Glycoconjugate cytochemistry of the rat fundic gland using lectin/colloidal-gold conjugates and Lowicryl K4M. Helix pomatia lectin is a specific marker for mucous neck cells in fundic glands of the rat gastric mucosa

The fundic gland of the rat stomach was studied using the low-temperature embedding resin Lowicryl K4M and postembedding staining with lectin/colloidal-gold (CG) conjugates. Intense labeling with Ricinus communis agglutinin I was observed not only in mucous-producing cells but also in parietal cells. In contrast, Helix pomatia agglutinin (HPA) only labeled mucous neck cells and intermediate cells between mucous neck cells and chief cells. The other epithelial cells present in the rat fundic gland showed virtually no reaction with this lectin. Our results indicate that HPA might be a marker lectin of mucous neck cells and their derivatives. The combination of embedding in the hydrophilic resin Lowicryl K4M and postembedding staining with lectin-CG conjugates provided satisfactory staining results, and made it possible to visualize the precise distribution of terminal glycoconjugates in intracellular components as well as on the plasma membrane.     

Lectin cytochemistry in the gastrointestinal tract with special reference to glycosylation in the Golgi apparatus of Brunner's gland cells.

Two hydrophilic, low temperature-embedding resins, Lowicryl K4M and LR White, were compared in lectin cytochemistry. Post-embedding staining of colloidal gold-labeled Griffonia symplicifolia agglutinin II (GSA-II) resulted in staining of the Golgi apparatus and mucous granules of mucous neck cells in the gastric fundic gland, pylorocytes, and Brunner's gland cells embedded in either resin, although it was much easier to make ultra-thin sections with LR White-embedded material than with the other. Post-fixation with uranyl acetate followed by LR White embedding improved general ultrastructure so that lectin binding sites were identified precisely. All examined lectins, soybean agglutinin (SBA), Maclura pomifera agglutinin (MPA), GSA-II, and Ulex europaeus agglutinin I (UEA-I), stained mucous granules and the Golgi apparatus, in which the staining pattern was characteristic of each lectin: cis cisternae were labeled with SBA and MPA, intermediate cisternae with GSA-II, and trans cisternae and mucous granules with SBA, GSA-II, UEA-I, and lightly with MPA. No labeling was observed in the rough endoplasmic reticulum with any lectin. These findings suggest that the Golgi apparatus is the site of O-linked glycosylation and can be divided into at least three distinct compartments with regard to the glycosylation.     

Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue

The recently developed low temperature embedding procedure with the resin Lowicryl K4M (Carlemalm E, Garavito M, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 656; Garavito M, Carlemalm E, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 658) was tested for its suitability for embedding of glutaraldehyde-fixed rat pancreatic tissue and for postembedding staining of thin sections with the protein A-gold (pAg) technique (Roth J, Bendayan M, Orci L: J Histochem Cytochem 26:1074, 1978) for amylase. Compared to conventional Epon embedding of glutaraldehyde fixed tissue, the low temperature embedding method with Lowicryl K4M resulted in a superior preservation of the general cellular fine structure, particularly in the Golgi apparatus. For low temperature embedded tissue, the quantitative evaluation of the immunocytochemical labeling for amylase showed a more specific staining of the rough endoplasmic reticulum, the Golgi apparatus, and the zymogen granules. This was due to a significant lowering of the background staining over all cellular organelles. The use of Lowicryl K4M at low temperature, due to the superior preservation, yields improved resolution and specificity in immunocytochemical postembedding staining.     

Glycoconjugate distribution in the human fundic mucosa revealed by lectin- and glycoprotein-gold cytochemistry

The glycoconjugates of the human fundic mucosa were characterized at the ultrastructural level by means of direct (Helix pomatia agglutinin-gold complex) and indirect lectin techniques (Concanavalin A and horseradish peroxidase-gold complex; wheat germ agglutinin and ovomucoid-gold complex). Surface mucous cells and mucous neck cells secreted O-glycoproteins with N-acetylgalactosamine and N-acetylglucosamine residues at the non reducing terminus of the saccharidic chain. The secretory granules of the mucous neck cells showed condensed areas slightly reactive to ConA. The results obtained in the chief cells suggest that these cells secrete N-glycoproteins rich in mannose and/or glucose residues. "Transitional cells", presenting both morphological characteristics and lectin binding pattern intermediate to the mucous neck and chief cells have been observed. The surface of the intracellular canaliculi of the parietal cell was labelled by HPA, WGA and ConA. In the neck region of the gastric glands, immature parietal cells containing abundant mucous granules reactive to HPA, WGA and ConA were observed. The present results further corroborate the existence of a common cell precursor for surface mucous, mucous neck and parietal cells. In a further step, mucous neck cells gradually differentiate into chief cells the transitional cells being an intermediate stage.     

