Cationic Dye Alcian Blue Research Articles

Detection of Glycosaminoglycans in Pancreatic Islets and Lymphoid Tissues.

In this chapter, we describe the detection of the glycosaminoglycans hyaluronan and heparan sulfate in pancreatic islets and lymphoid tissues. The identification of hyaluronan in tissues is achieved by utilizing a highly specific hyaluronan binding protein (HABP) probe that interacts with hyaluronan in tissue sections. The HABP probe is prepared by enzymatic digestion of the chondroitin sulfate proteoglycan aggrecan which is present in bovine nasal cartilage and is then biotinylated in the presence of bound hyaluronan and the link protein. Hyaluronan is then removed by gel filtration chromatography. The biotinylated HABP-link protein complex is applied to tissue sections, and binding of the complex to tissue hyaluronan is visualized by enzymatic precipitation of chromogenic substrates.To determine hyaluronan content in tissues, tissues are first proteolytically digested to release hyaluronan from the macromolecular complexes that this molecule forms with other extracellular matrix constituents. Digested tissue is then incubated with HABP . The hyaluronan-HABP complexes are extracted, and the hyaluronan concentration in the tissue is determined using an ELISA-like assay.Historically, heparan sulfate was identified in tissue sections using the cationic dye Alcian blue and histochemistry based on the critical electrolyte concentration principle of differential staining of glycosaminoglycans using salt solutions. For both human and mouse pancreas sections, the current optimal method for detecting heparan sulfate is by indirect immunohistochemistry using a specific anti-heparan sulfate monoclonal antibody. A peroxidase-conjugated secondary antibody is then applied, and its binding to the anti-heparan sulfate antibody is visualized by oxidation and precipitation of a chromogenic substrate.

Rsc14-controlled expression ofMNN6, MNN4andMNN1regulates mannosylphosphorylation ofSaccharomyces cerevisiaecell wall mannoproteins

ybl006cDelta was selected using an Alcian-blue-based screen aimed to identify nonessential genes involved in the regulation of mannosylphosphorylation. When cells of this deletant were mixed with the cationic dye Alcian blue in a typical assay, they remained white, indicating a low number of mannosylphosphate groups on the cell surface. ybl006cDelta cells did not show any defect in growth rate nor in the glycosylation or secretion rate of the major exoglucanase Exg1. Transcriptome analysis of ybl006cDelta using macroarrays showed at least two-fold changes in the expression of 52 genes (<0.9% of the genome). Three of these have previously been reported to be directly (MNN6 and MNN4) or indirectly (MNN1) implicated in the transfer of mannosylphosphate to the N- and O-oligosaccharides. Alterations in the expression of these genes were confirmed by Northern analysis. YBL006C product was recently identified as a subunit (Rsc14) of the RSC chromatin-remodelling complex of Saccharomyces cerevisiae. It follows that remodelling of chromatin can be an important regulatory mechanism for the maturation of cell wall mannoproteins.

Demonstration of polysaccharide capsule in Campylobacter jejuni using electron microscopy.

Recently, we reported that Campylobacter jejuni, an important gastrointestinal pathogen, has the genetic determinants to produce a capsular polysaccharide (Karlyshev et al., Mol. Microbiol. 35:529-541, 2000). Despite these data, the presence of a capsule in these bacteria has remained controversial. In this study we stain C. jejuni cells with the cationic dye Alcian blue and demonstrate for the first time by electron microscopy that C. jejuni cells produce a polysaccharide capsule that is retained in the coccoid form but is absent in a kpsM mutant.

Quantitation of Proteoglycans as Glycosaminoglycans in Biological Fluids Using an Alcian Blue Dot Blot Analysis

A method for quantitation of intact proteoglycans as GAGs in biological fluids (blood plasma, synovial fluid) or 4 M guanidine extracts of tissues has been published previously (S. Björnsson,Anal. Biochem.210, 282–291, 1993). The method is based on the specific interaction between sulfated polymers and the tetravalent cationic dye Alcian blue at pH 1.5 in 0.4 M guanidine-HCl and in the presence of 0.25% Triton. The absorbance assay has a measuring range of 1–20 μg of glycosaminoglycan (GAG) which is not sensitive enough to measure the low contents of proteoglycans in blood plasma, urine, or wound fluid. A dot blot assay is now described in which the Alcian blue–GAG complexes are collected on a polyvinylidene fluoride membrane, by filtration in a dot blot apparatus, and the stain is quantitated as reflectance by scanning and densitometry. The assay requires 10 μl of sample and has a measuring range of 10–800 ng of GAG, corresponding to a concentration of 1–80 mg/liter, suitable for proteoglycans in biological fluids. The procedures for chemistry, scanning, densitometry, and curve fitting were each evaluated separately. The error contributed by chemistry accounted for a minor portion of the imprecision. The imprecision contributed by scanning was the most important source of within-run and between-run imprecision, and was caused by inequalities of the charge-coupled device along the scanning arm. Unexpectedly, curve fitting was also a major source of total imprecision in dot blot quantitation and differed with the type of equation used. The between-run imprecision calculated as CV (SD/mean · 100) was 13.0% at 8 mg/liter. The response of the assay was identical for six different commercial preparations of GAGs (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and heparin) despite differences in degree of sulfation known to exist. There was no positive or negative interference by blood plasma, apart from a slight negative interference on the quantitation of heparan sulfate. Analysis of 319 paired blood plasma and urine specimens from hospitalized patients showed a variation of plasma GAGs of 0.1–17.6 and urine–GAGs of 0.0–45.6 mg/liter. There was no correlation between plasma and urine GAG concentrations.

