Identification of mannose moieties in N- and O-linked oligosaccharides of the primordial germ cells of Xenopus embryos

The presence of mannose (Man) in the glycoconjugates of primordial germ cells (PGCs) of Xenopus embryos was elucidated by lectin histochemistry with Concanavalin A (Con A) and snowdrop (Galanthus nivalis) bulb lectin (GNA), in combination with deglycosylative pretreatments: beta-elimination, which removes O-linked oligosaccharides, and incubation with Peptide N glycosidase F (PNGase F), which removes N-linked glycan chains. In addition, histochemistry with Con A, which binds to Man and glucose (Glc), was also performed after glucose-oxidase incubation, which converts Glc into gluconic acid, and GNA was carried out after acid hydrolysis, which removes terminal sialic acid (NeuAc) moieties. PGCs were analyzed during their migration over the mesentery until the genital ridge, and after colonization of this gonad anlage. The results showed that for both lectins: (1) the PGCs and other surrounding tissue showed a similar binding pattern, and (2) the staining in the PGCs was similar in the developmental stages studied. Labeling with Con A was due to Man, and not to Glc, as shown after incubation with glucose-oxidase, and it was assumed that Man was in N-linked oligosaccharides. However, GNA labeling was mainly due to O-linked oligosaccharides, because the pretreatment of beta-elimination turned cells negative. Moreover, acid hydrolysis pretreatment gave rise to a stronger GNA-staining, suggesting that either Man was also in subterminal position to NeuAc or some Man-containing glycans were unmasked after removal of NeuAc from other oligosaccharide chains.     

Specific techniques for the identification of O-acylated sialic acids in colonic mucins

The development of methodology for the histochemistry of mucins based upon their PAS reactivity is discussed in terms of mechanism, specificity and application. Two new histochemical methods (PB/KOH/PAS and PAT/KOH/PAS), supplemented by a variety of new and standard histochemical techniques, and correlated by parallel chemical studies, were used to demonstrate and identify C₄ and side chain O-acylated sialic acids in colonic epithelial mucins. The application of these methods in the field of histopathology is discussed.     

Glycoconjugates of the urodele amphibian testis shown by lectin cytochemical methods

Lectin histochemistry is a useful method that allows the in situ identification of the terminal sugar moieties of the carbohydrates that form the glycoconjugates. Moreover, when it is combined with chemical or enzymatic deglycosylation pretreatments, lectin histochemistry can be employed to determine if carbohydrates are linked to the protein core by means of an N- or O-glycosidic linkage or, indeed, to partially sequence the sugar chains. One of the most interesting model organs for the study of spermatogenesis is the amphibian urodele testis. However, this organ has not been very widely investigated with lectin histochemical research. In the last few years, we have carried out a research project to identify and locate glycoconjugates in the testis of the urodele Pleurodeles waltl, the Spanish newt, as a first approach to identify possible carbohydrates with key roles in spermatogenesis. Our findings reveal some glycan chains located in a fusome-like structure in early (diploid) germ cells, oligosaccharides with terminal GalNAc in the acrosome, the occurrence of glycan modifications in the acrosomal contents during spermiogenesis, and changes in glycan composition of follicle and interstitial cells during the spermatogenetic cycle. Furthermore, the similar labeling pattern of follicle and duct cells supports the hypothesis for a common origin of both cell types.     

A histochemical approach to glycan diversity in the urothelium of pig urinary bladder

Intracellular glycans in the urothelium of urinary bladder of 10 adult male Landrace pigs were characterized in situ by immunohistochemical detection of Muc1 mucin by anti MUC1 from rabbit, conventional histochemical techniques (Periodic‐Acid Schiff, Alcian Blue pH 2.5, High‐Iron Diamine), and binding with 13 lectins (PNA, DBA, RCA‐I, WGA, SBA, BSI‐B4, ConA, AAA, UEA‐I, LTA, LFA, MAA‐II, SNA) combined with chemical and enzymatic pre‐treatments (β‐elimination, desulfation and neuraminidase) to gather reference data for this model animal. Muc1 mucin was detected in the secreting granules of superficial cells and the underlying layer of intermediate cells. The secreting granules in both intermediate cells and superficial cells were rich in carbohydrates, with the oligosaccharidic chains mostly O‐linked to proteins. Glycoproteins were prevailing over glycosaminoglycans (GAGs). In both superficial and intermediate cells sulfated and/or sialylated glycans were present, sulfation decreasing in the deeper layers. Lectin‐binding detected presence of terminal sialic acid linked mostly in α2,6 to GalNAc, Gal terminal or subterminal to sulfates, GalNAc, GlcNAc, and Fuc, mostly linked in α1,6, α1,3 α1,4 and α1,2 to GlcNAc or Gal, but not to lactosamine chains. Except for fucosylation, the oligosaccharidic chains in the glycoproteins of the urothelium of pig urinary bladder were similar to those linked to human MUC1, which is fundamental in cell adhesion and immunological processes in the urothelium. The co‐distribution of Muc1 and saccharidic residues suggests that many of them are linked to the glycoprotein.     