Cationic colloidal gold — a probe for light- and electron-microscopic characterization of acidic glycoconjugates using poly-l -lysine gold complex

Summary Cationic colloidal gold (CCG) was used to characterize acidic glycoconjugates in semithin and ultrathin sections of rat large intestine and salivary glands embedded in hydrophilic Lowicryl K4M resin. It was prepared from poly-l-lysine and 10 nm colloidal gold solution. The staining of CCG in semithin sections was amplified after photochemical silver reaction using silver acetate as a silver ion donor and examined under bright-field and epi-illumination microscopy. CCG adjusted to various pH levels was tested on various rat tissues whose histochemical characteristics with regard to acidic glycoconjugates are well known. At pH 2.5 CCG labelled tissues containing sialylated and sulphated acidic glycoconjugates such as the apical cell surface, mucous cells in the distal and proximal colon, and acinar cells of the sublingual gland. In contrast, CCG at pH 1.0 labelled tissues containing sulphated acidic glycoconjugates such as mucous cells in the upper crypt of the proximal colon and mucous cells in the whole crypt of the distal colon. This specificity of CCG was verified by the alteration of CCG staining following several types of cytochemical pretreatment. These results were further confirmed by electron microscopy. CCG staining is thus a useful postembedding procedure for the characterization of acidic glycoconjugates at both the light- and electron-microscopic levels.     

Glycoconjugate distribution in the human fundic mucosa revealed by lectin- and glycoprotein-gold cytochemistry

Summary The glycoconjugates of the human fundic mucosa were characterized at the ultrastructural level by means of direct (Helix pomatia agglutinin-gold complex) and indirect lectin techniques (Concanavalin A and horseradish peroxidase-gold complex; wheat germ agglutinin and ovomucoid-gold complex). Surface mucous cells and mucous neck cells secreted O-glycoproteins with N-acetylgalactosamine and N-acetylglucosamine residues at the non reducing terminus of the saccharidic chain. The secretory granules of the mucous neck cells showed condensed areas slightly reactive to ConA. The results obtained in the chief cells suggest that these cells secrete N-glycoproteins rich in mannose and/or glucose residues. Transitional cells, presenting both morphological characteristics and lectin binding pattern intermediate to the mucous neck and chief cells have been observed. The surface of the intracellular canaliculi of the parietal cell was labelled by HPA, WGA and ConA. In the neck region of the gastric glands, immature parietal cells containing abundant mucous granules reactive to HPA, WGA and ConA were observed. The present results further corroborate the existence of a common cell precursor for surface mucous, mucous neck and parietal cells. In a further step, mucous neck cells gradually differentiate into chief cells the transitional cells being an intermediate stage.     

Localization of pepsinogen and cellular differentiation in the human gastric mucosa using lowicryl K4M

The localization of pepsinogens (PG A and PG C) was studied intracellularly in human gastric biopsies embedded in Lowicryl K4M, using affinity purified antibodies and protein A-gold. The homogeneous secretory granules of the chief cells contained both PG A and PG C, as was proved in serial sections. Identical reaction was seen in the core of the biphasic mocous neck cell granules, whereas the mantle did not label. Even the rough endoplasmic reticulum (RER) and Golgi complex of the chief- and mucous neck cells contained label. Transitional cells identified by the presence of granules of both chief- and mucous neck cells were seen. This type of mucous neck cell is thought to transform into a chief cell. However an increase of RER that could explain an increase of the pepsinogen production was not observed. A mixture of these granules were also found in morphologically characterized young parietal cells, suggesting a common precursor for these three cell-types. These observations makes the transformation from mucous neck- into chief cells questionable. In conclusion Lowicryl K4M appeared to be a significant improvement compared to the Epon 812. Its shows a better preservation of both cytoplasmic antigens and cellular fine structure. This improvement adds information on the transformation hypothesis. Lowicryl K4M enables us, firstly to distinguish PG A and C synthesizing RER in different types of cell and secondly to recognize immature cells with the characteristics of chief-, mucous neck-, and parietal cells in the fundic gland. Very likely these three cell-types all arise from a common precursor. It is questionable that in normal human gastric mucosa the mucous neck cells transform into chief cells.     

