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.     

The Bacteroides glycocalyx as visualized by differential interference contrast microscopy

The glycocalyx of eight strains representing six species of Bacteroides was examined by differential interference contrast microscopy. Wet mounts in India ink were prepared from bacteria cultured in broth and on an agar medium; the wet mounts were observed by phase-contrast microscopy and differential interference contrast microscopy. With differential interference contrast microscopy, all bacteria demonstrated a glycocalyx, which included capsules surrounding single cells and microcolonies, strands of glycocalyx connecting cells and microcolonies, detached slime, and solid masses of glycocalyx in which innumerable bacteria were enmeshed. Bacteria showed comparable amounts of glycocalyx by visual observation with differential interference contrast microscopy whether grown on plates or in broth. Serial transfers of cultures did not diminish the amount of glycocalyx. Differential interference contrast microscopy proved to be a superior method to phase contrast for examining wet preparations of Bacteroides.     

人毛乳头细胞组织化学研究

毛乳头细胞是一种高度特殊化的成纤维细胞。本文通过对体外培养的毛乳头细胞进行组织化学染色研究发现,它对阿新蓝、甲苯胺蓝和PAS染色均呈阳性,并对甲苯胺蓝显异染性．与原位时的细胞染色结果相同,表明在体外培养下．毛乳头细胞合成和分泌酸性、中性粘多糖的能力仍能维持较长时间;在细胞聚集区和多层化细胞团中有丰富的细胞外基质,阿新蓝和PAS染色呈强阳性,说明细胞外基质的存在与毛乳头细胞的聚集有很大关系;另外毛囊真皮鞘细胞对阿新蓝、甲苯胺蓝染色呈阳性反应．无甲苯胺蓝的异染性,PAS染色阴性,而真皮成纤维细胞这些染色均阴性,说明它与毛乳头细胞关系密切。     

Alcian blue as a protein stain for sodium alkyl sulfate-polyacrylamide gels: Application to analysis of polypeptide components of the human platelet

The cationic dye, Alcian blue, previously used as a glycoprotein-specific stain on cellulose acetate and polyacrylamide gels, was found to be capable of staining a variety of purified proteins and each of the components of the human platelet presently identifiable with Coomassie blue R or periodic acid-Schiff (PAS) reagent in sodium alkyl sulfate-polyacrylamide gel electrophoretic preparations. Evidence was obtained to indicate that staining of detergent-protein complexes by Alcian blue occurs by virtue of the affinity of the stain for accessible sulfate groups of detergent molecules, especially sodium tetradecyl sulfate, hydrophobically associated with polypeptide chains. Thus, Alcian blue fails to stain nonglycosylated proteins when pure sodium dodecyl sulfate (C₁₂) is used as the detergent, but does so readily when small quantities of sodium tetradecyl sulfate are also present. The advantages of using Alcian blue to determine platelet protein composition and to make quantitative comparisons between bands in sodium alkyl sulfate gels are discussed.     

Ultrastructural characteristics of the external surfaces of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits

The surface structures of the cells of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits were examined by transmission electron microscopy after ruthenium red staining and polycationic ferritin labelling. P. pneumotropica strains ATCC 35149 and K 79114 had slight extracellular fibrous materials associated with cell walls with ruthenium red staining. Ferritin labelling method revealed thick strands or sparsely ferritin-labelled materials on the cell surface of the strains. P. multocida strains Pm-78 and P-2440 had ferritin-labelled capsules surrounded with the cell wall. Strain Pm-78, which was serotyped as A:12, had a thick capsule, whereas serotype -:3 strain P-2440 had a thin and irregular capsule.     

