The quantitative measurement of Alcian Blue–glycosaminoglycan complexes

1. A simple new quantitative micro method was developed to study the interaction of the cationic dye Alcian Blue 8GX and acid glycosaminoglycans under different conditions. After washing with ethanol the precipitated Alcian Blue-glycosaminoglycan complex was dissociated in Manoxol IB solution and the amount of bound dye measured spectrophotometrically. 2. Reaction profiles of complex-formation were determined in the presence of different concentrations of MgCl(2) at pH5.8, and could be used to study the critical electrolyte concentrations of glycosaminoglycans. At least 50mm-MgCl(2) was required to produce maximum precipitation of, and maximum uptake of, Alcian Blue by standard glycosaminoglycans. Maximum uptake of Alcian Blue by glycosaminoglycans in the urine of a patient with Hurler's syndrome required the presence of 25-50mm-MgCl(2). 3. Under standard conditions of maximum interaction, calibration curves for the quantitative determination of a series of standard glycosaminoglycans in 20mul volumes were nearly linear over the range 1-10mug. 4. The technique was used to determine the molecular binding ratios of Alcian Blue to glycosaminoglycans under controlled conditions.     

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 histochemical specificity of Streptomyces hyaluronidase and chondroitinase ABC.

Synopsis Treatment of tissue sections with enzymes which degrade specific types of glycosaminoglycans should provide a means for localizing glycosaminoglycans in tissue sections. The feasibility of this technique was examined by utilizing endogenously labelled glycosaminoglycans in chick and quail embryos. Less than 8% of the total glycosaminoglycans appear to be lost non-specifically during fixation and dehydration. BothStreptomyces hyaluronidase and chondroitinase ABC degraded more than 90% of their respective substrates and demonstrated minimal non-specific extraction of other glycosaminoglycans. The selectivity of chondroitinase ABC for sulphated glycosaminoglycans was substantially increased by raising the pH of the incubation buffer to 8.6. At this pH, chondroitinase ABC degraded negligible amounts of hyaluronic acid. Use of bothStreptomyces hyaluronidase and chondroitinase ABC confirmed that embryonic hyaluronic acid binds Alcian Blue under conditions that were previously believed specific for sulphated glycosaminoglycans. We suggest that this may be due to the increased molecular weight of embryonic hyaluronic acid compared to the hyaluronic acid in adult tissues. The results presented suggest that treatment of adjacent sections with buffer, chondroitinase ABC at pH 8.6, andStreptomyces hyaluronidase and subsequent staining with Alcian Blue provides a method for localizing and quantitating glycosaminoglycans in tissue sections.     

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.     

Colocalization of TGF-beta 1 and collagen I and III, fibronectin and glycosaminoglycans during lung branching morphogenesis

The possible in vivo role of TGF-beta 1 in regulating various proteins of the extracellular matrix, including fibronectin, collagen I and III, and glycosaminoglycans, was examined by immunohistochemical methods during critical stages of lung morphogenesis in the 11- to 18-day-old mouse embryo. Sections of Bouin-fixed, paraffin-embedded whole embryos were exposed to polyclonal antibodies specific to synthetic peptides present in the precursor part of TGF-beta 1 (pro-TGF-beta 1), in the processed TGF-beta 1 (antibody CC), collagen I and III, fibronectin, followed by the PAP or ABC technique to visualize the location of the antibody. GAG were stained with Alcian Blue 8GX. Our results indicate colocalization of TGF-beta 1 expression and that of matrix proteins in the developing lung when branching morphogenesis (cleft formation) and tissue stabilization occur. The presence of TGF-beta 1 at the epithelial-mesenchymal interfaces of stalks and clefts at a time when matrix proteins can first be visualized in these areas, suggests a direct participation of the growth factor in the development of the basic architecture of the lung.     