Cytochemistry of sialoglycoconjugates,lysozyme, and β‐defensin in eccrine glands of porcine snout skin as studied by electron microscopy

In most mammals except for humanoid primates, eccrine glands are confined to the skin of a series of specific body regions. Sialic acids and antimicrobial substances exhibit various functional properties and serve as a component of nonspecific defense against micro‐organisms, respectively. In this study, the distribution of these moieties was studied by electron microscopic histochemical methods. The eccrine glandular acini consisted of two types of dark cells as well as clear cells. The secretory granules and Golgi apparatus of both types of dark cells contained sialic acid residues linked to α2‐6Gal/GalNAc. On the other hand, sialoglycoconjugates with Siα2‐3Galβ1‐4GlcNAc sequence were confined to those of the Type II dark cells. In addition, lysozyme and β‐defensin were mainly detected in the secretory granules of the Type II dark cells. These secretory products may create a defensive barrier against microbial invasion and play an essential role in preservation of the integrity of porcine snout skin as a sensory organ. Microsc. Res. Tech. 2013. © 2012 Wiley Periodicals, Inc.     

Prenatal development and histochemical characteristics of gastrointestinal mucins in sheep fetuses

The object of this study was to describe the prenatal development and histochemical properties of mucins in the sheep gastrointestinal tract. To determine changes in the mucin profile, the sections were stained with specific histochemical stains for carbohydrates. While neutral and mixed mucins were observed in the superficial epithelial cells of the abomasal pyloric region, acidic mucins were detected in the secretory ducts and corpus of the glands. Acidic mucins consisted predominantly of sialomucins. In the duodenal villi, the number of goblet cells containing neutral mucins increased toward the end of gestation, whereas Brunner's glands contained acidic mucins until the 95th day of gestation and both acidic and neutral mucins thereafter. The jejunal goblet cells contained either acidic, neutral, or mixed mucins. Goblet cells containing acidic mucins, which were mainly localized to the ileal crypts and villi, mostly contained sulfated mucins. While villi were observed in the proximal colon until the 115th day of gestation, later the typical crypt structure emerged. During the period in which the villi were found in the proximal colon, the goblet cells containing sulphomucins were predominant, whereas the goblet cells containing sialomucins were predominant after the typical crypt structure was formed. In conclusion, gastrointestinal mucins may be involved in the formation of meconium during the prenatal period, and acidic mucins may contribute to the strength of the intestinal barrier against pathogens and digestive enzymes, as the barrier is not fully functional after birth.     

An argyrophil III method for the demonstration of elastic fibres and membranes

An esterification with isopropyl alcohol containing 0.2% periodic acid and 2% acetone (at 56 degrees C for 16 hours) followed by a treatment in a special physical developer, similarly, an acetylation with a 3:2 mixture of pyridine and acetic anhydride (at room temperature for 16 hours) followed by the same development, render the elastic fibres and membranes visible. Both pretreatments (esterification and acetylation) serve to make the catalytic points more active in the elastic elements than in the other components of tissue.     

Dimethyl‐2‐[(acridin‐9‐yl)methylidene]‐malonate as fluorescent probe for histochemical analysis

Fluorescent compounds have been widely used for biomolecule labeling in cytochemistry and histochemistry analysis. Here, it is described the optical properties of dimethyl 2‐[(acridin‐9‐yl)methylidene]‐malonate (LPSF/IP‐81), an acridine derivative. This compound was conjugated to Concanavalin A (Con A) lectin and applied as sugar probe in lectin histochemistry. Evaluation of luminescent properties showed that LPSF/IP‐81 is photoluminescent with excitation at 360 nm and emission at 428 nm. Con A hemagglutinating activity and LPSF/IP81 photoluminescence were unaltered after conjugation. Circular dichroism of Con A‐LPSF/IP81 conjugate showed the maintenance of the Con A structure. Lectin histochemistry with Con A‐LPSF/IP81 conjugate demonstrated different pattern recognition studying normal, fibroadenoma, and invasive ductal carcinoma of human breast. These findings indicate that LPSF/IP‐81 can be proposed as an alternative probe for histochemical analysis.     

