Histochemistry of glycosaminoglycans in cartilage ground substance. Alcian-blue staining and lectin-binding affinities in semithin Epon sections

The critical-electrolyte-concentration staining method using Alcian blue (AB) was applied to etched semithin Epon-embedded sections. The distribution of various glycosaminoglycans (GAGs) was studied in hyaline, elastic, cellular and fibrous cartilage obtained from humans and rodents. The staining patterns in semithin sections were found to correspond to those obtained using paraffin-embedded material. Lectin histochemistry was performed on consecutive sections. The following peroxidase-labelled lectins were used: Ricinus communis A I, Arachis hypogaea, Ulex europaeus A I, Triticum vulgaris, Helix pomatia, Limax flavus, and concanavalin A. The lectin-binding capacity of cartilaginous ground substance was found to be low, as was expected on account of the few free sugar residues of GAGs. Chondroitin sulphate, the most widely distributed GAG, did not exhibit lectin staining. The lectin-binding sites (positive staining for all lectins tested except H. pomatia) observed corresponded to areas positive for keratan sulphate, as shown by AB staining in preceding or following sections. The pronounced lectin binding seen in cellular structures and the inner territorial matrix regions is considered to be due to higher concentrations of oligosaccharides involved in the metabolism of GAGs.     

Immunohistochemical localization of articular cartilage proteoglycan and link protein in situ using monoclonal antibodies and lectin-binding methods

Lectins have specificity for certain carbohydrate structures in macromolecules. Lectins are, therefore, useful histochemical tools for demonstrating the composition and localization of components of connective tissue matrices, such as articular cartilage. In order to assess the significance of observed lectin-binding patterns, experiments were performed in which monoclonal antibodies against chondroitin sulphate- and keratan sulphate-containing proteolgycans and link proteins were applied to sections of bovine articular cartilage after enzymatic digestion with chondroitinase ABC and keratanase. The following conclusions were made: (1) Binding of peanut agglutinin (PNA) in the interterritorial matrix predominantly indicates the presence of keratan sulphate, but may also detectO-linked oligosaccharides of proteoglycans. (2) In normal cartilage wheat germ agglutinin (WGA) binds nearly exclusively to keratan sulphate. In cartilage degraded with chondroitinase ABC and keratanase this lectin may also detect carbohydrates in link protein due to enhanced accessibility. Binding of WGA toO-linked oligosaccharides may eventually occur. (3) In enzymatically digested cartilage matrix, staining with soybean agglutinin (SBA) may be due to link protein, but not to chondroitin sulphate, because specific breakdown of the glycosaminoglycan chain is required for binding of SBA. (4)Ulex europaeus agglutinin I (UEA I) binding sites are only detectable in digested cartilage matrix.     

Lectin-binding patterns in the developing tooth

Summary The lectin-binding patterns of the cells involved in amelogenesis and dentinogenesis in developing teeth of rats, were studied. Undifferentiated odontogenic epithelia exhibited very slight staining with almost all of the lectins examined. The lectin-staining affinities of secretory ameloblasts could be divided into two categories: Concanavalin-A (Con-A), Wheat germ agglutinin (WGA) and Soybean agglutinin (SBA) binding occurred from the middle to apical cytoplasm, whereas Ricinus communis agglutinin-I (RCA-I) and Ulex europeus I (UEA-I) binding predominated in the basal regions. The cells of the stratum intermedium exhibited relatively stranges lectin staining, which appeared to be dependent on ameloblastic maturation. The basement membranes in undifferentiated epithelia were markedly positive for lectin binding. Odontoblasts showed moderate Con-A staining on the apical side of the cells, as well as slight-to-moderate reactions with WGA and SBA. Pulp cells and dental papillae showed slight-to-moderate lectin staining, and predentin and dentin were also moderately positive for Con-A and RCA-I binding and slightly so for WGA and SBA. The lectin-binding affinities were enhanced during the formation of enamel and dentin, and appeared to be dependent on the degree of cellular differentiation in ameloblasts and odontoblasts.     

