Alcian Blue Pyridine Variant–a Superior Alternative to Alcian Blue 8GX: Staining Performance and Stability

We compared the staining performance, dye content, solubility, and visual absorption maximum of two batches of alcian blue pyridine variant and of five batches of alcian blue 8GX (C.I. 74240). Whenever possible, we also compared results to those obtained with the same dye batches produced at an earlier date to provide information concerning dye stability. Both alcian blue pyridine variant batches were of high dye content, stable, of satisfactory solubility, and performed well in both the routine Mowry mucin stain and in the critical electrolyte concentration (CEC) stain. Of the five alcian blue 8GX samples, some were also of appropriate dye content, were sufficiently stable, and gave good staining in the two procedures. Two batches, however, were unstable, and three batches were unsatisfactory in staining performance and solubility in the CEC stain. Consequently alcian blue pyridine variant is a superior substitute for alcian blue 8GX.     

Alcian Blue Pyridine Variant-a Superior Alternative to Alcian Blue 8GX: Staining Performance and Stability

We compared the staining performance, dye content, solubility, and visual absorption maximum of two batches of alcian blue pyridine variant and of five batches of alcian blue 8GX (C.I. 74240). Whenever possible, we also compared results to those obtained with the same dye batches produced at an earlier date to provide information concerning dye stability. Both alcian blue pyridine variant batches were of high dye content, stable, of satisfactory solubility, and performed well in both the routine Mowry mucin stain and in the critical electrolyte concentration (CEC) stain. Of the five alcian blue 8GX samples, some were also of appropriate dye content, were sufficiently stable, and gave good staining in the two procedures. Two batches, however, were unstable, and three batches were unsatisfactory in staining performance and solubility in the CEC stain. Consequently alcian blue pyridine variant is a superior substitute for alcian blue 8GX.     

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.     

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.     

Side effects of reaction media in histochemical blocking procedures

Synopsis 0.2 N NaOH, the reaction medium for 1,2-cyclohexanedione, a specific reagent for arginyl residues in proteins, was found to intensify, at some sites in rat abdominal skin and human gingiva, the Sakaguchi reaction, staining with Pauly's reagent, and anionic dye binding at pH 6.4; at other sites these reactions were reduced, presumably due to extraction of material from sections. 0.2 N NaOH slightly reduced staining after the ninhydrin-Schiff procedure at all sites in rat skin. The interpretation of this finding is obscure, because some sites giving a positive Sakaguchi reaction and staining with anionic dyes failed to stain after the ninhydrin-Schiff procedure. There were also alterations in staining, with the cationic dyes Alcian Blue and Alcian Yellow. It is suggested that 0.2 N NaOH ruptures linkages between polycationic residues of proteins and polanions, demonstrable by Alican Blue. The blockade produced by acetic anhydride-pyridine (4060 v/v) mixtures was stable, in the alkaline conditions required for staining with Pauly's reagent. Pretreatment with pyridine alone reduced tissue binding of anionic dyes.     

Electron microscopic visualization of proteoglycans with Alcian Blue.

The intercellular matrices of bovine nasal cartilage, chick embryo perichordal cartilage, and chick embryo mesenchymal cells cultured in vitro have been examined by electron microscopy after staining them with Alcian Blue in salt solutions according to Scott & Dorling (1965). Matrix granules, which are typical components of cartilage at the ultrastructural level, are not visible after Alcian Blue staining and are replaced by alcianophilic rod-like particles, varying in length and width. With tissue cultures, Alcian Blue stains 40-120 A thick filaments which display an orthogonal and longitudinal relationship to collagen fibrils. We assume that cartilage matrix granules represent linear proteoglycans that are coiled as a consequence of the usual glutaraldehyde-osmium fixation. It is thought that Alcian Blue, on the other hand, contributes to the stabilization of the proteoglycans in their original structural arrangement. This stabilizing property presumably also results in the sharp visualization of fine filaments in the tissue culture matrix.     

LANTHANUM STAINING OF THE SURFACE COAT OF CELLS : Its Enhancement by the Use of Fixatives Containing Alcian Blue or Cetylpyridinium Chloride

Among the techniques which have been reported to stain the surface coat of cells, for electron microscopy, is lanthanum staining en bloc. Similarly, the presence of the cationic dye, Alcian blue 8GX, in a primary glutaraldehyde fixative has been reported to improve the preservation of the surface coat of cells of many types; however, the preserved coat is not very electron opaque unless thin sections are counterstained. The present paper shows that for several rat tissues lanthanum staining en bloc is an effective electron stain for the cell surface, giving excellent contrast, if combined sequentially with prefixation in an aldehyde fixative containing Alcian blue. The cationic substance cetylpyridinium chloride was found to have a similar effect to that of Alcian blue in enhancing the lanthanum staining of the surface coat material of the brush border of intestinal epithelial cells. The patterns of lanthanum staining obtained for the tissues studied strikingly resemble those reported in the literature where tissues are stained by several standard methods for demonstrating mucosubstances at the ultrastructural level. This fact and the reproduction of the effect of Alcian blue by cetylpyridinium chloride constitute a persuasive empirical argument that the material visualized is a mucopolysaccharide or mucopolysaccharide-protein complex.     