Freeze-substitution staining of rat growth plate cartilage with alcian blue for electron microscopic study of proteoglycans.

To selectively stain polyanionic macromolecules of growth plate cartilage and to prevent artifacts induced by aqueous fixation, proximal tibial growth plates were excised from rats, slam-frozen, and freeze-substituted in 100% methanol containing the cationic dye Alcian blue. Electron microscopic examination showed the tissue stained with Alcian blue to be comparable in ultrastructural preservation to tissues slam-frozen and freeze-substituted in the absence of Alcian blue. The extracellular matrix exhibited a characteristic staining pattern when stained by this method. The pericellular rim was identified as a band of varying width encircling the chondrocyte and its cell processes. Peripheral to the pericellular rim the heterogeneity of staining within the extracellular matrix increased, taking the form of polymorphic densities. X-ray microanalysis showed that the visual interpretation of electron density was related to the concentration of copper present, and that the concentration of sulfur was variable in the pericellular rim and in the interterritorial matrix. The difficulties associated with aqueous fixation and staining procedures are discussed in contrast to the improved preservation achieved by cryogenic methods.

Quantification of arabinogalactan-protein in plant extracts by single radial gel diffusion

The amount of arabinogalactan-protein in whole plant extracts can be quantified by single radial diffusion in agarose gels containing a dye known as the β-glucosyl-Yariv reagent which specifically interacts with and precipitates arabinogalactan-proteins. The lower limit of quantification is 0.04 μg of arabinogalactan-protein; gum arabic is used as a standard reference arabinogalactan-protein. In principle, this method can be adapted to measure levels of any dyeprecipitating macromolecule; for example, acidic polysaccharides can be estimated by their binding to the cationic dye Alcian blue.

Application of Gaboul Method to the Ultracytochemical Study of Mouse Blood-Brain Barrier

The cationic dye Alcian blue has been used to demonstrate protein-polysaccharide com plexes at the level of nerve tissue (1,2). The results obtained in the capillary wall when this dye was applied to mouse brain and examined in the electron microscope are the object of the present report.The central nervous system of normal adult mice was fixed either by vascular perfusion or by immersion with 4% glutaraldehyde-0.1M sodium cacodylate-0.1 M sucrose at pH 6.8 followed by the same procedure using the same fixative but with the addition of 0.5% Alcian blue. After fixation, tissue was immersed in 1% OsO4-0.1M phosphate buffer pH 7.2, dehydrated by graded ethanol and embedded in Araldite. Ultrathin sections were doubly stained with uranyl acetate and lead citrate. The complete procedure has been denominated GABOUL method (3).The submicroscopic study of cerebral and cerebellar capillaries showed the presence of a thin line of GABOUL positive material attached to the outer layer of the asymmetric luminal membrane of endothelial cells (endocapillary layer) (Fig. 1).

Ultrastructural studies on the external surface coat of epidermal cells: alcian blue-lanthanum nitrate staining during wound healing.

The external surface coat on mouse epidermal cells has been studied during epidermal wound healing in suction-induced intact subepidermal blisters. By sequentially incorporating the cationic dye Alcian blue and the electron opaque tracer lanthanum nitrate into the primary and secondary fixatives for electron microscopy (AB/LN staining), we have demonstrated a selective staining of the epidermal surface coat. At 12 and 24 h after wounding, AB/LN staining always occurred on opposing cell membranes of adjacent epidermal cells. No staining was seen along epidermal cell plasmalemmas exposed to the blister cavity or opposite the basal lamina. The full significance of this differential AB/LN staining during wound healing is not presently known. It is suggested that the presumed anionic sites that are binding the AB/LN stain may play a role in maintaining integrity during wound healing.

Preservation of intercellular substances by the cationic dye alcian blue in preparative procedures for electron microscopy

Various rat tissues (small intestine, kidney, suprarenal gland, epithelium of the tongue, endothelium, mesothelium, blood cells, and bone marrow) were examined in the electron microscope, after fixation with glutaraldehyde in the presence of the cationic dye Alcian blue. The procedure resulted in improved preservation of the “cell coat” of all cell types examined. Alcian blue appears to bind nonspecifically to anionic sites in cell coat mucosubstances, with the formation of a rather insoluble, osmiophilic dye-mucosubstance complex. The results are virtually identical to those obtained when tissues are stained with ruthenium red or other methods in current use for the demonstration of glycoproteins and acid mucopolysaccharides.

Electron microscopical observations on the surface coating of human blood platelets

The plasma membrane (trilaminar lipoprotein membrane) of human blood platelets exhibits, in conventional glutaraldehyde—osmium fixation, a layer of an amorphous substance coating its outer surface. The coat has a thickness of about 150–200 . It is considered to be an integral part of the cell membrane, which thus consists of the plasma membrane plus the coating material. The preservation of the coat is much improved if the cationic dye Alcian blue is added to the glutaraldehyde fixative. Ferritin, Thorotrast, and horseradish peroxidase are adsorbed to the coat in vitro. The coat reacts with ruthenium red, colloidal iron, lanthanum ions, and Thorotrast. Its stainability with ruthenium red is abolished by digestion with trypsin and pronase, whereas the stainability with colloidal iron and Thorotrast is not. Platelets incubated with trypsin and soybean trypsin inhibitor show an unimpaired stainability with ruthenium red. It is concluded that the coat probably consists of a sulfated acid mucopolysaccharide and that the coat furnishes the platelet with a zone of interest and with a mechanism for producing rapid changes in the quality of adhesion to surfaces.