Early events of secretory granule formation in the rat parotid acinar cell under the influence of isoproterenol. An ultrastructural and lectin cytochemical study

The events involved in the maturation process of acinar secretory granules of rat parotid gland were investigated ultrastructurally and cytochemically by using a battery of four lectins [Triticum vulgaris agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), Glycine max agglutinin (SBA), Arachys hypogaea agglutinin (PNA)]. In order to facilitate the study, parotid glands were chronically stimulated with isoproterenol to induce secretion. Specimens were embedded in the Lowicryl K4M resin. The trans-Golgi network (TGN) derived secretory granules, which we refer to as immature secretory granules, were found to be intermediate structures in the biogenesis process of the secretory granules in the rat parotid acinar cell. These early structures do not seem to be the immediate precursor of the mature secretory granules: in fact, a subsequent interaction process between these early immature granule forms and TGN elements seems to occur, leading, finally, to the mature granules. These findings could explain the origin of the polymorphic subpopulations of the secretory granules in the normal acinar cells of the rat parotid gland. The lectin staining patterns were characteristic of each lectin. Immature and mature secretory granules were labelled with WGA, SBA, PNA, and lightly with UEA-I. Cis and intermediate cisternae of the Golgi apparatus were labelled with WGA, and trans cisternae with WGA and SBA.     

Expression of N-acetylglucosamine residues in developing rat fundic gland cells

The development of rat fundic gland was studied by immunohistochemistry using a recently developed monoclonal antibody, HIK 1083, at both light and electron microscope levels. Antibody HIK 1083 recognized oligosaccharides with a non-reducing terminal -linked N-acetylglucosamine (GlcNAc) residue. In the developing rat fundic gland, cells expressing -GlcNAc residues were discernible from day 19.5 of gestation and continued to exist till adult. The distribution of the -GlcNAc expressing cells was consistent with that described previously for cells reacting to Griffonia simplicifolia lectin (GSA-II) in all developmental stages. These cells were located at the bottom of the fundic gland when they first appeared. With the elongation and maturation of the gland, these cells moved upwards and were finally restricted in the neck region of the gland. Combining previous reports and the present electron microscopical observations, HIK 1083-positive cells in the adult rat fundic gland are mucous neck cells. The interaction between antibody HIK 1083 and GSA-II lectin was investigated. GSA-II prevented the subsequent binding of HIK 1083, while HIK 1083 did not prevent GSA-II binding to mucous neck cells. Our results suggested that -GlcNAc residues exist in rat fundic gland from day 19.5 of gestation and continue to exist till adult. Cells expressing -GlcNAc residues appeared as typical mucous neck cells from postnatal four weeks.     

Ultrastructural demonstration of lectin binding sites in the Golgi apparatus of rat epiphyseal chondrocytes

Summary Binding sites for wheat germ agglutinin (WGA), Dolichos biflorus agglutinin (DBA), Ricinus communis I agglutinin (RCA I) and Limax flavus agglutinin (LFA) have been ultrastructurally detected in rat epiphyseal chondrocytes by a post-embedding cytochemical technique using colloidal gold as marker. The four lectins labelled exclusively the Golgi apparatus of chondrocytes embedded in Lowicryl K4M resin by two different methods. WGA binding sites were localized in medial and trans cisternae as well as in immature secretory vesicles, whereas those for DBA were seen concentrated in cis and medial cisternae. Labelling with both RCA I and LFA lectins was distributed throughout all the cisternac of the Golgi stack, and the latter also in vesicles and tubules at the trans face. Neuraminidase pretreatment of the sections abolished LFA staining, decreased reaction with WGA and increased that with RCA I, while it did not affect DBA staining. After chondroitinase ABC treatment only the RCA I reaction was modified, revealing new binding sites in the trans Golgi face, secretory granules and extracellular matrix. These results indicate that the distribution of subcompartments in the Golgi apparatus of chondrocytes is different from that in cells secreting glycoproteins as major products.     

