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.     

Demonstration of sialic acid groups in the glomerular basement membrane of the rat with phosphotungstic acid at low pH

Summary This paper reports an unrecognized aspect of phosphotungstic acid staining at low pH. It provides an on-section staining method in which sialic acid-containing molecules can be demonstrated in the laminae rarae of the rat glomerular basement membrane. The staining in the basement membrane became negative after perfusion with the following cations: protamine sulphate, hexadimethrine, Alcian Blue, Ruthenium Red and Toluidine Blue. Blocking ws not achieved with Alcian Blue at about pH 1. The staining was also abolished after mild methylation and demethylation restored the contrast. This is suggestive of the involvement of carboxyl groups. Prior digestion with pronase, trypsin and neuraminidase rendered the laminae rarae negative, whereas hyaluronidase, chondroitinase ABC and crude heparinase were without effect. This indicates that sialic acid groups are detected by this method and that heparan sulphate does not interfere. The staining of the epithelial plasma membrane, also carrying sialic acid groups, remained positive after neuraminidase treatment. It is presumed that this method can be applied successfully for detecting changes in the sialic acid content of the laminae rarae in rat glomerular basement membranes under normal and pathological conditions.     

Cationic dyes reveal proteoglycans on the surface of epithelial and endothelial kidney cells

The glomerular epithelial cells of the rat kidney fixed by vascular perfusion with an aldehyde solution containing either safranine O or alcian blue (and 0.3 M MgCl2) display filaments which are located close to the outer surface of the plasma membrane. These filaments are similar to those revealed by the same methods in the laminae rarae of the glomerular basement membrane. Alcian blue (and MgCl2) further demonstrates the presence of anionic sites inside the endothelial cell pores of the glomerular and peritubular capillaries, on the luminal surface of endothelial cells of large renal vessels and along the basolateral surface of the epithelial cells of the Bowman capsule and of the proximal convoluted tubule.     

Cationic dyes reveal proteoglycans on the surface of epithelial and endothelial kidney cells

Summary The glomerular epithelial cells of the rat kidney fixed by vascular perfusion with an aldehyde solution containing either safranine O or alcian blue (and 0.3 M MgCl₂) display filaments which are located close to the outer surface of the plasma membrane. These filaments are similar to those revealed by the same methods in the laminae rarae of the glomerular basement membrane. Alcian blue (and MgCl₂) further demonstrates the presence of anionic sites inside the endothelial cell pores of the glomerular and peritubular capillaries, on the luminal surface of endothelial cells of large renal vessels and along the basolateral surface of the epithelial cells of the Bowman capsule and of the proximal convoluted tubule.Supported in part by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 146)     

C3d fragment of complement interacts with laminin and binds to basement membranes of glomerulus and trophoblast

Two mouse monoclonal antibodies generated against human placental homogenate were found to react specifically with human complement component C3. In immunofluorescence of human tissues, these antibodies gave a bright linear staining outlining the glomerular basement membrane of the adult kidney and the trophoblast basement membrane of placenta. An identical staining pattern was observed with a rabbit C3d antiserum which also prevented binding of the monoclonal antibodies to tissue sections. Only negligible basement membrane staining was observed in the same tissues with antisera to human C3c, C5, IgG, IgA, or IgM. When interactions of C3 with basement membrane proteins were tested in enzyme immunoassays and column chromatography, C3(H2O) was found to bind efficiently to solid-phase laminin. Native C3 from fresh plasma did not bind to laminin but C3 from plasma treated with methylamine bound efficiently. When C3 was cleaved with trypsin, C3b and C3d but not C3c bound to laminin-Sepharose. The interaction of C3 and laminin was inhibited by soluble laminin and by high ionic strength. The results indicate that C3d, a biologically active breakdown product of C3, can be found in glomerular and placental basement membranes in the absence of signs for ongoing local complement activation or immune complex deposition. It is possible that binding affinities between C3 and basement membrane molecules, especially laminin, are involved in the retention of C3d at these sites. Such interactions between C3 and components of the glomerular basement membrane could play important roles in complement-related pathological processes of the glomerulus.     

