Histochemistry of carbohydrates in the epithelium lining the bulbourethral gland of the rat

The histochemistry of carbohydrates has been studied in the epithelium lining the bulbourethral gland of the rat by means of light- and electron-microscopic methods. The results obtained show that the cytoplasms of the epithelial cells contain neutral and acidic carbohydrates. The neutral carbohydrates exhibited positive reactions with periodic acid-Schiff and periodic acid-thiosemicarbazide-silver proteinate, whereas the acidic carbohydrates reacted positively with alcian blue (AB; pH 1.0 and 2.5) and dialyzed iron. Most neutral carbohydrates were found to be glycoproteins localized within the secretory granules. The acidic carbohydrates consist of at least two types, AB (pH 1.0)-reactive sulfated and AB (pH 2.5)-reactive nonsulfated carbohydrates; most nonsulfated carbohydrates were determined to be sialic acid. The acidic carbohydrates were also localized within the secretory granules.     

A postembedding staining method of intensifying alcian blue reactions of acidic glycoconjugates with phosphotungstic acid in electron microscopy

For the effective visualization of acidic glycoconjugates in electron microscopy, a post-embedding staining method has been devised for intensifying their alcian blue (AB) reactions by means of phosphotungstic acid (PTA). Tissue samples were prepared by glutaraldehyde-paraformaldehyde fixation of pieces of the trachea, aorta, and colon from adult rats. LR-White resin-embedded ultrathin sections were stained first with AB (pH = 1.0 or 2.5) and then reacted for PTA. In the tissues examined, the AB reaction of acidic glycoconjugates involved was effectively intensified by subsequent PTA staining in nearly all of the ultrastructures known to contain such carbohydrates. The majority of these ultrastructures failed to show any pronounced densities, if stained singly with PTA under the identical staining conditions. In all the ultrastructures, a series of selective methods such as active methylation and digestion with testicular hyaluronidase or neuraminidase have substantiated the selectivity of the PTA intensified AB reactions for acidic glycoconjugates involved. The present PTA intensified AB method resulted virtually in no contaminations of the backgrounds and can be regarded as a reliable and useful technique for the effective visualization of both intra- and extracellular acidic glycoconjugates in electron microscopy.     

Tunicate Endostyles: Histochemistry of Acidic Glycoproteins Re-examined in Ciona intestinalis and Styela plicata

Previous reports of tunicate endostyles have suggested that they contain little or no acidic glycoproteins in the glandular zones. The endostyles of Ciona intestinalis and Styela plicata were examined after anhydrous fixation with cyanuric chloride. Polyanions were stained with alcian blue (AB) at pH 2.5 or azure A, while sulfomucins were stained with high-iron diamine (HID) or AB at pH 1.0. Endostyles were also tested for sensitivity to acid hydrolysis (AH) and saponification. In Ciona zones 2 and 4 sometimes demonstrated positive HID and AB 1.0 responses. Almost invariably zone 6 was AB+ at pH 2.5; zones 2 and 4 were frequently responsive to AB, but less intense. Each of these 3 zones, when AB+, was sensitive to AH. Responses by zones 3 and 5 to AB (pH 2.5), azure A and saponification suggest that these zones contain mostly nuclear material. In secretory zones 2, 4 and 6 histological responses are consistent with the histochemistry of sialomucins. Zones 1 and 8 had sulfated material in the apical edges in both animal groups. Among the fixatives used for Ciona, only anhydrous fixation demonstrated most of the positive responses to polyanion-sensitive stains.     

The histochemistry of urea-unmasked glycosaminoglycans in the skin of the eel, Anguilla japonica

In the connective tissues of the dermis and subcutis of the eel skin, the histochemistry of urea-unmasked glycosaminoglycans has been studied by means of combined staining and enzyme digestion procedures. The staining procedures employed were alcian blue (AB) pH 1.0, AB pH 2.5, aldehyde fuchsin (AF), periodic acid-Schiff (PAS), AB pH 2.5-PAS, high iron diamine (HID) and low iron diamine (LID) methods, whereas the enzymes used were Streptomyces and testicular hyaluronidases, chondroitinases ABC and AC and keratanase. The results obtained have shown that a substantial amount of dermatan sulfate and a relatively small amount of hyaluronic acid, chondroitin, chondroitin sulfate A and/or C were the glycosaminoglycans involved in the connective tissues of the eel skin and that the tissues were devoid of keratan sulfate.     

An interesting case of colloidal iron positivity

Summary An interesting case of a colloidal iron (CI) positive basophilic substance in the adrenal medullary cells of amphibia and reptilia is reported here. The substance, however, does not stain by alcian blue (AB). It is negative to PAS, Azure A, aldehyde fuchsin, AB at pH 1 and MgCl₂ — AB though orthochromatically stained by toluidine blue at pH 3. More work is needed to establish the exact nature of the CI positive material.     

