Alcian Blue Dye Research Articles

Visualization of an extracellular mucoid layer of Treponema denticola ATCC 35405 and surface sugar lectin analysis of some Treponema species.

Slime layers and capsules are common amongst medically relevant bacteria. We herein report that Treponema denticola, which has been associated with periodontitis, synthesizes or acquires an extracellular polysaccharide layer that we have observed through electron microscopy using the polysaccharide-specific dye Alcian blue and phosphotungstate. We have also visualized this extracellular layer by dark-field microscopy of Alcian blue-stained spirochete cells. A representative strain of each of the oral spirochete species T. denticola, Treponema vincentii and Treponema socranskii were differentiated by concanavalin A, phaseolus, lotus A and arachis lectins in a microtiter plate immunoassay for the detection of surface sugars.

Isolation of new nonconditional Saccharomyces cerevisiae mutants defective in asparagine-linked glycosylation.

We describe the isolation and partial characterization of Saccharomyces cerevisiae nonconditional mutants that show defects in N-glycosylation of proteins. The selection method is based on the reduction of affinity for the ion exchanger QAE-Sephadex as a consequence of the decrease in the negative charge of the cell surface. This characteristic reflects a decrease in the incorporation of mannosylphosphate units into the N-linked oligosaccharides of the mannoproteins. The mutants exhibit low affinity for the basic dye alcian blue and for that reason we have called them Idb (low dye binding) mutants. Eight of the complementation groups seem to be new as shown by complementation studies with previously isolated mutants of similar phenotype. Four of the groups showed a significant reduction in the number and/or size of the N-linked oligosaccharides attached to secreted invertase. We have analyzed the N-linked oligosaccharides of Idb1 and Idb2, the mutants that show the most drastic reduction in the affinity for the alcian blue dye. In both cases, the purified endo H-released oligosaccharides from the mannoproteins lacked detectable amounts of phosphate groups as shown by ion exchange chromatography and the 1H NMR spectra. In addition, Ibd1 synthesizes a truncated and unbranched outer chain lacking any alpha (1,2) linked mannoses attached to the alpha (1,6) linear backbone.

Age and growth of yellow eel, Anguilla anguilla (L.), determined by two different methods

Abstract– Ageing and calculation of growth rate of eels has always been controversial, and remarkably little validation has been reported. This article describes the growth of yellow eels from three lakes in the Burrishoole system, Ireland, as determined by back‐calculation from otoliths prepared by burning and cracking. Eels captured by fyke net ranged in length from 25.0 cm to 98.5 cm and the ages ranged from 6 to 49 years. Back‐calculated growth was slow, with mean annual increments of 2.08 cm, 1.40 cm and 1.97 cm for the three lakes. Yellow eels captured in the Burrishoole system in 1987 and 1988 were marked by Pan Jet inoculated alcian blue dye and released back into the three lakes. Recaptures made over the following three years yielded considerable growth data. Mean annual growth rates recorded were 2.30 cm, 1.07 cm and 0.97 cm. These data compare favorably with the calculated growth rates, thereby validating the otolith analysis for the eels in the Burrishoole system.

Alcian blue. Now you see it, now you don't.

A brief outline of the history and chemistry of the copper pthalocyanin-based Alcian blue dyes highlights the adverse effects of commercial secrecy and entrepreneurial dishonesty on the use of these valuable histochemical and biochemical reagents in the biological sciences. The acquisition of detailed and validated chemical structures required the avoidable expenditure of much time that otherwise would have been spent on new research. The combination of this chemical knowledge with experiments on isolated polyanions, using critical electrolyte concentration (CEC) techniques, established a molecular recognition paradigm of Alcian blue usage in histochemistry. New reagents (Cuprolinic and Cupromeronic blues) were synthesised to investigate and support the hypothesis that Alcian blue did not intercalate with DNA. They were made with the important additional aim of using them in electron microscopy of polynucleotides (in the case of Cuprolinic blue) and non-nucleotide polyanions, such as sulphated proteoglycans in extracellular matrices (in the case of Cupromeronic blue). The cationic groups of Alcian blue are easily removed in very mild conditions, producing insoluble blue pigment in situ (ingrain dying). This reaction underlies its unique capability to stain in repeated cycles, thus greatly amplifying sensitivity of detection of substrates present in very small amounts in tissues and on electrophoretic strips etc.