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.     

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.     

Metachromatic staining and electron dense reaction of glycosaminoglycans by means of Cuprolinic Blue

Summary The cationic phthalocyanin-like dye Cuprolinic Blue, unlike phthalocyanin dyes such as Alcian Blue or Astra Blue, can definitely exhibit a clear metachromatic reaction with appropriate substrates, The application of Cuprolinic Blue to epoxy-embedded semithin sections revealed that mast cell cytoplasmic granules, goblet cell mucin and cartilage matrix stained in violet shades (metachromatic), whereas nuclear chromatin presented a bright blue coloration (orthochromatic). The metachromatic structures showed a high degree of contrast when ultrathin sections treated with Cuprolinic Blue were examined by electron microscopy.Cytophotometric measurements of stained components from the large intestine showed different absorption maxima: at 580 nm for mucin and at 640 nm for nuclei. The spectroscopical analysis revealed a clear-cut metachromatic shift when the dye was in the presence of chondroitin—4-sulphate. The addition of aluminium metal to Cuprolinic Blue solutions resulted in a striking spectral change; under such conditions the dye showed absorption maximum at 530 nm.     

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.     

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.     

Sialic acid in colonic mucin: An evaluation of modified PAS reactions in single and combination histochemical procedures

Summary Two new histochemical procedures for detecting sulphated and non-sulphated sialomucin in colonic mucosa were assessed: the saponification—Alcian Blue pH 1—periodic acid—phenylhydrazine—Schiff method (KOH—AB pH 1—PAPS) and the mild periodic acid modification of this (KOH—AB pH 1—mPAS). Using normal colonic mucosa obtained from 11 non-cancer patients, the mPAS and PAPS techniques were tested for specificity and reproducibility for staining sialic acid, either alone or in combination with Alcian Blue. A spectrophotometric method was devised to quantify the uptake of both Schiff and Alcian Blue stain by sections. At low temperature and pH5.5, the mPAS procedure had improved specificity over the PAPS procedure, and after saponification it could be used to stainO-acetyl-substituted sialic acid. When used in combination with Alcian Blue at pH 1, however, underestimation of the sialic acid content occurred owing to interference between Alcian Blue and Schiff dyes. Interference was even greater with KOH—AB pH1—PAPS procedure for both sialic acid and sulphate components. We conclude that caution must be exercised in interpretation of the staining results obtained with these new combination methods and that more accurate information on the sialic acid and sulphate content of colonic mucin is obtained by staining serial sections with the mPAS technique and Alcian Blue pH 1 alone.     

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.     

The quantitative determination of glycosaminoglycans in urine with Alcian Blue 8GX

1. The effect of MgCl(2) concentration on the interaction of Alcian Blue 8GX and glycosaminoglycans in the urine of patients with mucopolysaccharidosis was studied by using a new quantitative micro method for the measurement of Alcian Blue-glycosaminoglycan complexes. This provided a means of measuring the critical electrolyte concentrations of urinary glycosaminoglycans. 2. Theoretical considerations based on the preceding paper (Whiteman, 1973) and experimental evidence provided here show that Alcian Blue 8GX may be used for the direct quantitative determination of total urinary glycosaminoglycans. The method is simple, requires sample volumes of 50mul or less, and gives results comparable with those obtained by other more complicated methods.     

Staining of interphase nuclei and mitotic figures in cultured cells with alcian blue 8GX

Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction of Heidenhain iron hematoxylin but without the latters' length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.     

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.     

New tetrachromic VOF stain (Type III-G.S) for normal and pathological fish tissues

A new VOF Type III-G.S stain was applied to histological sections of different organs and tissues of healthy and pathological larvae, juvenile and adult fish species (Solea senegalensis; Sparus aurata; Diplodus sargo; Pagrus auriga; Argyrosomus regius and Halobatrachus didactylus). In comparison to the original Gutiérrez VOF stain, more acid dyes of contrasting colours and polychromatic/metachromatic properties were incorporated as essential constituents of the tetrachromic VOF stain. This facilitates the selective staining of different basic tissues and improves the morphological analysis of histochemical approaches of the cell components. The VOF Type III -6.5 stain is composed of a mixture of several dyes of varying size and molecular weight (Orange G相似文献