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.     

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.     

A Note on the Purification of Alcian Blue

Alcian blue 8GX is a copper phthalocyanin dye that shows a high degree of specificity for polyanionic substances such as hyaluronic acid, sialic acid and the chondroitin sulfates. This dye has proved useful for both histochemical and electrophoretic staining of these substances. The Biological Stain Commission has recently begun to certify Alcian blue (Schenk 1981). Commercially available lots contain approximately 50% dye. The remaining constituents have been identified as primarily boric acid, as well as sulfates and dextrins (Scott 1972, Horobin and Goldstein 1972). Horobin and Goldstein (1972) have pointed out that these contaminants may adversely affect staining in the critical electrolyte concentration procedure. Scott (1972), while not ascribing any adverse effects to the presence of boric acid, recommends its removal by differential precipitation with acetone. In this procedure one part of a 2-5% aqueous solution of the dye is added to 5-10 parts of acetone. The precipitated dye is approximately 80% pure. While this method is relatively simple, it does have several drawbacks. Low concentrations of Alcian blue (i.e., 2%) must be used to obtain purities near 80%. Thus a minimum of 250 ml of acetone is needed to purify 1 gram of dye. Furthermore, Horobin and Goldstein (1972) have reported that contamination by dextrin or unknown organic substances (detergent?) interferes with precipitation of the dye enough to make purification by Scott's method impossible. When difficulty in the precipitation of Alcian blue by Scott's method was encountered, the following simple method for the purification of the dye was developed.     

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.     

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

Summary 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 or 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.     

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.     

A Note on the Purification of Alcian Blue

Alcian blue 8GX is a copper phthalocyanin dye that shows a high degree of specificity for polyanionic substances such as hyaluronic acid, sialic acid and the chondroitin sulfates. This dye has proved useful for both histochemical and electrophoretic staining of these substances. The Biological Stain Commission has recently begun to certify Alcian blue (Schenk 1981). Commercially available lots contain approximately 50% dye. The remaining constituents have been identified as primarily boric acid, as well as sulfates and dextrins (Scott 1972, Horobin and Goldstein 1972). Horobin and Goldstein (1972) have pointed out that these contaminants may adversely affect staining in the critical electrolyte concentration procedure. Scott (1972), while not ascribing any adverse effects to the presence of boric acid, recommends its removal by differential precipitation with acetone. In this procedure one part of a 2-5% aqueous solution of the dye is added to 5-10 parts of acetone. The precipitated dye is approximately 80% pure. While this method is relatively simple, it does have several drawbacks. Low concentrations of Alcian blue (i.e., 2%) must be used to obtain purities near 80%. Thus a minimum of 250 ml of acetone is needed to purify 1 gram of dye. Furthermore, Horobin and Goldstein (1972) have reported that contamination by dextrin or unknown organic substances (detergent?) interferes with precipitation of the dye enough to make purification by Scott's method impossible. When difficulty in the precipitation of Alcian blue by Scott's method was encountered, the following simple method for the purification of the dye was developed.     

Histochemical analysis of extracellular matrix material in embryonic trisomy 1 mouse eye

Trisomic animals produced from mice doubly heterozygous for Robertsonian translocation chromosomes [Rb(1.3)/Rb(1.10)]consistently show eye defects (e.g., aphakia, microphakia, and retention of lens stalk). To determine if changes in distribution or composition of extracellular matrix material may be a factor in development of these defects, eye structures of trisomy (ts) 1 embryos and normal littermates were studied histochemically using the following methods: Alcian blue 8GX, pH 2.5; periodic acid-Schiff (PAS), Alcian blue/PAS combined; high-iron diamine (HID), and HID/Alcian blue combined. Eye development was divided into stages to account for the known delay in ts 1 mouse development. Differences were found in staining patterns as early as stage 1. In later stages, the most consistent difference was an increased period of contact between lens and optic cup due to retardation of interface matrix dissolution between these rudiments in ts 1 embryos. Eyes in which this occurred had abnormally shaped lenses. Overall, the ts 1 optic cup appeared to have fewer staining abnormalities and dysmorphology than did the lens or interface matrix. Triplication of a chromosome may indirectly alter temporal and spatial organization of extracellular matrix through action on cells responsible for the production of this material. Possible mechanisms of action are discussed.     

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.     

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.     

An improved method of sequential alcian blue and ammoniacal silver staining of chondroitin sulfate proteoglycan in polyacrylamide gels

Partially deglycosylated chondroitin sulfate proteoglycan (CSPG) or peptide fragments obtained from CSPG are not readily detectable in gels by staining with Alcian blue 8GX or ammoniacal silver using the technique of Oakley et al. (B. Oakley, D. Kirsh, and N. Morris (1980) Anal. Biochem. 105, 361). Sequencial staining with both reagents allows visualization of intact CSPG or peptides derived from proteoglycans in polyacrylamide gels at protein concentrations as low as 2 ng/mm2, or glucuronic acid and galactosamine concentrations of 1 ng/mm2 or less. This method is significantly more sensitive and has broader applicability than that described by H. Min and M. Cowman (1986) Anal. Biochem. 155, 275) for staining glycosaminoglycan fragments in polyacrylamide gels.     