Lectin localization in human nerve by biochemically defined lectin-binding glycoproteins,neoglycoprotein and lectin-specific antibody

Summary Molecular recognition can be mediated by protein (lectin)-carbohydrate interaction, explaining the interest in this topic. Plant lectins and, more recently, chemically glycosylated neoglycoproteins principally allow to map the occurrence of components of this putative recognition system. Labelled endogenous lectins and the lectin-binding ligands can add to the panel of glycohistochemical tools. They may be helpful to derive physicologically valid conclusions in this field for mammalian tissues. Consequently, experiments were prompted to employ the abundant -galactoside-specific lectin of human nerves in affinity chromatography and in histochemistry to purify and to localize its specific glycoprotein ligands. In comparison to the -galactoside-specific plant lectins fromRicinus communis andErythrina cristagalli, notable similarities were especially detectable in the respective profiles of the mammalian and the Erythrina lectin. They appear to account for rather indistinguishable staining patterns in fixed tissue sections. Inhibitory controls within affinity chromatography, within solid-phase assays for each fraction of lectin-binding glycoproteins and within histochemistry as well as the demonstration of crossreactivity of the three fractions of lectin-binding glycoproteins with the biotinylated Erythrina lectin in blotting ascertained the specificity of the lectin-glycoprotein interaction. In addition to monitoring the accessible cellular ligand part by the endogenous lectin as probe, the comparison of immunohistochemical and glycohistochemical detection of the lectin in serial sections proved these methods for receptor analysis to be rather equally effective. The observation that the biotinylated lectin-binding glycoproteins are also appropriate ligands in glycohistochemical analysis warrants emphasis. Overall, the introduction of biotinylated mammalian lectins as well as the lectin-binding glycoproteins will aid to critically evaluate the physiological significance of the glycobiological interplay between endogenous lectins and distinct carbohydrate parts of cellular glycoconjugates.     

Localized agglutinin staining in muscle capillaries from normal and very old atrophic human muscle using winged bean (Psophocarpus tetragonolobus) lectin

 The winged bean (Psophocarpus tetragonolobus) agglutinin (total lectin) and its basic (WBA I) and acidic isoform (WBA II) were used to analyze capillaries in sections from human muscle. The microvessels were clearly labeled after incubation with the lectins in both normal muscle and in old muscles with age-related type II atrophy or muscle fiber grouping. Muscle fibers, nerves, and connective tissue remained unstained. The total lectin detected muscle capillaries from all blood group AB0 individuals. The isoform WBA I reacted only with blood vessels in blood group A and B individuals, while the blood vessels in blood group 0 individuals were demonstrated with WBA II. WBA I staining was inhibited by p-nitrophenyl α-galactopyranoside and N-acetylgalactosamine, whereas 2′-fucosyllactose and preincubation with an antibody against type-1 chain H abolished capillary staining with WBA II. The study demonstrates the usefulness of WBA as a marker of capillaries in human muscle. Accepted: 2 September 1996     

Morphological evidence of sperm maturation in the ampulla ductus deferentis of the bull

The present lectin histochemical comparison of cauda epididymal and ampullary bovine sperm was conducted to investigate whether ampullary secretions are involved in altering the plasma-membrane glycoconjugates of sperm. A marked redistribution of glycoconjugates between sperm from these two regions was indeed revealed on the basis of changes in binding patterns of the following fluoroscein-isothiocyanate (FITC)-labelled lectins: wheat-germ agglutinin (WGA), Maclura pomifera agglutinin (MPA), Griffonia simplicifolia I agglutinin (GS I) and Bauhinea purpurea agglutinin (BPA). This was evidenced in the first three cases by a relative reversal of staining intensity between the acrosomal and post-acrosomal regions, and by a pronounced increase in the staining of the midpiece. Changes in the distribution of BPA binding sites were limited to the latter phenomenon. The results are compared with previous findings, discussed in the context of the hypermotility characteristic of ampullary sperm and related to previously reported differences in the lectin-binding patterns of the luminal and glandular epithelia.     

