The effect of pH on Alcian Blue staining of epithelial acid glycoproteins. II. Human bronchial submucosal gland

Synopsis The effect of pH on Alcian Blue staining of sialomucins and sulphomucins in human bronchial submucosal glands has been analysed. Using Alcian Blue combined with periodic acid-Schiff, lowering the pH was associated with a decrease in the area staining with Alcian Blue and an increase in that staining with periodic acid-Schiff, save in one bronchus with a large sulphomucin content, in which an increase in the area staining with Alcian Blue was found at pH1.0. In all bronchi, an increase in the intensity of Alcian Blue staining was found at this pH. Sialomucin sensitive to sialidase was found to lose Alcian Blue staining at a higher pH than sialomucin resistant to the enzyme. Some sulphomucins stained with Alcian Blue throughout the pH range studied and some only at the more acid pH levels. At pH1.0 Alcian Blue stained only sulphomucins, thus distinguishing them from sialomucins. Alcian Blue staining combined with the high iron diamine technique has enabled three sulphate groups to be identified: one stained with high iron diamine, the other two did not, and, of the latter, one stained with Alcian Blue at pH 2.6 and1.0, and the other only at pH1.0.     

Alcian Blue-stained particles in a eutrophic lake

We used a neutral solution of Alcian Blue to stain transparentparticles in eutrophic Lake Frederiksborg Slotss0, Denmark.Alcian Blue-stained particles (ABSP) appeared to be similarto the so-called transparent exopolymer particles (TEP) identifiedwith an acidic solution of Alcian Blue. Our results on the abundance,size distribution and bacterial colonization of ABSP thereforereflect general patterns of TEP. The abundance of ABSP in thesize range 3–162 µm and retained by 3mu;m pore sizefilters averaged 3.62.4910⁵ ml^–1 (SD), which is amongthe highest concentrations reported for comparable size spectraof TEP. On average, 35 % of ABSP (by number) were colonizedby bacteria and 8.610⁵ bacteria ml^–1 lake water wereattached to ABSP, which corresponds to 7% of the total bacterialabundance.     

Type, location and role of glycosaminoglycans in cloned differentiated chick retinal pigmented epithelium

In clonal culture, colonies of 3–4 week old chick retinal pigmented epithelial cells exhibit Alcian Blue positive extracellular matrix (ECM) material on the surface of the cells. Alcian blue positive ECM is located between undifferentiated cells at the edges of the disc-shaped colonies and beneath the differentiated cells in the colony center. The latter material is associated with the basement membrane. The staining properties suggest that glycosaminoglycans (GAG) are present in these regions. Extraction of GAG from homogenates of colonies, followed by electrophoresis on cellulose acetate strips, results in three bands with mobilities similar to those of hyaluronic acid, heparan sulfate, and chondroitin sulfate, respectively. All three bands label with [³H]glucosamine, and the last two also label with [³⁵S]sulfate. The composition appeared to differ when colonies were grown in different media. Digestion of the GAG preparations with various enzymes suggests that bands II and III represent heparan sulfate and chondroitin sulfate, respectively, in colonies grown in Ham's F10g medium. The composition of band I is as yet undetermined. In minimal Eagle's medium (MEM), bands I and III consisted of hyaluronic acid and chondroitin sulfate, respectively, while band II had properties suggestive of a copolymer of heparan sulfate and an unidentified GAG. Cells release only one [³H]glucosamine-labelled GAG into the medium. This material has a mobility similar to hyaluronic acid and is digested by Streptomyces hyaluronidase, suggesting that it is hyaluronic acid. Staining with Alcian Blue at different pH suggests that it may represent the material associated with the upper surface of the cells. Some of the ECM located between the undifferentiated cells and associated with the basement membrane in the differentiated regions of the colonies stains with Alcian Blue at pH 1.0 and 0.2 suggesting that it may contain GAGs found in bands I and II. Colonies treated with medium containing 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of GAG synthesis, for 48 hr showed a reduced Alcian Blue staining of the ECM in the undifferentiated regions. After 72 hr of treatment with DON, the undifferentiated cells had detached from the plate, whereas the differentiated cells remained intact. The results suggest that the GAG may be involved in cellular adhesion, particularly of the undifferentiated cells.     

A cytochemical approach to the wound repair mechanism inUdotea petiolata (Siphonales)

Summary When injured, the thalli of the coenocytic algaUdotea petiolata undergo a rapid sealing process mainly due to the extrusion of two successive plugs. In the first, external and transitory plug, sulphated polysaccharides are the predominant components. In the second, permanent and internal plug, roundish bodies having a complex polysaccharidic composition are embedded in a fibrillar matrix of still unknown nature. The sulphated sugars were identified and located by means of Alcian Blue staining and X-ray microanalysis. A periodic acid-thiocarbohydrazide-silver proteinate technique proved useful especially in the study of the roundish bodies and in the compositional and structural comparison of the siphon wall with the wound wall. Phosphotungstic acid at low pH was used to evidentiate an extensive plasma membrane activity in the repairing cytoplasm.Supported by a grant of C.N.R.     