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.     

The extracellular matrix between the optic vesicle and presumptive lens during lens morphogenesis in an anophthalmic strain of mice

Extracellular matrix material present during early lens morphogenesis in anophthalmic strain ZRDCT-Ch mice was studied histochemically 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. Observed staining patterns were compared with results from an analysis of a normal strain of mice (E.H. Webster, Jr., A.F. Silver, and N.I. Gonsalves, 1983, Develop. Biol. 100, 147-157). No differences in constituents were found between the strains in staining patterns of the ectodermal basal lamina. However, the optic vesicle basal lamina in the anophthalmic strain was found to have a relatively lower staining intensity for sulfated glycosaminoglycan associated with it than was observed in the normal strain, although these mutant optic vesicles were morphologically normal. Results from this and the earlier study on normal mice indicate that one function of sulfated glycosaminoglycan in early lens morphogenesis may be to serve as a cementing medium between the optic and lens rudiments. This sulfated glycosaminoglycan deficiency on the anophthalmic optic vesicle basal lamina is temporally correlated with and may be causally related to precocious lens cup formation and frequently observed separation of the normally adherent eye rudiments. Conclusions drawn from this study are consistent with the speculation of H.B. Chase and E.B. Chase (1941, J. Morphol. 68, 279-301) that there may be abnormal contact between the optic vesicle and presumptive lens ectoderm in the mutant strain, although there is a differing view on the cause of the abnormal contact.     

Haematoxylin-Phloxine-Alcian Blue-Orange G Differential Staining of Prekeratin, Keratin and Mucin

This is a modification of Kreyberg's stain with Alcian blue 8GS used to stain acid much while phloxine B and orange G stain keratin and prekeratin. Procedure: Dewax formalin-fixed paraffin sections in xylene and hydrate through alcohol. Stain in Mayer's haemalum, 10 min; blue in tap water; wash in distilled water; stain in 1% phloxine, 3 min; wash in running water, 1 min; wash in distilled water; stain in 0.5% aqueous Alcian blue in 0.5 acetic acid, 5 min; wash in distilled water; stain in 0.5% orange G dissolved in 2.0% phosphotungstic acid, 13 min; dehydrate quickly in 2 changes of 95% alcohol and 2 changes of absolute alcohol; clear in several changes of xylene; mount in a synthetic resin. Acid mucopolysaccharides are stained turquois blue; prekeratin and keratin are orange to red orange.     

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.     

Histochemistry of Alcian blue

Summary Two new cationic copper phthalocyanins, isomers of each other, have been synthesised, and compared with Alcian blue 8GX as stains for a variety of polyanions. Analysis of dye-substrate affinities has been carried out using the critical electrolyte concentration (CEC) approach. Recently acquired data on the complete structure of Alcian blue permit interpretations of the affinities in terms of clearly defined interactions with known features of the secondary structures of natural and synthetic nucleic acids. Molecular models and the CEC analysis complement each other in providing an integrated picture of the staining mechanism. A size criterion for the intra-helical binding of phthalocyanins has been established, which explains the histochemical staining behaviour of Alcian blue and its analogues. Evidence is produced to show that appropriate dyes may act as templates on which very flexible polyanions containing aromatic rings may take up conformations, in complexes which are very stable.One of the new phthalocyanins based on quinolinic acid, has been shown to have a very specific affinity for RNA in vitro and in tissues, in concentrated salt solutions.The general problem of the analysis of histochemical staining by dyes is discussed. The CEC analysis is obtained in a form which is immediately applicable to the investigation of tissue polyanions.Future developments of cationic phthalocyanin dyes, to perform specific histochemical tasks, are indicated.     

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.     

Evaluation of improved zirconyl hematoxylin compared to the classical pH 2.5 Alcian blue method for demonstrating acid mucins

Abstract

Acid mucins have diagnostic significance for many pathological conditions, especially in certain tumors. We compared the classical pH 2.5 Alcian blue method to a new, improved zirconyl hematoxylin (IZH) method for demonstrating acid mucins using two fixatives: Bouin`s solution and 10% neutral buffered formalin (NBF). We used rabbit small intestine, large intestine and trachea. Specimens were fixed in Bouin`s solution and NBF. A total of 160 paraffin sections were prepared and stained with pH 2.5 Alcian blue and IZH. The stained acid mucins were assessed using digital image analysis software. Stained mucins were quantified for each staining procedure and fixative. No important differences were observed in acid mucin staining by either method after either fixative. The IZH method provides results as good as pH 2.5 Alcian blue and can be used to obtain reliable staining for acid mucins.     

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.