Applications of immunocolloids in light microscopy. III. Demonstration of antigenic and lectin-binding sites in semithin resin sections

Previous studies have demonstrated that antigens or lectin-binding sites can be localized in sections from paraffin-embedded tissues with protein A or lectins bound to colloidal gold or colloidal silver (Roth J: J Histochem Cytochem 30:691, 1982 and 31:547, 1983). In the present study the protein A-gold technique and lectin-gold complexes have been applied to semithin sections (0.5-1.5 micron) of Epon- or low temperature Lowicryl K4M-embedded rat pancreas, kidney and submandibular gland. The results show that an increase in resolution and, therefore, in amount of information can be obtained. The optimal mode of imaging was determined on sections without counterstaining. Bright-field illumination gives the maximum information about the staining signal, while phase-contrast and Nomarski differential interference contrast give predominantly structural and, to a lesser extent, staining information. Polarization epi- and transillumination microscopy is inferior in all aspects. The application of a battery of lectin-gold complexes to rat submandibular gland revealed a specific staining pattern for each lectin in acinar and excretory duct cells.     

Immunocytochemical Localisation of Glycosaminoglycan-Like Activity in the Mucus and Associated Tissues of Terrestrial Slug Species (Gastropoda: Pulmonata)

Indirect immunofluorescence assays were conducted on cryotome sections of four terrestrial slug species from three distinct phylogenetic groups, Arion ater (L.), Arion hortensis (Férussac), Tandonia (Milax) budapestensis (Hazay), and Deroceras reticulatum (Müller) using monoclonal antibodies for two glycosaminoglycans (GAGs), heparan sulphate, and chondroitin sulphate. Specific staining for a heparan sulphate-like component was demonstrated in the foot and tail regions of each species and was particularly intense in A. ater and A. hortensis, notably in the epidermis and associated mucus-like material, and in mucus-like material from the pedal gland region of the latter species. Subsequent studies with A. ater confirmed the presence of heparan-sulphate–like activity in the caudal gland duct region. No evidence of specific staining for chondroitin sulphate-like activity was found in any of the slug species. This work suggests that a specific GAG, or a group of closely related GAGs, is a common component of mucus in a range of slug species and of different types of mucus, including trail (pedal) mucus and the more viscous mucus produced by the caudal gland.     

Post-embedding localization of glycoconjugates by means of lectins on thin sections of tissues embedded in LR white

Summary A simple post-embedding technique for the electron microscopical detection of lectin-binding sites using thin sections of tissues embedded in the resin LR White is described. With this technique, no prior etching of the sections is necessary. The cellular fine structure is well preserved and permits close correlation of the labelling to distinct cellular compartments. After mild aldehyde fixation (4% formaldehyde and 0.5% glutaraldehyde for 30 min), enterocyte brush border, vesicles and lysosomes as well as goblet cell Golgi apparatus and mucin are intensely stained after 30–60 min.The hydrophilia and penetrability of LR White is shown by the formation of oxidized diaminobenzidine reaction product arising from horseradish peroxidase-conjugated lectins. The precipitate not only covers the surface of the sections but is also formed within the resin, as is revealed on cross-sections through thin and semithin sections. The addition of 0.2m solutions of the appropriate inhibitory sugars prevented staining, which indicates a specific binding. Examples are given of the binding of gold-, ferritin- and peroxidase-conjugated lectins for the purpose of detecting glycoconjugates in various intracellular compartments.     

Lectin-binding patterns in the developing tooth

The lectin-binding patterns of the cells involved in amelogenesis and dentinogenesis in developing teeth of rats were studied. Undifferentiated odontogenic epithelia exhibited very slight staining with almost all of the lectins examined. The lectin-staining affinities of secretory ameloblasts could be divided into two categories: Concanavalin-A (Con-A), Wheat germ agglutinin (WGA) and Soybean agglutinin (SBA) binding occurred from the middle to apical cytoplasm, whereas Ricinus communis agglutinin-I (RCA-I) and Ulex europeus I (UEA-I) binding predominated in the basal regions. The cells of the stratum intermedium exhibited relatively strange lectin staining, which appeared to be dependent on ameloblastic maturation. The basement membranes in undifferentiated epithelia were markedly positive for lectin binding. Odontoblasts showed moderate Con-A staining on the apical side of the cells, as well as slight-to-moderate reactions with WGA and SBA. Pulp cells and dental papillae showed slight-to-moderate lectin staining, and predentin and dentin were also moderately positive for Con-A and RCA-I binding and slightly so for WGA and SBA. The lectin-binding affinities were enhanced during the formation of enamel and dentin, and appeared to be dependent on the degree of cellular differentiation in ameloblasts and odontoblasts.     