Metachromatic staining and electron dense reaction of glycosaminoglycans by means of Cuprolinic Blue

Summary The cationic phthalocyanin-like dye Cuprolinic Blue, unlike phthalocyanin dyes such as Alcian Blue or Astra Blue, can definitely exhibit a clear metachromatic reaction with appropriate substrates, The application of Cuprolinic Blue to epoxy-embedded semithin sections revealed that mast cell cytoplasmic granules, goblet cell mucin and cartilage matrix stained in violet shades (metachromatic), whereas nuclear chromatin presented a bright blue coloration (orthochromatic). The metachromatic structures showed a high degree of contrast when ultrathin sections treated with Cuprolinic Blue were examined by electron microscopy.Cytophotometric measurements of stained components from the large intestine showed different absorption maxima: at 580 nm for mucin and at 640 nm for nuclei. The spectroscopical analysis revealed a clear-cut metachromatic shift when the dye was in the presence of chondroitin—4-sulphate. The addition of aluminium metal to Cuprolinic Blue solutions resulted in a striking spectral change; under such conditions the dye showed absorption maximum at 530 nm.     

Detecting proteoglycans immobilized on positively charged nylon

Proteoglycans (PG) immobilized on positively charged Nylon 66 are detected readily by staining with Alcian blue. With the exception of hyaluronic acid, free glycosaminoglycans appear unreactive when treated similarly. Immobilization was performed by dot blotting or by electrophoretic transblotting from various gel supports. When transblotted to positively charged Nylon 66 from large-pore agarose-acrylamide gels, levels of 10-50 ng of PG could be detected by Alcian blue staining. This procedure appeared to be nearly 10(2) times more sensitive than staining of gels with toluidine blue. The transblot and staining procedure also appears to be effective with PG separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was applied to a preparation enriched in basement membrane components.     

Alcian blue: an agent for quantitative production of erythrocyte preparations and for understanding erythrocyte changes during aging

The polycationic dye Alcian blue 8 GS fits for a simple quantitative preparation of erythrocyte membrane as well as a tool for judgement of erythrocyte membrane alterations in blood preserves in different age of stage.     

Histochemical localization of protein-polysaccharides in renal tissue

The purpose of this study was to investigate the distribution of protein-polysaccharides in the glomerular and non-glomerular regions of the nephron. The techniques used include the digestion of kidney slices with specific polysaccharidases: neuraminidase, hyaluronidase, chondroitinase ABC, and collagenase followed by several cytochemical techniques to identify the glycosaminoglycans and glycoproteins at the light and electron microscope levels. Differential staining of hyaluronic acid and sulphated glycosaminoglycans was accomplished with Alcian Blue at pH 2.5 and pH 0.5, respectively. Sialoproteins were stained with Alcian Blue at pH 2.5. The periodic acid Schiff’s reaction technique was employed for the visualization of collagen. At the electron microscope level the polysaccharides were identified with the periodic acid-chromic acid-silver methenamine reaction. Our results indicated that the major polysaccharide components of the glomerular basement membrane were sialoproteins and collagen, with smaller amounts of hyaluronic acid and various sulphated glycosaminoglycans. Hyaluronidase digestion resulted in partial detachment of epithelial processes from the glomerular basement membrane indicating the hyaluronic acid may have a role in the stability of the attachment of these processes. Tubular basement membranes also contain sialoproteins and sulphated glycosaminoglycans but in considerably lower concentrations than the glomerular basement membrane. Bowman’s capsule appears to contain mostly sulphated glycosaminoglycans and has a lower concentration of sialoproteins and hyaluronic acid.     

Staining of proteoglycans in mouse lung alveoli. I. Ultrastructural localization of anionic sites