Retention of Jet-Injected Marks on Juvenile Coho and Sockeye Salmon

Abstract Juvenile coho salmon Oncorhynchus kisutch and sockeye salmon O. nerka were marked by jet injection to determine mark retention of three substances: Alcian Blue dye, black india ink, and acrylic paint (red, fluorescent yellow, and fluorescent orange). Fish were marked on the caudal fin and then held for 40 weeks in flow-through freshwater tanks. Coho and sockeye salmon both retained Alcian Blue and fluorescent orange marks best. The highest mark retention after 40 weeks was 73% for Alcian Blue marks on sockeye salmon. Overall mark retention was greater for orange-, yellow-, and black-marked coho salmon than for similarly marked sockeye salmon. Differences in growth rate—sockeye salmon grew about 7 mm more than coho salmon—probably contributed to the differences in mark retention between species. All colors appear suitable for short-term ( 6 weeks) marks, we recommend Alcian Blue dye or fluorescent orange or yellow acrylic paint for coho salmon and Alcian Blue dye fo...

High Molecular Weight Organic Compounds in the Xylem Sap of Mangroves: Implications for Long‐Distance Water Transport

AbstractThe rise of sap in mangroves has puzzled plant physiologists for many decades. The current consensus is that negative pressures in the xylem exist which are sufficiently high to exceed the osmotic pressure of seawater (2.5 MPa). This implies that the radial reflection coefficients of the mangrove roots are equal to unity. However, direct pressure probe measurements in xylem vessels of the roots and stems of mangrove (Rhizophora mangle) grown in the laboratory or in the field yielded below‐atmospheric, positive (absolute) pressure values. Slightly negative pressure values were recorded only occasionally. Xylem pressure did not change significantly when the plants were transferred from tap water to solutions containing up to 1700 mOsmol kg−1 NaCl. This indicates that the radial reflection coefficient of the roots for salt, and therefore the effective osmotic pressure of the external solution, was essentially zero as already reported for other halophytes. The low values of xylem tension measured with the xylem pressure probe were consistent with previously published data obtained using the vacuum/leafy twig technique. Values of xylem tension determined with these two methods were nearly two orders of magnitude smaller than those estimated for mangrove using the pressure chamber technique (−3 to −6MPa). Xylem pressure probe measurements and staining experiments with alcian blue and other dyes gave strong evidence that the xylem vessels contained viscous, mucilage‐ and/or protein‐related compounds. Production of these compounds resulting from wound or other artifactual reactions was excluded. The very low sap flow rates of about 20–50 cm h−1 measured in these mangrove plants were consistent with the presence of high molecular weight polymeric substances in the xylem sap. The presence of viscous substances in the xylem sap of mangroves has the following implications for traditional xylem pressure measurement techniques, development of xylem tension, and longdistance water transport: (1) high external balancing pressures in the pressure chamber are needed to force xylem sap to the cut surface of the twig; (2) stable tensions much larger than 0.1 MPa can be developed only occasionally because viscous solutions provide nucleation sites for gas bubble formation; (3) the frequent presence of small gas bubbles in viscous solutions allows water transport by interfacial, gravity‐independent streaming at gas/water interfaces and (4) the increased density of viscous solutions creates (gravity‐dependent) convectional flows. Density‐driven convectional flows and interfacial streaming, but also the very low radial reflection coefficient of the roots to NaCl are apparently the means by which R. mangle maintains water transport to its leaves despite the high salinity of the environment.

