquantitative histochemical estimation of glycosaminoglycans by microspectrophotometry in sections of articular cartilage. The methods were carried out on 3 m-thick paraffin and 1 m-thick glycolmethacrylate sections of bovine articular cartilage. The staining intensity of the sections was measured from spots 25 m apart using a leitz MPV 3 microspectrophotometer, starting at the surface of the cartilage and ending up at the tidemark. The result was compared with the fixed-charge density graph determined from the adjacent articular cartilage.Of the dyes tested, Thionin and Safranin O proved to be excellent cationic dyes for the histochemical quantification of cartilage matrix proteoglycans, since the staining intensity curves showed a linear correlation (r=0.900–0.995) with the fixed charge density curves from the adjacent cartilage. Also, the stain distribution was consistently uniform across the sections. In 1 m-thick glycolmethacrylate sections, the Safranin O staining gradient showed almost perfect identity with the fixed-charge density curve. Cuprolinic Blue and Cupromeronic Blue combined with the critical electrolyte concentration technique were also useful for the microspectrophotometric assays of glycosaminoglycans, but the presence of metachromasia should be checked prior to the measurements. The reliability of blocking procedures for quantitative histochemical work was not convincing.