SOLUBLE AND MEMBRANE LECTIN-BINDING GLYCOPROTEINS OF THE CHROMAFFIN GRANULE

Abstract— Fluorescein isothiocyanate-labelled lectins were used to identify lectin-binding glycoproteins of the chromaffin granule after electrophoresis of the membrane and soluble granule proteins on sodium dodecyl sulphate polyacrylamide slab gels. The glycoprotein nature of all lectin-binding bands was confirmed by staining the gels for carbohydrates, and the specificity of the lectin-binding was demonstrated by hapten sugar inhibition of binding. In samples of granule membrane proteins reduced with dithiothreitol 10 concanavalin A (Con A), 5 wheat germ agglutinin, 8 Ricinus communis agglutinin-60, and 7 Ricinus communis agglutinin-120 (RCA-120) binding glycoproteins were identified. Molecular weights of these glycoproteins varied from 20,000 to 200,000 daltons. All but two of the Con A-binding bands and one of the RCA-120 binding bands appeared to react with more than one lectin, suggesting possible carbohydrate heterogeneity in these membrane glycoproteins. The band identified as dopamine β-hydroxylase reacted most intensely with all four lectin tested, and in the soluble core material this enzyme was the sole significant lectin binding glycoprotein.     

Use of semithin sections for the histochemical study of carbohydrate-containing biopolymers in cells and tissues with the aid of lectins

A sensitive method of histochemical analysis of cellular and tissue glycoconjugates on semithin sections using lectins is suggested. For fixation tissue bioptates were incubated for 4 h in a 2.5% glutaraldehyde in phosphate buffered saline (PBS) at 4 degrees C, then washed for 1 h in 0.2 M glycine in PBS. After epon-araldite embedment and preparation of semithin sections, the resin was removed in saturated ethanol-KOH solution during 5-10 s. Endogenous perooxidase was inactivated in methanol containing 0.3% H2O2. For identification of lectin-binding sites semithin sections were incubated for 30 min in a 0.005% solution of lectin-peroxidase conjugate in PBS and visualized by 0.05% diaminobezidine solution in PBS, containing 0.015% H2O2. The method described ensures good preservation of cellular and tissue glycoconjugates and is highly specific and sensitive.     

Studies of lectin binding to the human cervix uteri: II. Cervical intraepithelial neoplasia and invasive squamous carcinoma

Summary The cell surface carbohydrate profile of formalin-fixed paraffin-embedded tissue sections of neoplastic cervical squamous epithelium was evaluated using lectins ofBauhinia purpurea (BPA),Canavalin ensiformis (Con A),Griffonia simplicifolia I (GS I),Griffonia simplicifolia II (GS II),Maclura pomifera (MPA),Archis hypogaea (PNA),Glycine max (SBA),Ulex europaeus I (UEA I) andTriticum vulgaris (WGA). Three lectins (BPA, Con A and PNA) showed a similar pattern of staining in both normal squamous epithelium and in cervical intraepithelial neoplasia (CIN). Variable alterations were seen in lectin-binding patterns in CIN with seven lectins (GS I, GS II, MPA, PNA, SBA, UEA I and WGA). A significant difference was seen between the intensity of staining of normal squamous epithelium and CIN with all lectins except WGA. The alteration in GS II-binding pattern and intensity was significantly related to grade of CIN. No correlation was found between lectin binding and the presence of koilocytes in squamous epithelium. Cases of invasive squamous carcinoma showed a heterogeneous lectin-binding pattern and no siginificant association was found between lectin binding and tumour differentiation or patient survival. These results indicate that neoplasia in cervical squamous epithelium is associated with alterations in terminal -Man residues, - and -GalNAc residues, - and -GlcNAc residues, - and -Gal residues, and -Fuc-containing residues, present in the outer parts of bothN-linked andO-linked glycoconjugates. The implications of these findings are discussed.     