Summary In order to contrast anionic sites, in mouse lung alveoli, two staining procedures were applied: (a) staining with Ruthenium Red and Alcian Blue and (b) staining with Cuprolinic Blue in a critical electrolyte concentration method. The Ruthenium Red-Alcian Blue staining procedure revealed electron-dense granules in the alveolar basement membrane. The granules were closely associated with the epithelial cell membrane and continued to stain even when the procedure was carried out at a low pH, indicating the presence of sulphate groups in the granules.After staining with Cuprolinic Blue, electron-dense filaments, also closely associated with the cell membrane, became visible in the basement membrane of type I epithelial cells. Their length depended on the MgCl₂ concentration used during staining. At 0.4m MgCl₂, the length was mostly within the range 100–180 nm. Using a modified Cuprolinic Blue method, the appearance of the filaments closely resembled that of spread proteoglycan monomers with their side-chains condensed. The basement membrane of type II epithelial cells also contained filaments positive towards Cuprolinic Blue; their length, however, was smaller in comparison with those of type I epithelial cells. The filaments lay in one plane and provided the whole alveolus with an almost continuous sheet of anionic sites. Cuprolinic Blue staining also revealed filaments in the basement membrane of the capillary endothelial cells. Furthermore, Cuprolinic Blue-positive filaments (average length about 40 nm) became apparent in close contact with collagen fibrils and separated from each other according to the main banding period of the collagen fibrils (about 60 nm), indicating a specific ultrastructural interaction between these two components. Filaments connecting collagen fibrils with each other were also detected.     

Proteoglycans in arterial smooth muscle cell cultures: an ultrastructural histochemical analysis

The extracellular matrix in cultures of arterial smooth muscle cells has been examined by ultrastructural histochemistry using each of the following cationic dyes: ruthenium red, Alcian blue, acridine orange, and safranin O. All dyes exhibited an affinity for a structural component that was either preserved as a granule with ruthenium red or Alcian blue, or as an extended filament or bottlebrush structure with acridine orange or safranin O. Both granules and filaments were removed when the cultures were pretreated with chondroitinase ABC, an enzyme that degrades the glycosaminoglycan moiety of some proteoglycans. These structural components of the extracellular matrix were not observed when cultures were prepared in the absence of the cationic dyes. Labeling experiments (35S-sulfate) revealed that approximately 40% of the total labeled proteoglycans were lost during routine processing for electron microscopy (i.e., fixation through dehydration). Inclusion of any one of the cationic dyes during fixation reduced the losses to less than 1%. The extended filamentous structure preserved by safranin O and acridine orange resembled the structure of purified proteoglycans prepared from the same cultures and spread on cytochrome c monolayer films. These observations suggest that proteoglycans exist as extended bottlebrush structures within the extracellular matrix, and support the interpretation that the granular deposits observed in the ruthenium red and Alcian blue preparations most likely represent individual proteoglycan monomers that have undergone molecular collapse during processing. In addition, the dyes also exhibited an affinity for chords of fine fibrils that contained small granules and/or filaments. Both the fibrillar material and the associated granular and filamentous structures enmeshed in the fibrils resisted digestion with chondroitinase ABC.     

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.     

Immunohistochemical distribution of type VI collagen in developing human kidney

Summary The distribution of type VI collagen was investigated immunohistochemically in the developing human kidney from 15 to 32 weeks gestational age and it was compared with that observed in the normal infantile and adult human kidney. In fetal kidney, type VI collagen was widely distributed as a fibrillar network in the subcapsularly undifferentiated mesenchyme and intertubular interstitium, and as a basement membrane-like structure around the ureteral bud branches, tubules, and collecting ducts. During nephrogenesis, type VI collagen disappeared from the induced mesenchyme close to the tips of ureteral branches, while it formed a distinct basement membrane-like structure around the early stages of nephron differentiation (comma-shaped and S-shaped bodies) and later along Bowman's capsule of capillary loop and maturing glomeruli. A strong immureactivity for type VI collagen was also found in the glomerular basement membrane and mesangial areas of capillary loop and maturing glomeruli. In infantile kidney, type VI collagen showed a distribution pattern similar to that observed during the fetal period. In adult human kidney, glomerular basement membrane showed a weak positivity for type VI collagen and the basement membrane-like staining around Bowman's capsule, tubules, and collecting ducts was less evident than in fetal and infantle kidney. Our immunohistochemical findings suggest that type VI collagen is a normal component of the glomerular and extraglomerular extracellular matrix of developing human kidney and that it undergoes changes in the expression during maturation.     