Immunocytochemical localization of pepsinogen in rat stomach

The localization of pepsinogen in rat stomachs was investigated by a postembedding immunoferritin method. When the preparations embedded in Epon were used, the secretory granules of chief cells were stained heavily and the granules of mucous neck cells were stained moderately. The secretory granules of cells intermediate between mucous neck cells and chief cells showed a bizonal staining; the electron dense parts were stained heavily and the electron lucent parts were stained moderately. The secretory granules of pyloric gland cells, on the other hand, were labeled faintly. However, the secretory granules of surface mucous cells, foveolar mucous cells, endocrine cells, cardiac mucous cells and cardiac serous cells were not stained by the method. The protein A-gold method showed a similar staining pattern of pepsinogen to that of the immunoferritin method. When the samples embedded in Lowicryl K4M were used to enhance the stainability of pepsinogen, essentially the same staining pattern as that of the samples embedded in Epon was obtained. In addition, the Golgi apparatus and the rough surfaced endoplasmic reticulum were more easily stained.     

Immunocytochemical demonstration of the secretory dynamics of zymogenic contents in rat gastric gland processed by high-pressure freezing/freeze substitution, with special references to phospholipase A2 and phospholipase Cγ1

High-pressure freezing/freeze substitution followed by Lowicryl K4M embedding provided an excellent morphology and antigenicity of the gastric glands, as well as the intraluminal fluid contents. Taking advantage of this, we histochemically investigated the secretory dynamics of the zymogenic contents in rat gastric gland, with special references to phospholipase A(2) (PLA(2)) and phospholipase Cgamma1 (PLCgamma1). The combination of immunogold labeling and KMnO4-uranyl acetate-lead citrate staining for zymogenic contents clearly demonstrated the rapid diffusion of PLA(2) molecules from the exocytosed zymogenic contents into the mucinous contents in gastric glandular lumens. In contrast, the exocytosed PLCgamma1 molecules remained within the zymogenic contents in the glandular lumens. These findings indicated the distinction between the exocytosed PLA(2) and PLCgamma1 in their diffusion rate. In addition, the mucinous contents surrounding the exocytosed zymogenic contents were intensely labeled with Griffonia simplicifolia II lectin which specifically recognizes the mucin of mucous neck cells. Interestingly, some of the PLA(2) immunolabeling on the mucinous contents was associated with the apical membranes of gastric epithelial cells, especially that of parietal cells. The secretory dynamics of the zymogenic contents in rat gastric glands, including their interaction with the mucinous contents are discussed.     

Changes in lectin-binding pattern in the digestive tract of Xenopus laevis during metamorphosis. I. Gastric region.

The distribution of structural and secretory glycoconjugates in the gastric region of metamorphosing Xenopus laevis was studied by the avidin-biotin-peroxidase (ABC) histochemical staining method using seven lectins (concanavalin A, Con A; Dolichos biflorus agglutinin, DBA; peanut agglutinin, PNA; Ricinus communis agglutinin I, RCA-I; soybean agglutinin, SBA; Ulex europeus agglutinin I, UEA-I; and wheat germ agglutinin, WGA). Throughout the larval period to stage 60, the epithelium consisting of surface cells and gland cells was stained in various patterns with all lectins examined, whereas the thin layer of connective tissue was positive only for RCA-I. At the beginning of metamorphic climax, the connective tissue became stained with Con A, SBA, and WGA, and its staining pattern varied with different lectins. The region just beneath the surface cells was strongly stained only with RCA-I. With the progression of development, both the epithelium and the connective tissue gradually changed their staining patterns. The surface cells, the gland cells, and the connective tissue conspicuously changed their staining patterns, respectively, for Con A and WGA; for Con A, PNA, RCA-I, SBA, and WGA; and for Con A, RCA-I, and WGA. At the completion of metamorphosis (stage 66), mucous neck cells became clearly identifiable in the epithelium, and their cytoplasm was strongly stained with DBA, PNA, RCA-I, and SBA. These results indicate that lectin histochemistry can provide good criteria for distinguishing among three epithelial cell types, namely, surface cells, gland cells, and mucous neck cells, and between adult and larval cells of each type.     