Lectin localization in human nerve by biochemically defined lectin-binding glycoproteins, neoglycoprotein and lectin-specific antibody

Molecular recognition can be mediated by protein (lectin)-carbohydrate interaction, explaining the interest in this topic. Plant lectins and, more recently, chemically glycosylated neoglycoproteins principally allow to map the occurrence of components of this putative recognition system. Labelled endogenous lectins and the lectin-binding ligands can add to the panel of glycohistochemical tools. They may be helpful to derive physiologically valid conclusions in this field for mammalian tissues. Consequently, experiments were prompted to employ the abundant beta-galactoside-specific lectin of human nerves in affinity chromatography and in histochemistry to purify and to localize its specific glycoprotein ligands. In comparison to the beta-galactoside-specific plant lectins from Ricinus communis and Erythrina cristagalli, notable similarities were especially detectable in the respective profiles of the mammalian and the Erythrina lectin. They appear to account for rather indistinguishable staining patterns in fixed tissue sections. Inhibitory controls within affinity chromatography, within solid-phase assays for each fraction of lectin-binding glycoproteins and within histochemistry as well as the demonstration of crossreactivity of the three fractions of lectin-binding glycoproteins with the biotinylated Erythrina lectin in blotting ascertained the specificity of the lectin-glycoprotein interaction. In addition to monitoring the accessible cellular ligand part by the endogenous lectin as probe, the comparison of immunohistochemical and glycohistochemical detection of the lectin in serial sections proved these methods for receptor analysis to be rather equally effective. The observation that the biotinylated lectin-binding glycoproteins are also appropriate ligands in glycohistochemical analysis warrants emphasis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light and electron microscopic detection of (3 Gal β1,4 GlcNAc β1) sequences in asparagine-linked oligosaccharides with the Datura stramonium lectin

Summary The Datura stramonium lectin recognizes with high affinity the disaccharide N-acetyllactosamine (Gal 1,4 GlcNAc). We have developed a highly specific cytochemical affinity technique in which an ovomucoid-gold complex serves as second step reagent for the visualization of this lectin bound to reactive sequences present in tissue sections. The lectin binding sites were detected in semithin and ultrathin sections of aldehyde-fixed and low temperature Lowicryl K4M embedded tissues. For light microscopical labeling the photochemical silver reaction for signal amplification was required. The application of this technique for the detection of N-acetyllactosamine containing asparagine-linked oligosaccharides in various intracellular organelles and the plasma membrane is demonstrated.This study was supported by the Swiss National Science Foundation grant nr. 31-26273.89 (to J.R.) and GM 29470 from the National Institutes of Health (to I.J.G.). Dr. G. Egea was a recipient of a European Molecular Biology Organization long term fellowship.     

Elderberry bark lectin-gold techniques for the detection of Neu5Ac (α2,6) Gal/GalNAc sequences: applications and limitations

Summary The lectin from the elderberry (Sambucus nigra L.) bark, shown to recognize the sequence neuraminic acid (2,6) galactose/N-acetylgalactosamine, was applied for detecting binding sites in Lowicryl K4M sections by light and electron microscopy. The lectin was used either directly complexed to colloidal gold or in a two-step cytochemical affinity technique. The lectin-gold complex proved to be superior and thus was extensively tested on rat liver, kidney and hepatoma cells as well as on sheep and bovine submandibular glands. Controls to establish specificity of lectin-gold binding included sugar and glycoprotein inhibition tests and enzymic removal of sialic acid. In agreement with biochemical data demonstrating the potentiating effect of sialic acid on the binding of the lectin to oligosaccharides, enzymic removal of sialic acid from liver sections resulted in abolition of lectin staining. However, in the submandibular glands, neuraminidase pretreatment of the sections had no effect on the subsequent lectin-gold binding. In rat kidney some structures became negative while others retained the lectin-gold staining due to binding to penultimate.N-acetylgalactosamine exposed after sialic acid removal. In line with this, spot blot analysis demonstrated that the lectin-gold complex reacted with both fetuin and asialofetuin. Taken together, these results suggest that, for cytochemical staining, the sialic acid and the galactose/N-acetylgalactosamine lectin combining subsites ofSambucus nigra L. lectin are equally reactive with cellular glycoconjugates and that neuraminidase predigestion of tissue sections is of utmost importance to ensure specificity of staining for the sequence neuraminic acid (2,6) galactose/N-acetylgalactosamine.     