Crystallization of the gastric H,K-ATPase

Crystalline arrays of the gastric H,K-ATPase were obtained in membrane preparations from hog and rabbit gastric mucosa. The lattice was formed rapidly in a medium containing K+, vanadate, Mg2+, and dimethyl sulfoxide at pH 6.0-6.9 in imidazole buffer from 4 to 22 degrees C. The crystal lattice exhibited P2 symmetry, and the unit cell dimension (a = 5.6, b = 11, and c = 10 nm) could accommodate 2 polypeptides of mass 116-129 kDa. In addition, the isolated preparation contained previously undescribed long cylindrical structures 16 nm thick. These structures consisted of a central core 6-7 nm wide from which particles spaced 5.5 nm apart protruded symmetrically.     

Abnormal development of the notochord and perinotochordal sheath in duplicitas posterior, patch and tail-short mice

Interest in developmental interactions involving the notochord and perinotochordal sheath led to a comparative investigation of these structures in three mouse mutants. Alcian blue or periodic acid-Schiff staining of 9 1/2-13 days' gestational age embryos revealed a supernumerary notochordal-like mass of cells or a deflected notochord in association with duplication of the neural tube in mice of the duplicitas posterior stock. The perinotochordal sheath and basement membrane of the accessory notochordal masses were frequently defective. Patch and Tail-short embryos were also utilized for study by means of light microscopy using Alcian blue staining. In Patch embryos, although the notochord was sometimes compressed dorso-ventrally, it had an intact perinotochordal sheath and a defined, but undulated, basement membrane. Mesenchymal cells between the notochord and neural tube were occasionally replaced by cell-free space. In contrast, in Tail-short embryos a poorly formed, lightly staining or totally absent notochordal sheath was revealed. Indeed, it was sometimes difficult to distinguish the notochord from surrounding mesenchymal cells. In both the Patch and Tail-short embryos the notochord was also deflected from its medial position. In the three mutants studied, the direct or indirect effect of gene action appeared to be on the notochord and perinotochordal sheath, and the important role of these structures in abnormal axial development was established.     

Ultrastructural organization of the glomerular basement membrane as revealed by a deep-etch replica method

Summary The fine structure of the glomerular basement membrane was re-evaluated by using a deep-etch replica method.The structure of the laminae rarae interna and externa of the rat glomerular basement membrane was basically identical in that 6 to 8 nm fibrils were interconnected to form a three-dimensional, polygonal network. The size of the mesh was quite variable but most often ranged from 20 to 25 nm in width. In addition, a zipper-like substructure of the epithelial slit diaphragm was observed. By contrast, the lamina densa was composed of closely packed particles.After exposure of the bovine glomerular basement membrane to ultrasonic waves or trypsin, the particles of the lamina densa were effectively removed. The underlying structure showed the fibrillar network closely resembled that seen in the laminae rarae of the rat glomerular basement membrane.The glomerular basement membrane thus revealed was as principally composed of a fibrillar network, which might be regularly arranged units of type-IV collagen. Numerous fine particles, most likely proper components of the glomerular basement membrane, were attached onto this basic fibrillar structure, giving rise to a morphologic appearance different from that of the laminae rarae.     

Ultrastructure of the secretory cells of the submucosal glands in the human maxillary sinus

Tissue samples obtained from the lateral wall of the maxillary sinuses of five patients were examined by light microscopical, histochemical, and ultrastructural techniques. Submucosal glands were tubulo-alveolar mixed glands. The acini consisted of either all serous or all mucous cells, or a mixture of both. Serous granules were stained by toluidine blue, or by hematoxylin and eosin (H and E), but showed little or no reaction with periodic acid-Schiff (PAS) or Alcian blue. Mucous granules were pale in toluidine blue or H and E preparations, and consisted primarily of acid mucosubstances, as demonstrated by their staining reaction with PAS and Alcian blue. At the electron microscope level, the serous granules were either homogeneously dense, or showed a substructure consisting of at least two layers of distinctly different electron-opacity. Typical mucous droplets consisted of a fibrillar network dispersed in a translucent matrix. A second secretory product was present in the mucous cells in the form of elongated, membrane-bounded structures containing numerous parallel filaments, which measured about 55 Å in diameter. The mucous droplets and the filamentous bodies appear to arise from the opposite faces of the Golgi complex in the mucous cells. The filamentous bodies showed a pronounced tendency to fuse with the mucous droplets. All acini were surrounded by a well-defined myoepithelial layer and contained intercellular nerve terminals.     