Analysis of Tetrahymena Mucocyst Material with Lectins and Alcian Blue1

ABSTRACT. There are numerous mucocysts in Tetrahymena; however, little is known about their composition, organization, biosynthesis, or function. Mucocysts of Tetrahymena are membrane-bounded vesicles located at the cell cortex. They are torpedo-shaped structures (0.9 μm x 0.3 μm) lined up in longitudinal rows along the surface. It is estimated here that each cell contains about 5000 mucocysts. Mucocyst contents are organized in a crystalline manner, but when that material is released by exocytosis, it swells and forms a gel. Using fluorescence microscopy, we demonstrate that mucocysts contain concanavalin A (Con A)-binding material. First, intracellular fluorescent particles in fixed cells incubated with fluorescein-derivatized Con A (F-Con A) have the same distribution, shape, and orientation as mucocysts in living cells. Also, mucocysts were induced to undergo synchronous exocytosis, and the released material formed a capsule around the cell. The capsule was fluorescent after incubation with F-Con A. In both cases fluorescence was abolished by competition with α methyl mannoside, indicating that Con A is binding specifically to a glycosidic component of the mucocyst. Mucocyst capsules also bind wheat germ agglutinin but not soybean agglutinin, pea lectin, or lentil lectin. Preparations of mucocyst material were analyzed by SDS-PAGE. Silver stain revealed a high molecular weight band that had not previously been detected by Coomassie blue staining. That band also stained with Alcian blue, indicating that it is a mucopolysaccharide. Finally, that same band was shown to be Con A binding. Thus the Con A-binding and Alcian blue-staining properties of mucocysts can be attributed to the same high molecular weight mucopolysaccharide component. This study indicates that it may be possible to purify a specific carbohydrate component of mucocysts which may be helpful in analyzing their function, biogenesis, and structural organization.     

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.     

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.     

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.     

Histochemistry of the duodenal glands of the cat and horse.

The duodenal glands of cat and horse were studied using PAS, Alcian blue, dialysed iron, aldehyde fuchsin-Alcian blue and high iron diamine stains. It was found that the duodenal glands of the horse reacted positively to Alcian blue, dialysed iron stains and also took the Alcian blue stain in the combined aldehyde fuchsin-Alcian blue and high iron diamine-Alcian blue stains. Those of the cat gave negative results. These results suggest the presence of acidic groups in the mucosubstances secreted by the horse's duodenal glands. A suggestion is put forward on the strength of the high iron diamine-Alcian blue combined stains that the acidity is due to the presence of carboxyl groups. It is suggested that the acidity may be significant in either cellulose metabolism or the digestion of the bacterial microflora from the stomach of herbivores.     

Fixation of mucus on rainbow trout (Oncorhynchus mykiss Walbaum) gills for light and electron microscopy

Fixation of mucus for the assessment of biofilms and surface associated pathogens often involves complex and expensive techniques. Rainbow trout killed by an overdose of MS 222 had their gills removed and immersion-fixed gently in buffered glutaraldehyde containing 2% Alcian blue. Control tissues consisted of gills fixed in Alcian blue-free fixative. Trout were also killed and directly immersed in liquid nitrogen and the gills freeze-dried then vapour fixed with osmium tetroxide at −50° C. Following fixation gill tissue was processed for light and electron microscopy. A continuous and intact mucous coat was not detected on tissue fixed by conventional methods but the addition of Alcian blue to the fixative preserved an extensive mucous coat trapped between the lamellae and overlying the epithelia. Electron microscopic examination revealed that mucus preservation with the conventional fixative was poor and intermittent whereas the addition of Alcian blue to the fixative greatly enhanced the preservation of the branchial mucous coat. Mucus appeared as interdispersed flocculant material between the epithelial microridges and formed extensive superficial sheets over the epithelium. Freeze-dried/vapour-fixed gill tissue also provides excellent preservation of the integrity of branchial mucous coats, the mucus appearing as a continuous sheet over the filament and secondary lamellae. However, freeze-dried tissue fails to preserve sufficient cellular integrity for this technique to be useful for light or transmission electron microscopy. The potential for use of glutaraldehyde-Alcian blue fixed-gill tissue diagnostically and in research are discussed.     