The histochemical properties of some periodate-reactive mucosubstances of the pregnant Syrian hamster before and after methylation with methanolic thionyl chloride

The mucosubstances secreted in various tissues of pregnant Syrian hamsters exhibit a wide range of histochemical characteristics. When sections of fixed tissues containing these mucosubstances are treated with a methanolic solution of thionyl chloride for four hours at room temperature, nucleic acid phosphate groups, protein and sialomucin carboxyl groups, and the sulphate groups of many sulphomucins are apparently esterified. In sulphomucins believed to contain carboxyl and sulphate groups, only the sulphate is esterified; the carboxyls (uronic acid groups ?) are left free. If such methylated mucins are saponified (to remove their ester methyl groups) some, but not all, of their histochemical reactions for “free” sulphate are restored. Some desulphation, however, usually occurs during the methylation process. A few sulphated mucins, such as those in the goblet cells of the small intestine, are desulphated completely. The ability to esterify many sulphated mucins without either desulphating them completely or extracting them from the tissue distinguishes methanolic thionyl chloride from the hot methanolic solutions of hydrochloric acid usually used for methylating tissue sections. Moreover, methanolic thionyl chloride solutions are much easier to use. The little chemistry that is at present known about the reactions between methanol and thionyl chloride is insufficient to explain how methanolic thionyl chloride solutions actually methylate polyanionic substances in tissue sections.     

Galactosides in glycoconjugates of Xenopus laevis testis shown by lectin histochemistry

The implication of galactosides and other glycoconjugates on spermatogenesis has been previously reported. Glycans show such a complex structure that it makes them very difficult to analyze. Lectin histochemistry is a helpful tool for the study of glycan composition. Lectin histochemistry can be combined with deglycosylation pretreatments to explore the glycan type to which carbohydrates are linked. The aim of the present work was the localization of galactose (Gal)-containing glycoconjugates in the testis of Xenopus laevis, a species widely used in cell, molecular and developmental biology. Gal specific lectins BPL, PNA, BSI-B4, MAA-I, and RCA-I, were used in combination with deglycosylation procedures. Except for BPL, all the lectins were reactive for several testicular tissues. Some of the lectins showed a different reactivity depending on the stage of spermatogenic development, suggesting that cell glycoconjugates are modified during spermatogenesis. The surface of primary spermatocytes was strongly labeled with lectins from peanut (PNA) and castor bean (RCA-I), which agrees with the presence of galactosyl-glycolipids reported in the cell membrane of mammalian spermatocytes. The acrosome was unexpectedly negative to all the lectins tested, whereas the acrosome of mammals and other amphibians has shown a high expression of glycoconjugates, including galactosides. The results obtained after deglycosylation by β-elimination or incubation with PNGase F, which respectively remove O- and N-linked oligosaccharides, allowed us to elucidate the nature of the labeled glycans. The strong expression of galactosides at the cell surface of spermatocytes and spermatids suggests the involvement of these glycans in cell adhesion mechanisms during spermatogenesis.     

Cytochemical characteristics of the golgi apparatus

Lectinocytochemistry provides a useful tool for localizing subcompartments of the complex reticular apparatus of Golgi. The technique is based on interactions of lectins with glycoconjugates present in the limiting membranes and luminal spaces of Golgi elements. Application of a series of lectins of different sugar specificities permits a differentiation between Golgi subcompartments containing glycoconjugates with different oligosaccharide side chains. These may be (a) different glycoconjugates or (b) glycoconjugates at different stages during synthesis or repair of their glycans. The lectinocytochemical studies with mannose-, glucose-, N-acetyl-glucosamine-, N-acetylgalactosamine-, galactose-, fucose-, and sialic acid-recognizing lectins revealed predominating patterns that labeled distinct, i.e., cis, medial, trans, and transmost, regions of the Golgi apparatus. A further refinement could be achieved by differential lectin-inhibition that enables a dissection of lectin binding reactions on the basis of their binding affinities. High-affinity binding reactions showed that subcompartments are not necessarily confined to one single Golgi subregion and may change their position from one to another subregion. Some of the patterns observed may be interpreted in relation to certain steps during synthesis and modifications of glycans.     