Histochemical analysis of urea-unmasked glycosaminoglycans in the skin of the rat and mouse

Summary Histochemical analysis of urea-unmasked glycosaminoglycans has been performed in connective tissues of the rat and mouse skin by means of combined staining and enzyme digestion procedures. The staining procedures used were Alcian Blue pH 1.0, Alcian Blue pH 2.5, Aldehyde Fuchsin, periodic acid-Schiff (PAS), Alcian Blue pH 2.5-PAS, high iron diamine and low iron diamine methods. The digestive enzymes employed wereStreptomyces and testicular hyaluronidases, chondroitinases ABC and AC and keratanase. The results obtained indicated that the major components of the glycosaminoglycans in the connective tissues of the skin were hyaluronic acid, dermatan sulphate and chondroitin sulphate A and/or C, whereas the tissues were devoid of keratan sulphate.     

The histochemistry of Alcian blue

Summary Boric acid has been identified as a major component of Alcian blue 8GX, comprising 40–50% by weight of commercial samples. Several methods are outlined whereby Alcian blue 8GX may be freed of boric acid. The simplest involves differential precipitation of Alcian blue 8GX from aqueous solution with acetone.     

Investigations of dye binding in the sequential staining of mucosaccharides by Alcian Yellow-Alcian Blue. II. Spot tests and experiments on dye-mucopolysaccharide (glycosaminoglycan) precipitates

Synopsis The amounts of dye bound to mucopolysaccharide in the histochemical sequential staining Alcian Yellow-Alcian Blue method were determined by studying dye-mucopolysaccharide (glycosaminoglycan) precipitates in test-tube and spot test experiments. In the second step (Alcian Blue) of the method, previously bound Alcian Yellow was released into the staining solution and simultaneously the precipitate took up Alcian Blue. The amounts of Alcian Yellow released and Alcian Blue taken up varied for different mucosaccharides, and depended on the staining time of the second step (Alcian Blue) of the sequence, as well as on the concentration of dye and salt in the Alcian Blue solution. It is thought that, among other things, the Alcian Blue in solution displaces some of the bound Alcian Yellow. Some observations could be explained by the aggregation of dye molecules. The results were in agreement with previous histochemical observations.     

Ultrastructural architecture of proteoglycans in the glomerular basement membrane. A cytochemical approach

Rat kidneys were perfused with fixative solutions containing either a) a polycationic dye (Alcian blue 8 GX, Astra blue 6 GLL, cuprolinic blue, ruthenium red), b) a monocationic dye (safranine 0), or c) Alcian blue in the presence of a 0.3 M MgCl2 concentration. Whereas solutions of a revealed the glomerular basement membrane proteoglycans as particles or threads 60 nm apart and arranged in a reticular pattern, solutions of b and c demonstrated new morphological aspects of these molecules. They appeared as tiny filamentous structures, about 100 to 160 nm long, ordered in a network-like pattern with a mesh of about 60-nm width. The filaments displayed lateral branches about 20 nm apart and about 25 nm long, projecting within the meshes. We suggest that the filamentous structures are the protein core, and the branches are the glycosaminoglycans of proteoglycan molecules. Because of this arrangement the negatively charged sites of the glomerular basement membrane would lie closer to each other than previously assumed.     

Etiology of the developing eye in myelencephalic blebs (my) mice.

The etiology of the eye defects in myelencephalic blebs (my) mutant mice has been poorly understood for almost seventy years. Embryos from 9 to 14 1/2 days of gestation were subjected to Alcian blue 8GX staining for acidic glycosaminoglycan deposition in basement membrane structures of the developing eye in my stock and control specimens. In addition 12 day embryos were subjected to avidinbiotin-peroxidase labelling for laminin. At 9-9 1/2 days of gestation more Alcian blue positive extracellular matrix was found in the region between the optic vesicle and the overlying putative lens ectoderm in the my stock embryos. By 12 days, there was an irregular and lesser amount of deposition of glycosaminoglycans in the len's capsule and in the "inner limiting membrane" of the presumptive neural retina; however, the deposition of laminin appeared to be greater in the inner limiting membrane of the my eye. By 14 days, the damage to the eye in the my embryos can be quite extensive, and the deposition of glycosaminoglycans was very meager in this situation. It appears that irregular deposition of glycosaminoglycans in the extracellular matrix and possible increase in the amount of laminin in basement structures in my embryos indicate disruption of the normal histochemistry involved in the development of the eye. Altered histochemistry may in turn indicate changes in permeability between cells of the developing tissues which result in the blebbing.     