Alcianblue/PAS or PAS/alcianblue? Remarks on a classical technic used in carbohydrate histochemistry

The combined alcian blue (AB)/PAS technic is widely used for the detection and characterization of mucosubstances in tissue sections. Mostly the sequence AB/PAS is used, occasionally also the reserved sequence PAS/AB. The present study shows clearly that the sequence of the combined technic, i.e. AB/PAS or PAS/AB is substantially influencing the results. So it could be demonstrated that by using the combination PAS/AB originally PAS-positive and AB-negative reacting mucosubstances become AB-positive. This could be caused by periodic acid oxidation followed by addition of hydrogen sulfite to aldehyde group thus providing secondary basophilic resp. AB positive material.     

Alcianblue/PAS or PAS/alcianblue?

Summary The combined alcian blue (AB)/PAS technic is widely used for the detection and characterization of mucosubstances in tissue sections. Mostly the sequence AB/PAS is used, occasionally also the reserved sequence PAS/AB. The present study shows clearly that the sequence of the combined technic, i.e. AB/PAS or PAS/AB is substantially influencing the results. So it could be demonstrated that by using the combination PAS/AB originally PAS-positive and AB-negative reacting mucosubstances become AB-posltive. This could be caused by periodic acid oxidation followed by addition of hydrogen sulfite to aldehyde group thus providing secondary basophilic resp. AB positive material.     

Isolation of a domain of the plasma membrane in Chinese hamster ovary cells which contains iodinatable, acidic glycoproteins of high molecular weight

A light vesicle fraction, apparently derived from the plasma membrane, was obtained following breakage of Chinese hamster ovary (CHO) cells by means of a fluid pump disrupting device. The final preparation was enriched approx. 40-fold over the homogenate in K+,Na+-stimulated ATPase and phosphodiesterase I, but only approx. 10-fold in 125I specific radioactivity after lactoperoxidase-catalyzed iodination. This preparation was compared with another plasma membrane fraction purified as large sheets via a two-phase centrifugation procedure. Two-dimensional polyacrylamide gel electrophoresis followed by Coomassie blue staining indicated that both fractions were fairly similar in polypeptide composition, although a few consistent differences were evident. However, staining of glycoproteins by the periodic acid-Schiff technique or by overlaying with 125I-labeled concanavalin A showed that the vesicle fraction was highly enriched in groups of high molecular weight, acidic glycoproteins which stain only weakly with Coomassie blue. These glycoproteins also bound 125I-labeled ricin I agglutinin and wheat germ agglutinin. They appear to be the major receptors for wheat germ agglutinin on the CHO cell surface. After surface labeling of cells by the 125I-lactoperoxidase technique, the membrane sheet fraction contained a large number of iodinated polypeptides, whereas labeling in the vesicle fraction was restricted almost entirely to the high molecular weight, acidic glycoproteins. It is proposed that the vesicle fraction constitutes a specific domain of the cell surface which is coated on its exterior by this group of glycoproteins. These components probably mask underlying proteins of the plasma membrane from external labeling.     

Concanavalin A-perioxidase-diaminobenzidine (Con-A-PO-DAB)-alcian blue (AB): a reliable method for dual staining of complex carbohydrates.

A method has been established for the dual staining of complex carbohydrates in light microscopy. It is a combined concanavalin A-peroxidase-diaminobenzidine (Con-A-PO-DAB)-alcian blue (AB) (pH 2.5) method, and with this method it is possible to color alpha-D-glucosyl and alpha-D-mannosyl residues and acidic groupings of complex carbohydrates in tissues brown and blue respectively. Histochemical experiments using histological sections with reactive complex carbohydrates and casein films containing carbohydrates of known chemical structure have substantiated the validity of the above significance of the dual staining. Thus, the present dual staining method is a reliable one and a new addition to a series of dual staining techniques hitherto employed in the light microscopic histochemistry of complex carbohydrates.     

The use of peroxidase-labelled Limulus polyphemus agglutinin for the histochemistry of sialic acid-containing glycoproteins in light microscopy.