Changes in Ricinus communis lectin binding to the cell surface of human liver cells in culture : The relationship between in vitro cellular aging and the age of the donor

Ricinus communis lectin RCA I-binding sites on the surface of cultured human liver cells have been studied with a view to establishing a relationship between the aging of the cells in vitro and the age of the donors. Fetal and new-born liver cells exhibited two classes of lectin-binding sites in the early in vitro passages, whereas adult cells in the early passages and fetal and new-born cells in the late passages had only one class of binding site. The modifications of the cell surface RCA I-binding sites would appear to occur either following masking of one of the classes of lectin-binding site during aging and/or be caused by a post-translational event rather than by glycoprotein mis-synthesis. A striking relationship was found between the in vitro cellular aging and human age in vivo, which would suggest that fetal human liver cells could prove to be a convenient model for studying the changes occurring during aging in vivo.     

Comparative study of procedures for histological detection of lectin binding by use of Griffonia simplicifolia agglutinin I and gastrointestinal mucosa of the rat

Summary The histological localisation of -D-galactopyranosyl residues in glycoconjugates of rat stomach and duodenal mucosae was studied by use of Griffonia simplicifolia agglutinin I, i.e. the isolectin mixture (A+B) and the isolectin B₄ (B₄). Cryostat sections which were either unfixed or acetone fixed and paraffin sections from both ethanolacetic acid and formaldehyde fixed tissue blocks were compared. Cellular details were better preserved in paraffin than in cryostat sections. Reactivity of cells binding GS I was less sensitive after formaldehyde than after ethanol-acetic acid fixation inasmuch as higher concentrations of lectins were needed. This drawback could be overcome by trypsinisation of the sections. The binding pattern of GS I (A+B) corresponded with that of GS I (B₄) in either cryostat or paraffin sections. GS I was detected in the cytoplasm of parietal cells and in Brunner's gland cells. In duodenal crypts and villi, lectin was bound to supranuclear regions in the cytoplasm of columnar and goblet cells. The staining efficiency of fluorescein (FITC), horseradish peroxidase (HRP) and colloidal gold particle (CGP) labels in both direct and indirect lectin stainings was compared. Under all experimental conditions, indirect methods required lower concentrations of lectins than direct ones; indirect procedures increased sensitivity about 5–10 fold. CGP labels were always of highest sensitivity when gold particles were further developed by a silver precipitation method. HRP was not as efficient in lectin localisation as CGP, but cytochemical staining was more convenient in routine work. Direct FITC labellings proved to be of lowest sensitivity.     

Studies of lectin binding to the human cervix uteri: I. Normal cervix

Summary Lectins ofBauhinia purpurea (BPA),Canavalin ensiformis (Con A),Griffonia simplicifolia I (GS I),Griffonia simplicifolia II (GS II),Maclura pomifera (MPA),Arachis hypogaea (PNA),Glycine max (SBA),Ulex europaeus I (UEA I) andTriticum vulgaris (WGA) were used to evaluate cell surface carbohydrates in formalin-fixed paraffin-embedded tissue sections of normal human cervix uteri. Consistent patterns of staining of the squamous epithelium were obtained in all 30 cases with BPA, GS II, MPA, PNA, SBA and WGA. A variable distribution of lectin binding was seen in squamous epithelium with Con A, GS I and UEA I. The patterns of GS I and GS II binding reflected squamous epithelial maturation. Columnar epithelium did not stain with GS II, stained variably with Con A, and stained consistently with the remaining seven lectins in all cases. No association between lectin binding and blood group or phase of the menstrual cycle was found. These findings may be used as a baseline for evaluation of lectin binding in both preinvasive and invasive lesions of the cervix uteri.     

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.