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 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 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.     

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.     

Untersuchungen zur Oberfläuchenstruktur von Pasteurella multocida

Detailed studies of the surface structures are an important requirement for the development of efficient vaccines against enzootic pneumonia in calves and piglets caused by Pasteurella multocida. Electron microscopical examination after Alcian Blue staining shows capsular material extending far into the surrounding medium. The extent of the capsules depicted by light microscopy did not correlate with virulence and immunogenicity. However, the extent of the capsules was effected by different culture conditions. The immunization with extract material resulted in a good protection against homologous infection. It has been shown that different cultivation conditions can result in heterogeneity of LPS and in altered OMP-profiles.     

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.     

Studies on exopolysaccharide release by diazotrophic batch cultures of Cyanospira capsulata

Summary Diazotrophic batch cultures of Cyanospira capsulata producing large amounts of a soluble exopolysaccharide (EPS) were studied over a period of about 30 days under continuous illumination. The thickness of the capsule surrounding the trichomes remained almost the same throughout the growth phases and the EPS was continuously released into the medium at a rate which was roughly constant throughout the culture period. A mean EPS productivity of about 6 g m^–2 day^–1 was attained. Purified EPS samples exhibited a saccharidic composition consisting of four neutral sugars (glucose, mannose, fucose and arabinose) and galacturonic acid in a molar ratio of 1:1:1:1:2, respectively. The EPS was also characterized by the presence of pyruvic residues and by a protein content of about 2%. O-Acetyl groups and sulphate residues were not detected. The massive release of this polysaccharidic material into the liquid medium made the cultures progressively more viscous. Offprint requests to: M. Vincenzini     

The use of alcian blue in vivo

Summary Parenteral use of Alcian Blue in rabbits leads to apparent staining of glomerular basement membrane acutely but there is a particularization of the dye in mesangial structures and endothelial cells during the second to fourteenth day following injection. Membranous structures in pancreas, stomach and small intestine are also colored, possibly stained. The effect in the kidney and perhaps in other structures may be a non-specific one, based on filtration of the dye through such a naturally positioned barrier as basement membrane.A comparison of in vivo and in vitro staining patterns showed marked similarity in the deposition of the dye, suggesting specificity of the in vivo staining pattern. But the alteration of staining patterns with time could be construed as arguing against specificity.In the generalized Shwartzman reaction, Alcian Blue stained glomerular occlusive deposits, lending credence to, but not proving, the hypothesis that an acid mucopolysaccharide, with altered fibrinogen, may be implicated in the formation of glomerular aggregates in the Shwartzmann phenomenon.Supported by Research Grant AM 05071, United States Public Health Service, Bethesda, Md., and by the Health Research Council of the City of New York, of which the authors are Career Scientists.     

Spatial and temporal dynamics of the steady-state phytoplankton assemblages in a temperate shallow hypertrophic lake (Lake Manyas,Turkey)

Spatial and temporal dynamics of phytoplankton biomass and species composition in the shallow hypertrophic Lake Manyas, Turkey, were studied biweekly from January 2003 to December 2004 to determine steady-state phases in phytoplankton assemblages. Steady-state phases were defined when one, two or three coexisting species contributed to at least 80% of the standing biomass for at least 2 weeks and during that time the total biomass did not change significantly. Ten steady-state phases were identified throughout the study peiod. During those periods, Achnanthes microcephala (Kützing) Cleve twice dominated the phytoplankton biomass alone and contributed to more than 50% of the total biomass in seven phases. Microcystis aeruginosa (Kützing) Kützing, Anabaena spiroides Klebahn, Cyclotella stylorum Brightwell, Pediastrum boryanum (Turpin) Meneghini and Phacus pusillus Lemmermann were also represented once in steady-state phytoplankton assemblages. A. microcephala was dominant usually during cold periods of the year, while M. aeruginosa and A. spiroides were usually dominant in warm seasons. The total number of species showed a clear decrease during steady-state phases at all stations. All stations were significantly different in terms of the measured physical and chemical parameters (P < 0.05) and phytoplankton biomass (F = 117, P < 0.05).     