Synopsis The usefulness of a lectin,Limulus polyphemus agglutinin (LPA) has been tested in a series of mammalian tissues with sialic acid-containing glycoproteins. In nearly all the tissues employed, the positive peroxidase-labelled LPA diaminobenzidine (LPA-PO-DAB) reaction of various histological structures was markedly diminished in intensity or abolished, following digestion with neuraminidase. In the same tissues, sialic acid added with LPA-PO abolished the LPA-PO-DAB reaction or notably suppressed its intensity. In the majority of the tissues tested, the LPA-PO-DAB-Alcian Blue (AB) (pH 1.0 or 2.5) procedures appear to be useful dual staining methods which enable one to colour selectively sialic acid-containing and other acidic carbohydrates. In view of the endogenous peroxidase activity in particular histological structures, however, appropriate control staining procedures should be performed when the LPA-PO-DAB procedure is employed, either alone or in combination with AB procedures, to determine the histochemical properties of sialic acid-containing glycoproteins.     

Mucopolysaccharide histochemistry of rat tooth germs

Summary The histochemical distribution and localization of acid mucopolysaccharides and glycoproteins have been studied in rat tooth germs and lower joints. After fixation, sections were stained with PAS, alcian blue-PAS, colloidal iron, colloidal iron-PAS, colloidal iron-Feulgen, azure A and safranin O. Prior to staining, sections were incubated in hyaluronidase, pepsin and papain. A 2 hour incubation in pepsin greatly enhanced AB or CI basophilia in the stellate reticulum and the enamel-dentinal junction. These findings demonstrated that some acidic radicals of the polysaccharides are partially blocked by basic proteins. Hyaluronidase did not completely destroy the alcian blue or colloidal iron positive material whereas azure A metachromasia was completely removed. These results indicated that AB and/or CI stained substrates different from those revealed by azure A.With 10 Figures in colourThis work was supported by Grant No. D-1325 of the National Institutes of Health, Bethesda, Maryland.On leave of absence at the University of Rome Medical School, Viale Regina Elena 287-A, Rome, Italy.     

Anisotropic staining of apurinic acid with toluidine blue.

Using normal rat liver imprints, studies were carried out on the effects of histone extraction and the formation of aldehyde groups from deoxyribose on anisotropic toluidine blue staining of depurinized DNA after sodium bisulfite treatment. The anisotropic effect of bisulfite was found to be determined by binding of bisulfite ions to the aldehyde groups of apurinic acid which, together with free phosphate groups of DNA ensure coparallel attachment of the dye molecules. It was also shown that at pH 5.0 toluidine blue binds with both the phosphate and aldehyde groups of apurinic acid, to give anisotropic staining.     

Anisotropic staining of apurinic acid with toluidine blue

Summary Using normal rat liver imprints, studies were carried out on the effects of histone extraction and the formation of aldehyde groups from deoxyribose on anisotropic toluidine blue staining of depurinized DNA after sodium bisulfite treatment. The anisotropic effect of bisulfite was found to be determined by binding of bisulfite ions to the aldehyde groups of apurinic acid which, together with free phosphate groups of DNA ensure coparallel attachment of the dye molecules. It was also shown that at pH 5.0 toluidine blue binds with both the phosphate and aldehyde groups of apurinic acid, to give anisotropic staining.     

The oxidative generation of sulfonic acid groups in neuromelanin and lipofuscin in the human brain

Sulfonic acid groups were oxidatively generated in the soluble lipid-free lipofuscin component of neuromelanin of human substantia nigra and in lipofuscin of human inferior olive. Exposure of these oxidized, intraneuronal pigments to low pH Alcian blue or aldehyde fuchsin demonstrated an intensity of staining that related to the type of oxidant and the conditions of its use. Utilization of the following oxidants generated increasingly strong staining reactions as signified by the following sequence; periodic acid under mild conditions, bromine in carbon tetrachloride, hydrogen peroxide, periodic acid under drastic conditions, potassium permanganate followed by oxalic acid, hydrogen peroxide followed by bromine in carbon tetrachloride, potassium permanganate followed by metabisulfite or bisulfite, and performic acid. Neither Alcian blue nor aldehyde fuchsin revealed oxidatively generated aldehyde as judged by 1) their failure or near failure to stain inferior olive lipofuscin following mildly applied periodic acid, and 2) the increase in staining intensity, from moderate to strong, displayed by the soluble lipid-free lipofuscin component of neuromelanin and by inferior olive lipofuscin when potassium permanganate was followed by a rinse with metabisulfite or bisulfite in place of one with oxalic acid.     