An Agarose Gel Electrophoretic Method for Analysis of Hyaluronan Molecular Weight Distribution

An electrophoretic method is described for determining the molecular weight distribution of hyaluronan (HA). The method involves separation of HA by electrophoresis on a 0.5% agarose gel, followed by detection of HA using the cationic dye Stains-All (3,3′-dimethyl-9-methyl-4,5,4′5′-dibenzothiacarbocyanine). The recommended sample load is 7 μg. Calibration of the method with HA standards of known molecular weight has established a linear relationship between electrophoretic mobility and the logarithm of the weight-average molecular weight over the range of approximately 0.2-6 × 106. The separated HA pattern may also be visualized after electrotransfer of HA from the agarose gel to a nylon membrane. The membrane may be stained with the dye alcian blue. Alternatively, specific detection of HA from impure samples can be achieved by probing the nylon membrane with biotin-labeled HA-binding protein and subsequent interaction with a streptavidin-linked gold reagent and silver staining for amplification. The electrophoretic method was used to analyze HA in two different liquid connective tissues. Normal human knee joint synovial fluid showed a narrow HA molecular weight distribution, with a peak at 6-7 × 106. Owl monkey vitreous HA also showed a narrow molecular weight distribution, with a peak at 5-6 × 106. These results agree well with available published data and indicate the applicability of the method to the analysis of impure HA samples which may be available in limited amounts.

Permeability of the fetal villous microvasculature in the isolated perfused term human placenta.

1. Capillary permeability-surface area (PS) products for the low molecular weight radioactive tracers, 22Na, 51Cr-EDTA (relative molecular mass 357) and 57Co-cyanocobalamin (relative molecular mass 1353) were measured in the fetal circulation of isolated dually perfused lobules of normal term human placentae using the single circulation, multiple-tracer dilution technique. 2. In lobules perfused with M199 medium, containing dextran and 5 g l-1 bovine albumin, the extractions of all three tracers decreased as the flow was increased over the range of 2-8 ml min-1, and PS products for 51Cr-EDTA and 57Co-cyanocobalamin, but not for 22Na, reached constant values at flows above 0.1 ml min-1 g-1. 3. Flow-independent PS products in the presence of albumin were 0.025 +/- 0.002 ml min-1 g-1 (mean +/- S.E.M., n = 25) for 57Co-cyanocobalamin and 0.057 +/- 0.003 ml min-1 g-1 (n = 25) for 51Cr-EDTA. The ratio of PS values (51Cr-EDTA/57Co-cyanocobalamin) was 2.28, while the ratio of the corresponding free diffusion coefficients was 1.79, indicating substantial restriction to the diffusion of the 57Co-cyanocobalamin. 4. In another series of lobules perfused in the absence of albumin, extraction values for all three test tracers were constant over the same flow range. Values at high flow rates were therefore about twice those measured in the presence of albumin, and PS products for all three tracers failed to reach diffusion-limited values. 6. Lobules perfused with and without albumin were fixed using a glutaraldehyde fixative containing 1% Alcian Blue dye. An ultrastructural examination of the endothelium showed no significant changes in cell or cleft morphology, or in the glycocalyx, in the absence of albumin which might account for the observed permeability change. 7. These data are the first physiological measurements specifically characterizing fetal microvascular permeability in the human placenta. The results suggest that permeability resembles that found in skeletal muscle and, as such, the endothelium presents a significant barrier to the diffusion of large solutes. The observed 'protein effect' indicates that albumin can interact with elements of the solute pathway to increase its restrictiveness.

Altered surface character of stretched condom latex

A new type of imperfection in condom latex, present during moderate stretching, was observed by low magnification scanning electron micropscopy. The normally smooth surface of relaxed natural latex was transformed into an accordion-like arrangement of ripples in addition to tears. A corollary experiment with Alcian blue dye placed into both stretched and unstretched condoms leaked no dye. These surface features are consistent with latex acting as a molecular barrier. The tears may represent areas of lowered mechanical resistance and raise questions concerning quality control.

Micro-PIXE quantification of histochemical reactions

The proton induced X-ray emission in combination with a proton microprobe (micro-PIXE) was used for the quantitative determination of a locally bound histochemical reaction end-product. The determination of proteoglycans (PG) content in the cartilage is described in detail. The cartilage from the femur and the skull of 17–19 days old embryos and from ribs of mature CBA strain mice was investigated. The 8 μm cryomicrotome sections, placed on a formvar backing, were incubated in colloidal iron or in alcian blue (dye molecule contains Cu) solutions. The determination of PG contents was made indirectly by assessment of the Fe or Cu concentrations. Differences in the PG contents were observed within all investigated cartilage types. The embryonal cartilage contains more PG than that obtained from mature animals. For the cartilage from the femur the highest content of PG was found in the mineralization front while fully formed bone trabeculae exhibited a PG level 10 times lower. In the case of intramembranaceous ossification the content of PG was also higher in the skull cartilage than in the loose connective tissue. On the basis of the performed studies some methodological conclusions can be drawn. For PG determination, the alcian blue method is more reliable than the colloidal iron technique. The review of the histochemical procedures which may be combined with the micro-PIXE method is also given.