Fluid dynamics of the excretory flow of zymogenic and mucin contents in rat gastric gland processed by high-pressure freezing/freeze substitution.

The high-pressure freezing/freeze substitution technique followed by Lowicryl K4M embedding provided an excellent ultrastructure and retention of antigenicity of rat gastric glands as well as the intraluminal fluid contents. By taking this advantage, we histochemically investigated the excretory flow of the zymogenic and mucin contents in rat gastric glandular lumen at the ultrastructural level. The combination of KMnO(4)-UA/Pb staining for zymogenic contents and Griffonia simplicifolia agglutinin-II (GSA-II) labeling for mucous neck cell (MNC) mucin distinguished the exocytosed zymogenic contents from the MNC mucin in the glandular lumen. Interestingly, at the base and neck regions, the zymogenic contents showed a droplet-like appearance, forming a distinct interface with the MNC mucin. At the pit region, the GSA-II labeling demonstrated restricted paths, designated as MNC mucous channels, which flowed into the surface mucous gel layer. It should be noted that the interface between exocytosed zymogenic contents and MNC mucin disappeared, and that the zymogenic contents merged into the MNC mucous channels. At the top pit region, the surface mucous gel layer showed laminated arrays of three types of gastric mucins. On the basis of these ultrastructural findings, we propose a model of the excretory flow in rat gastric gland.     

Bromodeoxyuridine-immunohistochemistry on cellular differentiation and migration in the fundic gland of Xenopus laevis during development

Summary Cellular differentiation and migration in the fundic glands of adult and larval Xenopus laevis have been examined using bromodeoxyuridine-immunohistochemistry. In the adult fundic gland, cumulative labeling with bromodeoxyuridine revealed a proliferative cell zone between the surface mucous cells and mucous neck cells, in what is referred to as the neck portion of the gland. The labeling-index of mucous neck cells had rapidly increased by week-5. The labeling-index of oxynticopeptic cells showed a more delayed increase until week-7, coincident with the decrease in the labeling of mucous neck cells. In the immature fundic glands of larvae, the labeled proliferating cells were randomly distributed throughout the developing gastric mucosa. During metamorphosis, the labeling-index of immature epithelial cells was highest at stage 63. Following administration of bromodeoxyurdine at this, stage, there was no significant loss of labeled epithelial cells during the metamorphosing period. Furthermore, there was no significant difference in the labeling-indices among the epithelial cells, such as surface mucous cells/generative cells, mucous neck cells, and oxynticopeptic cells, 7 days after administration. Cellular differentiation and migration pathways of epithelial cells in the fundic gland of adult X. laevis and its larvae are discussed.     

Angiotensin I converting enzyme in gastric mucosa of the rabbit: localization by autoradiography, immunofluorescence, and immunoelectron microscopy.

To localize angiotensin converting enzyme (ACE) in the fundic mucosa of the rabbit, we used autoradiography with the ACE inhibitor [3H]-trandolaprilate and post-embedding immunocytochemical techniques with a goat anti-rabbit ACE, using fluorescence and electron microscopy. Autoradiographic localization of [3H]-trandolaprilate in rabbit fundus sections shows that ACE is present in the fundic mucosa and mainly in the gland area. Fundic mucosa was fixed with 4% formaldehyde and embedded in Lowicryl K4M. Semi-thin (1 micron) or thin sections (800-900 A) were stained with anti-rabbit ACE followed by fluorescein isothyocyanate-labeled rabbit anti-goat IgG or protein A-gold reagent, respectively. Label was present on endothelium of all blood vessels running through the mucosa. Label was prominently localized in the granules of mucous surface and neck cells and on the granules of chief cells. The intracellular localization of ACE, and particularly its intragranular presence within chief and mucous cells, suggests that the enzyme, at the fundic level, is involved in the intragranular processing of a peptide, the nature of which remains to be determined.     