Morphology and lectin‐binding sites of pyloric caeca epithelium in normal and GnRH‐treated Atlantic bluefin tuna (Thunnus thynnus) Linnaeus 1758

Mucosal epithelium of pyloric caeca was studied in normal and in GnRH‐treated Atlantic bluefin tuna Thunnus thynnus L., using morphological analysis, conventional and lectin glycohistochemistry. The lining epithelium consisted of columnar (absorptive) cells, goblet cells and intraepithelial leucocytes. The epithelium from normal animals was significantly taller than GnRH‐treated samples. Conventional histochemistry displayed the same staining pattern in normal and hormone‐treated specimens which showed a mixture of neutral and sulphated acidic glycoconjugates in the luminal surface and goblet cells, and neutral glycans in apical granules of enterocytes. Lectin histochemistry revealed a different glycoconjugate pattern in normal and GnRH‐treated tunas. In normal specimens the luminal surface expressed sialoglycoconjugates which bound MAL II, SNA, KOH‐sialidase‐PNA, KOH‐sialidase‐SBA as well as asialoglycans stained with HPA, SBA, GSA I‐B₄, LTA. N‐linked glycans were highlighted by Con A and KOH‐sialidase‐WGA. In GnRH‐treated tunas the luminal surface did not react with SNA, SBA and LTA. The columnar cells of normal tunas bound KOH‐sialisase‐PNA in the apical region, KOH‐sialidase‐PNA, KOH‐sialidase‐DBA, HPA, SBA, KOH‐sialidase‐SBA and KOH‐sialidase‐WGA in apical granules, GSA I‐B₄ and LTA in the supranuclear region. GnRH‐treated specimens showed some columnar cells that stained with KOH‐sialidase‐WGA in the apical granules and with GSA I‐B₄ in the supranuclear region. The goblet cells of normal animals produced mucins positive to PNA, HPA, KOH‐sialidase‐DBA, SBA, GSA II. The latter three binding sites lacked in GnRH‐treated tunas. The results suggest that the mucosal epithelium of Thunnus thynnus L. pyloric caeca expresses a complex glycan pattern that is affected by GnRH‐treatment. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Molecular histochemistry and plant biology: a review

This review considers some of the recent advances in the applications of molecular histochemical techniques to the analysis of the temporal and spatial patterns of gene expression associated with differentiation and development in plant tissues. A range of methods is outlined, and those for tissue preparation and the isotopic and non-isotopic labelling of probes for in situ hybridization studies are discussed, taking account of the various approaches which can be used for the localization of specifically bound probes. An overview of some successful applications of molecular histochemistry includes the elucidation of the roles of homeotic genes in floral biology, the ontogeny and differentiation of the photosynthetic apparatus, and aspects of seed development, including storage protein gene regulation during embryogenesis.     

Mucin profiles of the abomasum in bulls and rams: A comparative study

Many pathogens require direct binding to mucosal cells to cause an infection. The mucosal epithelium of the digestive tract, which is covered by a mucin layer, fulfills several protective functions that are essential to maintaining the health of the digestive tract. Mucins are glycoproteins, which are found on membranes and in mucus gels and protect the underlying mucosal cells. Both membrane‐associated mucins and secreted mucins are critical components of mucosal defense. The aim of this study was to determine the localization and expression of mucin profile of the abomasum via histochemistry and immunohistochemistry. The abomasums of 20 bulls and 20 rams were evaluated. Histochemical examination showed that neutral and acidic mucins were present in the mucosa and the glands of the pars cardiaca, fundus, and pars pylorica of the abomasums of both bulls and rams. However, the expression of acidic mucins was weak in the superficial glands and strong in the deep glands of the abomasum of rams. In both bulls and rams, MUC1, MUC5AC, and MUC6 were expressed in the glandular epithelial cells in all regions of the abomasum. Interestingly, while MUC2 was not expressed in the pars cardiaca and fundus, it was weakly expressed in the parietal cells of the pars pylorica in both species. In conclusion, the presence of neutral and acidic mucins and MUC1, MUC2, MUC5AC, and MUC6 proteins in luminal epithelial and glandular cells of abomasum in the bulls and rams support the hypothesis that mucins play a key role in the protection of the abomasal mucosa against infectious agents.     