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.     

Simultaneous Preparation and Quantitation of Proteoglycans by Precipitation with Alcian Blue

Conditions for specific interaction between Alcian blue and proteoglycans were optimized by comparing the differential spectra of Alcian blue obtained with purified chondroitin sulfate dissolved in water with the spectra obtained with nasal cartilage proteoglycans dissolved in synovial fluid. A method was then designed that provides specific precipitation of proteoglycans or glycosaminoglycans in 4 M guanidine-HCl in the presence of protein, hyaluronic acid, or nucleic acids. The specificity is achieved by using a low pH in combination with detergent and high salt concentration. Stepwise addition of reagents is necessary to avoid binding of Alcian blue to proteins and nucleic acids. All polyanions, except polysulfates, are first neutralized by lowering the pH to 1.5. By including detergent in this step, the hydrophobic protein regions are blocked and not accessible for binding with the dye. These regions could otherwise bind Alcian blue by hydrophobic interaction. When the Alcian blue reagent is added after, only the polysulfated molecules will remain charged and free to interact with Alcian blue. At least 0.4 M guanidine-HCl is required to abolish the negative interference by proteins. All sulfated glycosaminoglycans are precipitated at 0.4 M guanidine-HCl. With increasing guanidine-HCl concentrations, the different glycosaminoglycans are precipitated in accordance with the critical electrolyte concentration of the respective glycosaminoglycan. The Alcian blue precipitation can be performed at different concentrations of guanidine-HCl in order to separate different classes of proteoglycans. Excess dye and contaminating proteins are removed by a wash in a DMSO-MgCl₂ solution and the precipitate is dissolved in a mixture of guanidine-HCl and propanol. For quantitation, the absorbance is recorded in a microplate reader with the 600-nm filter, the assay being linear between 0.5 and 20 μg proteoglycan. Since no digestion of samples with protease is needed, the proteoglycans are recovered in native form. The proteoglycan-Alcian blue complexes dissociate in the guanidine-HCl/propanol mixture and the proteoglycans can be selectivelyprecipitated with propanol. The dye is used for quantitation and the proteoglycans can be utilized for further analysis.     

Effect of brown spider venom on basement membrane structures

Loxoscelism or necrotic arachnidism are terms used to describe lesions and reactions induced by bites (envenomation) from spiders of the genus Loxosceles. Envenomation has been reported to provoke dermonecrosis and haemorrhage at the bite site and haemolysis, disseminated intravascular coagulation and renal failure. The purpose of this work was to study the effect of the venom of the brown spider Loxosceles intermedia on basement membrane structures and on its major constituent molecules. Light microscopy observations showed that L. intermedia venom obtained through electric shock, which reproduces two major signals of Loxoscelism in the laboratory, exhibits activity toward basement membrane structures in mouse Engelbreth-Holm-Swarm (EHS) sarcoma. Basement degradation was seen by a reduced periodic acid-Schiff (PAS) and alcian blue staining as well as by a reduced immunostaining for laminin when compared to control experiments. Electron microscopy studies confirmed the above results, showing the action of the venom on EHS-basement membranes and demonstrating that these tissue structures are susceptible to the venom. Using purified components of the basement membrane, we determined through SDS-PAGE and agarose gel that the venom is not active toward laminin or type IV collagen, but is capable of cleaving entactin and endothelial heparan sulphate proteoglycan. In addition, when EHS tissue was incubated with venom we detected a release of laminin into the supernatant, corroborating the occurrence of some basement membrane disruption. The venom-degrading effect on entactin was blocked by 1,10-phenanthroline, but not by other protease inhibitors such as PMSF, NEM or pepstatin-A. By using light microscopy associated with PAS staining we were able to identify that 1,10-phenanthroline also inhibits EHS-basement membrane disruption evoked by venom, corroborating that a metalloprotease of venom is involved in these effects. Degradation of these extracellular matrix molecules and the observed susceptibility of the basement membrane could lead to loss of vessel and glomerular integrity, resulting in haemorrhage and renal problems after envenomation.