Cytochemical characterization of basement membranes in the enamel organ of the rat incisor

Ameloblasts are unique epithelial cells, in that once they have deposited the entire thickness of enamel and the process of maturation begins, they reform a basal lamina-like structure at their apical surface. In order to characterize further this basal lamina, its composition was analysed using (1) lectin-gold cytochemistry for glycoconjugates, (2) high-iron diamine (HID) staining for sulfated glycoconjugates and (3) immunogold labeling for collagen type IV and laminin. The labeling patterns were compared to that of other more typical basement membranes found in the enamel organ. Sections of rat incisor enamel organs embedded in Lowicryl K4M were stained with Helix pomatia agglutinin (HPA), Ricinus communis I agglutinin (RCA), wheat germ agglutinin (WGA) and Ulex europaeus I agglutinin (UEA). Samples from the late maturation stage were also reacted en bloc with lectins and embedded in Epon for transmission electron microscopic examination or prepared for scanning electron microscopy. Such samples were also stained with HID and conventionally processed for Epon embedding. Tissue sections were then reacted with thiocarbohydrazide-silver proteinate (TCH-SP). Analysis of the lectin labeling suggested that the region of extracellular matrix immediately adjacent to ameloblasts, where the basal lamina is situated, was intensely reactive with HPA and RCA, moderately reactive with WGA, and weakly reactive with UEA. In general, other basement membranes were mildly reactive with all lectins used. No HID-TCH-SP staining was observed directly over the basal lamina while numerous stain deposits were present over other basement membranes of the enamel organ. Immunolocalization of collagen type IV and laminin yielded a weak and variable labeling over the basal lamina. These results are consistent with the concept of basement membrane heterogeneity and, although the precise nature and composition of the basal lamina associated with maturation stage ameloblasts remain to be determined, they suggest that it may possibly function as a specialized basement membrane with particular compositional characteristics.     

Improved structural preservation in freeze-substituted sporidia ofUstilago avenae—a comparison with low-temperature embedding

Summary In the present study the cellular fine structure of freeze-substituted sporidia of the phytopathogenic fungusUstilago avenae is investigated by means of thin-section electron microscopy. A conventional embedding method using Spurr's low viscosity resin is compared with the recently developed methacrylate mixtures Lowicryl^® K 4 M and HM 20 resin. Generally, freeze-substitution yields improved preservation of fine structural details of the fungus compared to previously applied conventional fixation methods. Using double fixation during freeze-substitution prior to conventional embedding the fungal membrane system (plasmalemma, endoplasmic reticulum), organelles (mitochondria, nucleus etc.) and other cytoplasmic features (ribosomes, cytoskeleton) appear well resolved and smoothly contoured. Aldehyde fixed and Lowicryl embedded sporidia ofU. avenae resemble these double fixed fungal specimens fairly closely. The complete low-temperature preparation produces visualization of distinct cellular details although contrast reversal of cellular membranes (er, mitochondria etc.) is sometimes observed. In particular, fine structure resolution is enhanced in Lowicryl HM 20 embedded fungal cells. This is due also to significant improvement in staining of the cellular membranes, cytoskeleton (microfilaments and microtubules) and Golgi apparatus-like areas, using tannic acid. In case of the fungusU. avenae, freeze-substitution in combination with mild glutaraldehyde fixation and final low-temperature embedding in HM 20 resin prove suitable for improved preservation of cellular ultrastructure.Abbreviations cw cell wall - cy cytoplasm - FS freeze-substitution - FS-A GA/OsO₄ freeze-substitution and Spurr's LV-embedding - FS-B GA freeze-substitution and Lowicryl K 4 M LT-embedding - FS-C GA freeze-substitution and Lowicryl HM 20 LT-embedding - go Golgi apparatus-like body - GA glutaraldehyde - g glycogen deposit - l lipid droplet - LT low temperature - Lowicryl LT-embedding Lowicryl low-temperature embedding - Lowicryl LT-resin Lowicryl low-temperature resin - MeOH methanol - mf microfilament - mt microtubule - m mitochondrion - mvb multivesicular body - ne nuclear envelope - np nuclear pore - npl nucleoplasm - nu nucleolus - n nucleus - OsO₄ osmium tetroxide - pl plasmalemma - pr polyribosomes - Pb-citrate Reynolds' lead citrate - r ribosome - RT room temperature - rer rough endoplasmic reticulum - Spurr's LV-embedding Spurr's low viscosity embedding - Spurr's LV-resin Spurr's low viscosity resin - t tonoplast - Uac uranyl acetate - v vacuole     