Secretory activity of hamster tracheal explants and isolated tracheal epithelial cells and the effects of cystic fibrosis serum

Serum from cystic fibrosis patients has been shown by scanning electron microscopy to cause release of large quantities of mucus from the cultured tracheal rings of 3–4-month-old male Golden Syrian hamsters. In order to study this phenomenon on single cells, an epithelial (HTE) cell culture has been established from the hamster tracheal rings using the cell rescue method of Goldman and Baseman (1980a, In Vitro, 16:313). The cells were demonstrated to be epithelial by histochemical staining and immunofluorescent detection of laminin. Proteins secreted by HTE cells were partially characterized and shown to consist, at least in part, of acidic glycoproteins. The proteins were precipitated by addition of buffered alcian blue (AB) to the cell-free medium under conditions in which all of a polyanionic protein [³H]-labeled mucin, was precipitated without carrier. [¹⁴C] galactosamine-labeled AB precipitate was β-eliminated and, after neutralization and centrifugation, the material in the supernatant was sized by chromatography on a calibrated Bio-Gel P₂ column. The label eluted with a molecular weight close to a disaccharide. HTE cells pulse--labeled for 1.0 hr with [³H] leucine or [¹⁴C] galactosamine secreted increasing amounts of labeled glycoprotein during the chase. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography of labeled AB precipitates revealed three major bands, two with molecular weights greater than 100 kd. Secretion was stimulated by retinoate (50% increase), but not by retinol. Exposure of HTE cells to whole sera from cystic fibrosis patients resulted in heightened secretion rates as compared to results obtained with normal sera. Heterozygote sera produced secretion rates intermediate between the two extremes.     

Quantitative determination of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Quantitative determination of the sulfated glycoproteins present in tissue and secretion fluid was performed. After digestion of the specimen with pronase in order to convert glycoproteins to glycopeptides, the sulfated glycopeptides were separated from a mixture of acidic glycans (glycosaminoglycans, sialoglycopeptides and sulfated glycopeptides) by two-dimensional electrophoresis on cellulose acetate membrane [(1986) J. Biochem. Biophys. Methods 12, 239-246]. After staining with alcian blue, the spot of sulfated glycopeptide on the cellulose acetate membrane was cut out, and then only the dye bound to the sulfated glycopeptide was extracted with a 5% cetylpyridinium chloride solution at 100 degrees C for 15 min. The extract was then measured by absorbance at 615 nm using an authentic sulfated glycopeptide as a standard. This method facilitated the determination of sulfated glycopeptides, which were separated from other acidic glycans, within the range 0-25 micrograms.     

Studies on the molecular arrangement of RNA in tissues with a selective topo-optical reaction of RNA.

Selective demonstration of RNA in tissues was achieved by treating tissue sections with potassium permanganate followed by bisulfite and toluidine blue at pH 1.0 (PBT reaction). It is suggested that this reaction is due to aldehyde groups which are formed by the oxidative cleavage of the pyrimidine rings of RNA which can be selectively demonstrated using bisulfite-toluidine blue as the aldehyde reagent. The specificity of the reaction was tested after RNAase treatment, after acid hydrolysis, and on pure RNA droplets. The aldehyde nature of the reacting groups was checked, after permanganate oxidation, by Schiff's leucofuchsin reagent, and by aldehyde blocking reactions. Two types of intracellular molecular arrangement of RNA molecules could be distinguished by polarization optics after application of the PBT reaction: 1) The strong birefringence, dichroism and metachromatic staining of membrane-bound RNA in ergastoplasm of pancreas, liver and plasma cells indicate a linear (planar) molecular order of RNA molecules on the surface of the membranes, and 2) the isotropic, basophilic staining of RNA not organized in membrane structures (Nissl substance, nucleoli) suggest a random distribution of their dye binding sites.     

Studies on the molecular arrangement of RNA in tissues with a selective topo-optical reaction of RNA

Summary Selective, demonstration of RNA in tissues was achieved by treating tissue sections with potassium permanganate followed by bisulfite and toluidine blue at pH. 1.0 (PBT reaction). It is suggested that this reaction is due to aldehyde groups which are formed by the oxidative cleavage of the pyrimidine rings of RNA which can be selectively demonstrated using bisulfite-toluidine blue as the aldehyde reagent.The specificity of the reaction was tested after RNAase treatment, after acid hydrolysis, and on pure RNA droplets. The aldehyde nature of the reacting groups was checked, after permanganate oxidation, by Schiff's leucofuchsin reagent, and by aldehyde blocking reactions.Two types of intracellular molecular arrangement of RNA molecules could be distinguished by polarization optics after application of the PBT reaction: 1) The strong birefringence, dichroism and metachromatic staining of membrane-bound RNA in ergastoplasm of pancreas, liver and plasma cells indicate a linear (planar) molecular order of RNA molecules on the surface of the membranes, and 2) the isotropic, basophilic staining of RNA not organized in membrane structures (Nissl substance, nucleoli) suggest a random distribution of their dye binding sites.     

Nuclear stains with soluble metachrome metal mordant dye lakes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues.

Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe2+ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO4 sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control. Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe2+ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results. Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes. The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes. Benzil at pH 13, which prevents the beta-naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.