Morphological and biochemical characterization of the exocellular investments of polysaccharide-producing Nostoc strains from the Pasteur Culture Collection

A total of 40 Nostoc strains, belonging to the Pasteur Culture Collection and originally isolated from different habitats, were photoautotrophically grown in liquid cultures and tested for the presence of exocellular polysaccharidic investments surrounding the trichomes. However, 25 of them showed a significant presence of these structures, coupled with the release of polysaccharidic material (RPS) into the medium. A rather large number of different morphological forms was observed in the cultures during growth, but at the time of harvesting the predominant morphological form was, in most cases, the vegetative trichome. With regard to the exocellular mucilaginous investments, three main types of morphologies were observed: (i) capsules surrounded by an external pellicle, (ii) capsules with sharp outlines but without an external pellicle, (iii) slimy investments that either loosely surrounded the trichomes without following their shape or were organized in large globular lumps. Among the twenty-five strains that released polysaccharides into the culture medium, three showed mean daily productivities ranging from 30 to 50 mg (RPS) l⁻¹ d⁻¹, values comparable with those of the most productive cyanobacterial strains so far described. The morphological characteristics of the polysaccharidic investments produced by the Nostoc strains seem not to be related to their original habitats. Furthermore, the differences in RPS productivities observed among the strains seem not to be related to the shape of the mucilaginous exocellular investments. Chemical analysis of purified samples of the polysaccharides demonstrated that all the polymers possess an acidic nature, due to the presence of uronic acids, and that they are characterized by the presence of a peptidic moiety and of amino sugars. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.     

Transparent exopolymer particle production and aggregation by a marine planktonic diatom (Thalassiosira weissflogii) at different growth rates

Transparent exopolymer particles (TEP) play an important role in the ocean carbon cycle as they are sticky and affect particle aggregation and the biological carbon pump. We investigated the effect of growth rate on TEP production in nitrogen limited semi‐continuous cultures of the diatom Thalassiosira weissflogii (Grunow) G. Fryxell & Hasle. Steady‐state diatom concentrations and other indicators of biomass (chl a, and total carbohydrate) were inversely related to growth rate, while individual cell volume increased with growth rate. There was no change in total TEP area with growth rate; however, individual TEP were larger at high growth rates and the number of individual TEP particles was lower. TEP concentration per cell was higher at higher growth rates. SYTOX Green staining showed that <5% of the diatom population had permeable cell membranes, with the proportion increasing at low growth rates. However, TEP production rates were greater at high growth rates, refuting our hypothesis that TEP formation is dependent on dying cells with compromised cell membranes in a diatom population. Measurements of particle size distribution in the cultures using laser scattering showed that they were most aggregated at high growth rates. These results indicate a coupling between TEP production and growth rate in diatoms under N limitation, with fast growing T. weissflogii producing more TEP and aggregates.     

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.     

Glycosaminoglycans of disaggregated foetal mouse brain tissue cultures

Synopsis Disaggregated foetal mouse brain tissue cultures were examined for glycosaminoglycans using Alcian Blue and periodic acid-Schiff staining techniques. It was found that spongioblasts (neuron and glial cell precursors) were rich in sulphated glycosaminoglycans, while astrocytes contained little or no sulphated polymers. The chief acid glycosaminoglycans of the brain reportedin vivo, hyaluronic acid, chondroitin sulphate and sialic acid-bearing polymers, were not demonstrated in the mouse brain cultures. There was a decline in glycosaminoglycan content over two weeks in culture, but during the corresponding periodin vivo an increase has been reported. These deficiencies are possibly correlated with the failure of the cultures to myelinate.     

An immunocytochemical study of pituitary cells of the Senegalese sole, Solea senegalensis (Kaup 1858)

Different antisera directed against mammalian and piscine pituitary hormones, as well as a battery of various conventional histochemical techniques (PAS, Alcian Blue pH 2.5, Bromophenol Blue) and lectins, were used to identify the different hormonal cell types in the pituitary of the Senegalese sole, Solea senegalensis. Prolactin and adrenocorticotrophic cells were located in the rostral pars distalis of the pituitary. Gonadotrophic, thyrotrophic and growth hormone cells were distributed in the proximal pars distalis, but gonadotrophic cells appear also at the border of the pars intermedia. Somatolactin cells, as well as α-melanotrophic cells were located in the pars intermedia of the Solea senegalensis pituitary. The PAS reaction was positive in somatolactin cells, which were unreactive with the lead--Haematoxylin technique, whereas melanotrophic cells were positive. Glycoproteins containing mannose and/or glucose, as well as N-acetyl-glucosamine and sialic acid sugar residues, are synthesized and secreted by gonadotrophic, thyrotrophic and somatolactin cells. Adrenocor ticotrophic cells and, especially, the amphiphilic somatolactin and acidophilic growth hormone cells were stained with the Bromophenol Blue technique that identifies proteins in general, but adrenocorticotrophic and growth hormone cells were unreactive towards PAS, Alcian Blue pH 2.5 and lectins (Con A and WGA) This revised version was published online in November 2006 with corrections to the Cover Date.