Rapid combined light and electron microscopy on large frozen biological samples

The use of large unfixed frozen tissue samples (10 × 10 × 5 mm³) for combined light microscopy (LM) and electron microscopy (EM) is described. First, cryostat sections are applied for various LM histochemical approaches including in situ hybridization, immunohistochemistry and metabolic mapping (enzyme histochemistry). When EM inspection is needed, the tissue blocks that were used for cryostat sectioning and are stored at −80 °C, are then fixed at 4 °C with glutaraldehyde/paraformaldehyde and prepared for EM according to standard procedures. Ultrastructurally, most morphological aspects of normal and pathological tissue are retained whereas cryostat sectioning at −25 °C does not have serious damaging effects on the ultrastructure. This approach allows simple and rapid combined LM and EM of relatively large tissue specimens with acceptable ultrastructure. Its use is demonstrated with the elucidation of transdifferentiated mouse stromal elements in human pancreatic adenocarcinoma explants grown subcutaneously in nude mice. Combined LM and EM analysis revealed that these elements resemble cartilage showing enchondral mineralization and aberrant muscle fibres with characteristics of skeletal muscle cells.     

Staining sections of water-miscible resins 2. Effects of staining-reagent lipophilicity on the staining of glycol-methacrylate-embedded tissues

Glycol methacrylate (GMA) sections of animal tissues were stained with a group of twenty-seven reagents of very varied chemical characteristics. The artefactual background staining of the resin was found to be dependent on the hydrophilic/lipophilic character of the staining reagent, as estimated from the logarithm of its octanol-water partition coefficient (log P). Intense background staining occurred with lipophilic stains, whose log P>2. In keeping with this, use of GMA semi-permeable membranes for enzyme histochemistry failed to give staining when using a lipophilic substrate, probably because the substrate was trapped in the membrane. An analysis of other routine histochemical stains—in terms of the probable occurrence of high resin background staining and low tissue sensitivity—is made. A numerical guide is provided to help avoid artefacts resulting from hydrophobic and size effects. Note: small, hydrophilic reagents (log P <0; molecular weight < 550 Da) are least likely to show either type of artefact. Conversely, reagents which are lipophilic, or/and of intermediate size (log P > 2; 550 < ionic weight < 1000 Da), give strong background staining.     

Nanostructural analysis by atomic force microscopy followed by light microscopy on the same archival slide

Integrated information on ultrastructural surface texture and chemistry increasingly plays a role in the biomedical sciences. Light microscopy provides access to biochemical data by the application of dyes. Ultrastructural representation of the surface structure of tissues, cells, or macromolecules can be obtained by scanning electron microscopy (SEM). However, SEM often requires gold or coal coating of biological samples, which makes a combined examination by light microscopy and SEM difficult. Conventional histochemical staining methods are not easily applicable to biological material subsequent to such treatment. Atomic force microscopy (AFM) gives access to surface textures down to ultrastructural dimensions without previous coating of the sample. A combination of AFM with conventional histochemical staining protocols for light microscopy on a single slide is therefore presented. Unstained cores were examined using AFM (tapping mode) and subsequently stained histochemically. The images obtained by AFM were compared with the results of histochemistry. AFM technology did not interfere with any of the histochemical staining protocols. Ultrastructurally analyzed regions could be identified in light microscopy and histochemical properties of ultrastructurally determined regions could be seen. AFM-generated ultrastructural information with subsequent staining gives way to novel findings in the biomedical sciences. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Leaf and stem micromorphology of Byrsonima sericea DC. by light and scanning electron microscopy

Micromorphological studies were carried out using multiple microscopic techniques on the leaves and stem bark of Byrsonima sericea DC. (Malpighiaceae), a species popularly known as “murici” and used medicinally, in order to identify both qualitative and quantitative features of leaf and stem anatomy and histochemistry as differential parameters to support both the quality control of its ethnodrugs and the taxonomy of the genus. The study was conducted using traditional techniques of plant anatomy, histochemical tests, and the stomatal index (SI). Byrsonima sericea has hypostomatic leaves, anomocytic stomata, and its epidermal walls are anticlinal and straight on the adaxial and curved on the abaxial faces. T‐shaped trichomes were observed mainly on the abaxial surface. The leaf epidermis showed waxes syntopism on both surfaces, with the occurrence of different crystalloid forms on a single phylloplane. The mesophyll is dorsiventral, with 3‐4 collateral vascular bundles. Phenolic compounds, starch, and proteins were identified in the petiole and stem. The SI was 14.5 ± 0.53% (p < .05), but did not showed significant variations. A set of characters were found to be distinctive for the studied species, however, constituting parameters that could be used to separate B. sericea from other species of the genus.