Dietary sodium, glomerular filtration rate autoregulation, and glomerular size distribution profiles in domestic fowl (Gallus gallus)

Mammalian glomerular filtration rate (GFR) autoregulation can be impaired by protocols that inhibit tubuloglomrular feedback, such as high sodium intake. Domestic fowl were fed diets containing either high sodium (0.39% Na: High-Na Group) or low sodium (0.03% Na: Low-Na Group). An arterial snare was used to reduce renal arterial perfusion pressure (RAPP) in a stepwise fashion to evaluate GFR autoregulation. Absolute sodium excretion, fractional sodium excretion (FENa), and ambient systemic arterial blood pressure were significantly elevated in the High-Na Group when compared with the Low-Na Group, and pressure natriuresis was abolished by the Low-Na diet. However, GFR autoregulatory profiles were identical in birds fed High-Na and Low-Na diets, suggesting that tubuloglomerular feed-back does not contribute significantly to avian GFR autoregulation. Filtering glomeruli were stained in vivo with alcian blue dye to determine if RAPP-induced reductions in GFR are associated with cessation of filtration (glomerular intermittency) by a portion of the nephron population. RAPP was held below the GFR autoregulatory range (experimental group) or was at ambient systemic arterial pressure (control group) during glomerular staining. Reducing RAPP below the autoregulatory range reduced GFR by 50%, but similar glomerular size distribution profiles were observed for experimental and control groups. These results indicate that sustained glomerular intermittency does not contribute to the decrease in GFR when RAPP is reduced below the autoregulatory range.

Silver staining of extensively glycosylated proteins on sodium dodecyl sulfate-polyacrylamide gels: Enhancement by carbohydrate-binding dyes

Two methods are described for detecting less than 1 μg of highly glycosylated proteins, such as mucins, on sodium dodecyl sulfate-polyacrylamide gels. They combine commonly employed periodic acid-Schiff (PAS) and Alcian blue dyes with silver stain. Carbohydrate prestaining renders mucins more cationic and favors greater complexation with ionic silver. Comparisons of different mucin samples stained either with PAS-silver or alcian blue-silver indicate differential staining between the two techniques. Such differences may, in part, be due to an affinity of Alcian blue for sulfated glycoproteins. These two staining protocols when used in conjunction with silver staining alone are particularly valuable for assessing sample purity and for detecting contaminating proteins during mucin purification protocols.

Isolation and recovery of acidic oligosaccharides from polyacrylamide gels by semi‐dry electrotransfer

Acidic oligosaccharides derived from glycosaminoglycan heparin were separated by polyacrylamide gradient gel electrophoresis (PAGE). The gel could be visualized using Alcian Blue dye to give a pattern of highly resolved, well defined bands. The particular banding pattern obtained was the result of a heparinase catalyzed depolymerization which afforded oligosaccharide products that differed in size by one disaccharide unit. The separated oligosaccharides could be recovered prior to staining by electroelution onto a positively charged nylon membrane by a semi-dry transfer procedure. Subsequent elution and quantitative recovery of individual oligosaccharides from the membrane was achieved. By using multiple membrane layers a second separation dimension was obtained, resulting in increased oligosaccharide purity proportional to transfer depth. Preparative gradient polyacrylamide gel electrophoresis followed by semi-dry electro-transfer and recovery represents a novel method for the preparation of homogeneous acidic oligosaccharides.

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

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 pH 5.5, the mPAS procedure had improved specificity over the PAPS procedure, and after saponification it could be used to stain O-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 pH 1-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.

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.