Histochemical evaluation of secretory glycoproteins in human salivary glands with lectin-horseradish peroxidase conjugates

Paraffin sections of submandibular, sublingual, minor salivary, and parotid glands from ten human autopsy cases were stained with a battery of ten lectins conjugated to horseradish peroxidase. Variable affinity for one or another lectin between mucous cells in a gland evidenced cellular heterogeneity in mucin production. Mucous cells of a given type of gland varied among individuals, but for a single individual appeared markedly but not completely similar from one type of salivary gland to another. The individual variation related, in part, to the ABO blood group and secretor status of the individual. For mucous cells in secretors of blood group A and B all antigens stained strongly for the presence of terminal alpha-N-acetylgalactosamine or alpha-galactose, respectively. Mucous cells in AB secretors contained both antigens, whereas those of O (H) secretors lacked both. Mucous cells of three presumed nonsecretors, two of whom were immature infants and possibly too young to produce ABO antigen, failed to stain. Mucous cells in glands from the presumed nonsecretors, however, revealed a staining pattern consistent with the presence of Lea antigen. Mucous cells of nonsecretors stained with Lotus tetragonolobus agglutinin but not with Ulex europeus I agglutinin, whereas mucous cells of ABO secretors stained with both lectins. This difference in lectin binding indicated that sites reactive only with Lotus tetragonolobus agglutinin contain 1----4 linked fucosyl residues and sites stained by both lectins contain fucose linked 1----2 to the oligosaccharide. Staining of mucous cells of nonsecretors with Pisum sativum agglutinin indicate that either the lectin binds to internal N-acetylglucosamine of Lea substance or the mucous cells contain an N-glycosidic glycoprotein of the type thought to bind this lectin. Serous cells stained less strongly than mucous cells and differed in lectin affinities from one type of gland to another in an individual. Staining of serous cells of a given gland varied markedly among different subjects. This individual variability did not relate to blood group as terminal sugars demonstrative of A or B blood group antigens were not detected in any serous cells. Serous cells in the submandibular glands from the two immature infants were unreactive with all lectin conjugates. Secretions in parotid and submandibular serous cells generally contained a higher content of fucose than those in sublingual serous cells, which contained higher levels of a terminal galactose-sialic acid dimer. Some but not other cells of striated and interlobular ducts of submandibular glands of one subject stained for alpha-N-acetylgalactosamine.     

Post-embedding staining of rat gastric mucous cells with lectins

Summary The mucous cells of the rat stomach were stained with lectins by two post-embedding staining methods for electron microscopy. The mucous granules of surface mucous cells and foveolar mucous cells were stained weakly by Ricinus communis agglutinin-ferritin and wheat germ agglutinin-ferritin. The mucous granules of mucous neck cells were stained by concanavalin A-ferritin, Ricinus communis agglutinin-ferritin and wheat germ agglutinin-ferritin. The mucous granules of pyloric gland cells showed an affinity for wheat germ agglutinin-ferritin and concanavalin A-ferritin, while Ricinuscommunis agglutinin-ferritin only slightly stained the granules. The granules of mucous neck cells and pyloric gland cells were also stained by the concanavalin A-horseradish peroxidase-colloidal gold method, but the granules of surface and foveolar mucous cells were not stained by this method. Periodic acid oxidation of the sections before the standard concanavalin A-ferritin procedure enhanced the staining of the granules of mucous neck cells and pyloric gland cells slightly. Reduction of the sections after the periodic acid oxidation weakened the staining. Similar results were obtained using the concanavalin A-horseradish peroxidase-colloidal gold method. Though the staining with Ricinus communis agglutinin-ferritin was inhibited by periodic acid oxidation of the sections before staining, the staining with wheat germ agglutinin-ferritin was not inhibited by the oxidation. It is suggested that the paradoxical staining is closely related to the position of the concanavalin A-binding sugar residues in the carbohydrate chains.This work was supported in part by a grant-in-aid (No. 457008) from the Ministry of Education, Science and Culture, Japan and a grant-in-aid for cancer research (55-21) from the Ministry of Health and Welfare, Japan