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.     

Histochemical analysis of extracellular matrix material during embryonic mouse lens morphogenesis in an aphakic strain of mice

Extracellular matrix material (ECM) present during early lens morphogenesis was analyzed histochemically in normal CFW mice and mutant strain aphakia by the Alcian blue 8GX, pH 2.5, Alcian blue 8GX, pH 2.5/periodic acid-Schiff combined, high-iron diamine, and van Gieson methods. At lens placode formation, the optic vesicle basal lamina in both strains was higher in sulfated glycosaminoglycan content than was the ectodermal basal lamina. In the aphakia strain, ECM components were observed intercellularly in the presumptive neural retina and lens rudiment of some specimens. This observation was peculiar to the aphakia strain. At the lens cup stage (10.5 days), the interface ECM became less uniformly dense in the CFW strain, resulting in the formation of a fibrillar structure in the widening interspace area. In contrast, the interface ECM in the mutant strain stained solidly and continuously for acidic materials, particularly sulfated glycosaminoglycans, for a full 2 days longer than in the normal strain. The optic cup and lens rudiment remained closely apposed and intercellular ECM components were observed in these tissues in most mutant specimens throughout these stages. The exact mechanism resulting in these intercellular deposits is unknown, although it is possible that they are either pulled along on the cell surface away from the interface ECM during cell shape changes related to the cell cycle or that they are secreted abnormally due to some disturbed cellular polarity. It is unclear at this time if these abnormalities of the ECM in the aphakia strain play a role in the pathogenesis of the multiple eye anomalies, or if they are a secondary effect of the gene mutation.     

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 localization of protein-polysaccharides in renal tissue

The purpose of this study was to investigate the distribution of protein-polysaccharides in the glomerular and non-glomerular regions of the nephron. The techniques used include the digestion of kidney slices with specific polysaccharidases: neuraminidase, hyaluronidase, chondroitinase ABC, and collagenase followed by several cytochemical techniques to identify the glycosaminoglycans and glycoproteins at the light and electron microscope levels. Differential staining of hyaluronic acid and sulphated glycosaminoglycans was accomplished with Alcian Blue at pH 2.5 and pH 0.5, respectively. Sialoproteins were stained with Alcian Blue at pH 2.5. The periodic acid Schiff’s reaction technique was employed for the visualization of collagen. At the electron microscope level the polysaccharides were identified with the periodic acid-chromic acid-silver methenamine reaction. Our results indicated that the major polysaccharide components of the glomerular basement membrane were sialoproteins and collagen, with smaller amounts of hyaluronic acid and various sulphated glycosaminoglycans. Hyaluronidase digestion resulted in partial detachment of epithelial processes from the glomerular basement membrane indicating the hyaluronic acid may have a role in the stability of the attachment of these processes. Tubular basement membranes also contain sialoproteins and sulphated glycosaminoglycans but in considerably lower concentrations than the glomerular basement membrane. Bowman’s capsule appears to contain mostly sulphated glycosaminoglycans and has a lower concentration of sialoproteins and hyaluronic acid.     

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.     

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.     

Immunohistochemical staining for chondroitin sulphate and keratan sulphate. An evaluation of two monoclonal antibodies

Summary Immunohistochemical staining with commercially available antibodies against chondroitin sulphate (clone CS-56) and keratan sulphate (clone 1/20/5-D-4) was compared with two conventional histochemical methods for the demonstration of glycosaminoglycans, namely Alcian Blue with varying pH and critical electrolyte concentrations, and a modified PAS stain. The antibodies were tested on sections from both frozen and fixed, paraffin embedded human material from umbilical cord, skin, and bronchus. The results showed immunostaining to function equally well on frozen and routine sections, and to be superior to Alcian Blue and PAS with regard to morphological detail. Thus, reactivity with anti-chondroitin sulphate was demonstrated in vessel walls, in small nerves, in the basal membrane zone of the skin, in perichondrium, and in and around chondrocytes. Reactivity with anti-keratan sulphate occurred in chondroid matrix and in perichondrial tissue; however, some cells of the bronchial epithelium and mucous glands also exhibited positivity.     