Vascular glycosaminoglycans in periventricular leukoencephalopathy

Three cases of adult dementia with periventricular leukoencephalopathy demonstrated staining with alcian blue dye (alcianophilia) in thickened cerebral arteries and arterioles of the abnormal white matter. The property of alcianophilia identifies glycosaminoglycans (GAGs); retention of alcian blue staining at high MgCl2 concentrations (0.7 M) in these cases indicates that the GAGs are highly sulfated and are likely to represent heparan sulfate. Arteriosclerotic risk factors (including hypertension) were absent in all cases, suggesting that GAG deposition occurred as part of a separate cerebral vasculopathic process. These findings suggest that cerebral white matter changes associated with dementia do not always represent Binswanger's disease.

Alcian Blue Staining of Cone Specimens: A Guide to Complete Excision of the Transformation Zone

Alcian blue dye in dilute acetic acid was applied to 54 opened cone biopsy specimens. The dye was found to highlight the position of the squamocolumnar junction by producing a line of demarcation between acetowhite and blue-stained tissue. Following application of the dye, a close correlation was observed between the naked eye and histologic assessments of complete disease excision. This rapid simple staining technique may allow the gynecologic surgeon to assess the adequacy of conization at the time of surgery. (J GYNECOL SURG 5:375, 1989)

Glycosaminoglycans in cortical autopsy samples from Alzheimer brain.

Each of the known classes of mammalian glycosaminoglycans, with the exception of keratan sulphate, was found in cerebral cortex samples from patients with Alzheimer-type dementia and age-matched controls. These molecules were quantitated, after electrophoresis and staining with Alcian Blue dye, by scanning densitometry. No significant differences were found between the mean levels of each of the above glycosaminoglycans in frontal cortex from patients with dementia compared with controls. An increase (26%; p less than 0.05) in the mean level of hyaluronate, but not of other glycosaminoglycans, was found in temporal cortex samples. On the other hand, the uronic acid content of hyaluronate degradation products following Streptomyces hyaluronidase treatment of brain glycosaminoglycans did not reveal any statistically significant changes in Alzheimer's disease. HPLC of disaccharide products from Arthrobacter chondroitinase AC digests did not reveal any significant changes in sulphate substitution of chondroitin sulphate in Alzheimer brain.

Detection and quantitation of proteoglycans extracted from cell culture medium and cultured cartilage slices

Detection and quantitation of extracted proteoglycans, by staining with the dye Alcian blue on cellulose acetate followed by dissolution of the stained cellulose acetate strips in dimethyl sulfoxide containing 0.5% ( v v ) sulfuric acid for absorbance measurement, is described. It is shown that, in the present system, the dye uptake by the proteoglycan is dependant only on the glycosaminoglycan content of the proteoglycan. The method is applied to the quantitation and characterization of proteoglycans and glycosaminoglycans, which have been extracted from radiolabeled bovine ankle cartilage and from mononuclear cell supernatant and which have been separated by DEAE-Sephacel column chromatography. The high sensitivity of the method allows detection of proteoglycans in 25-μl samples of solutions containing as little as 1 μg of glycosaminoglycan per milliliter of solution.

The Effects of γ Radiation on Chondrogenic Development in Vitro

Gamma radiation (0.9-8.0 Gy) was used as a perturbing agent to study factors influencing in vitro chondrogenesis of embryonic chick limb bud cell culture. Chondrogenesis was measured using a number of criteria, including (1) cartilage nodule production, (2) spectrophotometric determination of the amount of bound Alcian blue dye, and (3) computer-assisted analysis of the spatial distribution (area) and density of Alcian blue present in individual micromass colonies. Gamma radiation inhibited both cell proliferation and chondrogenesis in a dose- and time-dependent fashion. Administration of benzamide caused a significant increase in cell proliferation at 0.9 and 2.7 Gy, and in chondrogenesis at all doses. Cartilage nodule production was affected during the first 2 days (prior to 48 h) of culture only, suggesting that chondrocytic commitment occurs during this period. Cultures irradiated at 48 and 72 h produced the same number of nodules as controls, but bound significantly less dye, presumably because of decreased cell numbers and/or cell synthesis products. Computer analysis of micromass colonies provided data similar to those collected spectrophotometrically, but displayed the advantages of (1) increased sensitivity to individual variations, (2) the ability to collect data sets without having to pool three or more colonies, and (3) long-term storage of raw images for later analysis.