Development of the major pathways for neurite outgrowth in the chick hindlimb

To elucidate mechanisms that may control development of the gross anatomical nerve pattern, motoneuron outgrowth into the chick hindlimb was examined using orthograde labeling, scanning and transmission electron microscopy, and Alcian blue staining. Results show that growth cones are not guided by contact with oriented extracellular fibrils, aligned mesenchyme cells, the myotome, or the vasculature. Pathways are not delineated by cell-free space or channels of lower cell density; however, densely packed mesenchyme may form barriers that channel outgrowth. In addition, abundant mesenchymal cell death was seen at the nerve front. This cell death may provide space that encourages growth cone advancement. Pathways often lie along interfaces between areas that stain darkly and lightly with Alcian blue, which specifically stains glycosaminoglycans, and growth cones never penetrate areas that stain intensely, such as the pelvic girdle, which is known to be a barrier to outgrowth. Leading growth cones form specialized contacts with mesenchyme cells, but the predominant contacts are interneuronal. It is proposed that the anatomical pattern of outgrowth is determined by the distribution of preferred substrata, the most preferred substratum being other neurites. Further, neurites tend to prefer loose mesenchyme to dense mesenchyme or areas rich in glycosaminoglycans.     

Haemocytic encapsulation in the locust Schistocerca gregaria (Orthoptera) and in the cockroach Periplaneta americana (Dictyoptera)

Summary The numbers and types of haemocytes in adult male Schistocerca gregaria and Periplaneta americana have been studied in an attempt to explain the differences in thickness of haemocytic capsules formed around abiotic particles in the 2 species. Total and differential haemocyte counts and measurements of blood volume using ³H-inulin indicate that there are 3–4 times more plasmatocytes in the cockroach than in the locust. Although the three main haemocyte types are easily recognised by phase-contrast microscopy, there are few distinguishing ultrastructural characteristics and thus defining the cell types that make up the capsule is difficult. In early capsules in the locust, but not in the cockroach, signs of coagulocyte lysis are apparent, and in both species the bulk of the capsule appears to be made up of granular plasmatocyte-like cells. The relatively thinner capsules formed in the locust might be due to the slow, and limited, recruitment of plasmatocytes to the developing capsule. The material coating completed capsules appears ultrastructurally similar to the subepidermal basement membrane, and both these layers stain with Alcian blue. Once the coating material has formed, the capsule appears to be treated as self by the immunorecognition system.     

The distribution of oxidizable mucosubstances and polysaccharides in the planarian Polycelis tenuis iijima

A chromic acid oxidation-silver technique was used to localize polysaccharide material in Polycelis tenuis at the electron microscope level. In the epithelium, staining was observed within apical vacuoles and on the free surfaces of the cells. A similar staining was observed in relation to the glycocalyx of the pharyngeal epithelia and that of the flame cells. Silver was deposited in the basement membrane. In the parenchyma, the major components giving a positive reaction were the cyanophil and mucous gland cells. Particularly strong silver staining (confirmed by X-ray microanalysis) was observed in the granules and Golgi apparatus of the cyanophil cells. IDPase activity was also found in relation to the Golgi apparatus and its secretory products. The overall distribution of mucopolysaccharide material was confirmed with the PAS and Alcian blue techniques. The fine structural localization of the Alcian blue was also determined using electron microscopy and X-ray microanalysis.     

Lack of correlation between transepithelial transport capacity and paracellular pathway ultrastructure in Alcian blue-treated rabbit gallbladders

The effects of mucosal application of 1 mg% Alcian blue (a trivalent cationic phthalocyanine dye) on functional and ultrastructural parameters of the isolated rabbit gallbladder have been studied. Apart from minor changes in the shape of the group of central microvilli observed in thin-section electron microscopy and scanning electron microscopy, the major ultrastructural change induced by Alcian blue was an almost complete collapse of intercellular spaces in the region above the tight junctions up to the bases of the marginal microvilli as revealed by thin-section electron microscopy. Freeze-fracture electron microscopy demonstrated a complete disappearance of intramembrane particles of neighboring cell membranes corresponding to the region of interspace collapse. Transepithelial electrical resistance (RT) increased from 44.5 to 58.7 ohm . cm2 upon treatment with Alcian blue. This increase could be well accounted for by the observed structural changes in the paracellular pathway if this pathway determines the low resistance of the rabbit gallbladder epithelium. Despite the increase in RT, net mucosa-to-serosa fluid transport and the spontaneous mucosa- positive potential difference of 3 mV were unaltered by Alcian blue treatment, supporting the hypothesis that the transepithelial transport mechanism per se is electroneutral. A calculation of the maximal paracellular mucosa-to-serosa waterflow in response to a lateral intercellular space hypertonicity of 20 mosM demonstrates that in the Alcian blue-treated gallbladder the resulting figure is about three orders of magnitude too low to keep up with the unaltered spontaneous transepithelial net fluid transport. It is therefore concluded that the tight junction pathway in rabbit gallbladders does not serve as a route for net fluid transport.     