Flow cytometric determination of basophils in whole blood with n-propyl Astra Blue iodide

Within the past year, it has become apparent, in connection with its use on automatic flow cytometers, that the quality of commercially available Alcian Blue has significantly declined. A homologous series of alkylated (C1-C7) Astra Blue quaternary ammonium halides was prepared, characterized, and evaluated for the detection of basophils in whole blood. On the Technicon H6000 flow cytometer, the resolution of the basophil cluster from the main population of unstained white blood cells was found to depend on the chain length of the quaternizing alkyl group. Optimal basophil resolution was observed for the n-propyl derivative. Correlation of the new method vs Alcian Blue as the reference on the H6000 was expressed as follows: %Baso (Astra Blue) = 0.89% Baso (Alcian Blue) + 0.12% for 180 fresh whole blood samples. Within-run precision at a basophil differential count of 0.73% was characterized by SD = 0.11, identical to that obtained for Alcian Blue. Aqueous solutions of n-propyl Astra Blue iodide, in contrast to Alcian Blue, are thermally stable. Heating the reagent for 1 h at 100 degrees C did not alter solubility or cytochemical behavior. In contrast, parallel treatment of Alcian Blue yielded insoluble material by hydrolysis of the isothiouronium groups. The reagent for basophil detection comprises n-propyl Astra Blue iodide, lanthanum chloride, sodium chloride, Tween 20, and cetylpyridinium chloride. The Astra Blue derivatives were characterized by uv-vis, ir, percentage halide, paper chromatography, and 13C NMR.     

Immunohistochemical staining for chondroitin sulphate and keratan sulphate. An evaluation of two monoclonal antibodies

Immunohistochemical staining with commercially available antibodies against chondroitin sulphate (clone CS-56) and keratan sulphate (clone 1/20/5-D-4) was compared with two conventional histochemical methods for the demonstration of glycosaminoglycans, namely Alcian Blue with varying pH and critical electrolyte concentrations, and a modified PAS stain. The antibodies were tested on sections from both frozen and fixed, paraffin embedded human material from umbilical cord, skin, and bronchus. The results showed immunostaining to function equally well on frozen and routine sections, and to be superior to Alcian Blue and PAS with regard to morphological detail. Thus, reactivity with anti-chondroitin sulphate was demonstrated in vessel walls, in small nerves, in the basal membrane zone of the skin, in perichondrium, and in and around chondrocytes. Reactivity with anti-keratan sulphate occurred in chondroid matrix and in perichondrial tissue; however, some cells of the bronchial epithelium and mucous glands also exhibited positivity.     

Histochemistry of Alcian blue

Summary The structure of Alcian blue 8GX has been investigated using chemical and physical methods. Elemental analyses of the boric acid-free dye and electrophoresis suggest a phthalocyanin with an average of slightly less than four S-methylene tetramethylisothiouronium side chains per molecule. Oxidative break-down in two stages to benzene carboxylic acids, leading to the identification of benzene 1, 2, 4 tricarboxylic acid (trimellitic acid) as the main product, prove the presence of predominantly 4-substitution by the side chains in each iso-indole residue. Paper chromatography was used to resolve and identify the carboxylic acids, but final confirmation of their identity came from mass spectrometry of the mixture. Nuclear magnetic resonance spectra of the phthalimides produced in high yield during the first stage of oxidation in sulphuric acid by hydrogen peroxide confirmed and extended these findings. The ultra-violet, visible and infra-red spectra of Alcian blue and similar phthalocyanin pigments are discussed, and related to the structure.A consideration of the possible distributions of four substituents in the eight possible positions which could be substituted leads to the conclusion that Alcian blue 8GX consists mainly of four geometrical isomers, the structures of which are presented.