Sustained Food Vacuole Formation by Axenic Paramecium tetraurelia and the Inhibition of Membrane Recycling by Alcian Blue

It is believed that the uptake mechanism of some nutrients by Paramecium tetraurelia primarily involves transport through the cell surface, whereas the uptake of other compounds appears to be restricted to bulk transport during food vacuole (phagosome) formation. In this study, we established that, in axenically grown cells, food vacuole formation occurred at continuous rates over long periods. This information allows quantitation of the volume of media taken up by bulk transport. India ink and latex beads were shown to be inert food vacuole markers and carmine was found to have an initial stimulatory effect on phagosome formation rates. Cultures grown for 3.5 h or longer with the glycocalyx stain Alcian Blue, contained only three phagosomes/cell, whereas cells cultured with the other markers contained 15 phagosomes/cell. Electron microscopy of fecal material that accumulated at the bottom of Alcian Blue-grown cells demonstrated the presence of membranes, suggesting that the vacuolar membrane was eliminated during defecation. Neither cell lysis nor the formation of autophagous vacuoles was detected in Alcian Blue-grown cells, indicating that the stain was not cytotoxic at the concentrations used. Thus it appeared that the binding of Alcian Blue to the digestive vacuole membrane resulted in a loss of the vacuole membranes from the cell which reduced the amount of membranes retrieved and recycled and hence eventually reduced the rate of phagosome formation. Alcian Blue-treated cultures exhibited decreased rate of growth and final density, which is consistent with a decrease in bulk transport of nutrients resulting from reduced membranes of digestive cycle organelles available in the cell.     

Sustained food vacuole formation by axenic Paramecium tetraurelia and the inhibition of membrane recycling by Alcian Blue.

It is believed that the uptake mechanism of some nutrients by Paramecium tetraurelia primarily involves transport through the cell surface, whereas the uptake of other compounds appears to be restricted to bulk transport during food vacuole (phagosome) formation. In this study, we established that, in axenically grown cells, food vacuole formation occurred at continuous rates over long periods. This information allows quantitation of the volume of media taken up by bulk transport. India ink and latex beads were shown to be inert food vacuole markers and carmine was found to have an initial stimulatory effect on phagosome formation rates. Cultures grown for 3.5 h or longer with the glycocalyx stain Alcian Blue, contained only three phagosomes/cell, whereas cells cultured with the other markers contained 15 phagosomes/cell. Electron microscopy of fecal material that accumulated at the bottom of Alcian Blue-grown cells demonstrated the presence of membranes, suggesting that the vacuolar membrane was eliminated during defecation. Neither cell lysis nor the formation of autophagous vacuoles was detected in Alcian Blue-grown cells, indicating that the stain was not cytotoxic at the concentrations used. Thus it appeared that the binding of Alcian Blue to the digestive vacuole membrane resulted in a loss of the vacuole membranes from the cell which reduced the amount of membranes retrieved and recycled and hence eventually reduced the rate of phagosome formation. Alcian Blue-treated cultures exhibited decreased rate of growth and final density, which is consistent with a decrease in bulk transport of nutrients resulting from reduced membranes of digestive cycle organelles available in the cell.     

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 microgram 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.     

Characteristics of a clone of endothelial cells derived from a line of normal adult rat lung cells

Summary A strain of endothelial cells derived from a single cell cloned from a line of normal adult rat lung parenchyma has been maintained in tissue culture for more than 3 years. These cells have been identified as endothelial cells based on the combination of their growth characteristics, cell morphology as observed with both light and electron microscopy, and their physiological properties. They have continued to produce granules, which stain specifically for glycosaminoglycans with Alcian blue, for over 2 1/2 years. During the same period of time, glycosaminoglycans were identified biochemically in both cells and medium. They have maintained the ability to degrade bradykinin over this period as well. This work was supported in part by NIH Program Project HL 15832.