Histochemical analysis of urea-unmasked glycosaminoglycans in the skin of the rat and mouse

Summary Histochemical analysis of urea-unmasked glycosaminoglycans has been performed in connective tissues of the rat and mouse skin by means of combined staining and enzyme digestion procedures. The staining procedures used were Alcian Blue pH 1.0, Alcian Blue pH 2.5, Aldehyde Fuchsin, periodic acid-Schiff (PAS), Alcian Blue pH 2.5-PAS, high iron diamine and low iron diamine methods. The digestive enzymes employed wereStreptomyces and testicular hyaluronidases, chondroitinases ABC and AC and keratanase. The results obtained indicated that the major components of the glycosaminoglycans in the connective tissues of the skin were hyaluronic acid, dermatan sulphate and chondroitin sulphate A and/or C, whereas the tissues were devoid of keratan sulphate.     

The effect of pH on Alcian Blue staining of epithelial acid glycoproteins. I. Sialomucins and sulphomucins (singly or in simple combinations)

Synopsis This study is concerned with the staining of epithelial acid glycoproteins by Alcian Blue at various pH levels. A detailed analysis of the effect of pH on Alcian Blue staining of epithelial tissues at selected sites was made. Alcian Blue was combined with the periodic acid-Schiff technique, the Alcian Blue being used at pH levels between 2.6 and 0.5.Animal salivary glands, human foetal tracheal gland and epithelial goblet cells of the adult bronchial mucosa were chosen for study because the nature of their acid glycoprotein is known and is relatively simple.In sites containing sialomucin alone, no Alcian Blue staining took place below pH 1.5. A difference was demonstrated between sialidase-sensitive sialomucin which stained only between pH 2.6 and 1.7, and sialidase-resistant sialomucin which stained between pH 2.6 and 1.5. Two types of sulphomucin were identified: the usual one stained with Alcian Blue at all the pH levels studied, and the other, in the canine gland, stained only at the most acid pH levels, that is, between pH 1.5 and 0.5.     

An immunocytochemical study of pituitary cells of the Senegalese sole, Solea senegalensis (Kaup 1858)

Different antisera directed against mammalian and piscine pituitary hormones, as well as a battery of various conventional histochemical techniques (PAS, Alcian Blue pH 2.5, Bromophenol Blue) and lectins, were used to identify the different hormonal cell types in the pituitary of the Senegalese sole, Solea senegalensis. Prolactin and adrenocorticotrophic cells were located in the rostral pars distalis of the pituitary. Gonadotrophic, thyrotrophic and growth hormone cells were distributed in the proximal pars distalis, but gonadotrophic cells appear also at the border of the pars intermedia. Somatolactin cells, as well as α-melanotrophic cells were located in the pars intermedia of the Solea senegalensis pituitary. The PAS reaction was positive in somatolactin cells, which were unreactive with the lead--Haematoxylin technique, whereas melanotrophic cells were positive. Glycoproteins containing mannose and/or glucose, as well as N-acetyl-glucosamine and sialic acid sugar residues, are synthesized and secreted by gonadotrophic, thyrotrophic and somatolactin cells. Adrenocor ticotrophic cells and, especially, the amphiphilic somatolactin and acidophilic growth hormone cells were stained with the Bromophenol Blue technique that identifies proteins in general, but adrenocorticotrophic and growth hormone cells were unreactive towards PAS, Alcian Blue pH 2.5 and lectins (Con A and WGA)     

An immunocytochemical study of pituitary cells of the Senegalese sole, Solea senegalensis (Kaup 1858)

Different antisera directed against mammalian and piscine pituitary hormones, as well as a battery of various conventional histochemical techniques (PAS, Alcian Blue pH 2.5, Bromophenol Blue) and lectins, were used to identify the different hormonal cell types in the pituitary of the Senegalese sole, Solea senegalensis. Prolactin and adrenocorticotrophic cells were located in the rostral pars distalis of the pituitary. Gonadotrophic, thyrotrophic and growth hormone cells were distributed in the proximal pars distalis, but gonadotrophic cells appear also at the border of the pars intermedia. Somatolactin cells, as well as α-melanotrophic cells were located in the pars intermedia of the Solea senegalensis pituitary. The PAS reaction was positive in somatolactin cells, which were unreactive with the lead--Haematoxylin technique, whereas melanotrophic cells were positive. Glycoproteins containing mannose and/or glucose, as well as N-acetyl-glucosamine and sialic acid sugar residues, are synthesized and secreted by gonadotrophic, thyrotrophic and somatolactin cells. Adrenocor ticotrophic cells and, especially, the amphiphilic somatolactin and acidophilic growth hormone cells were stained with the Bromophenol Blue technique that identifies proteins in general, but adrenocorticotrophic and growth hormone cells were unreactive towards PAS, Alcian Blue pH 2.5 and lectins (Con A and WGA) This revised version was published online in November 2006 with corrections to the Cover Date.     

The effect of pH on Alcian Blue staining of epithelial acid glycoproteins. II. Human bronchial submucosal gland

Synopsis The effect of pH on Alcian Blue staining of sialomucins and sulphomucins in human bronchial submucosal glands has been analysed. Using Alcian Blue combined with periodic acid-Schiff, lowering the pH was associated with a decrease in the area staining with Alcian Blue and an increase in that staining with periodic acid-Schiff, save in one bronchus with a large sulphomucin content, in which an increase in the area staining with Alcian Blue was found at pH1.0. In all bronchi, an increase in the intensity of Alcian Blue staining was found at this pH. Sialomucin sensitive to sialidase was found to lose Alcian Blue staining at a higher pH than sialomucin resistant to the enzyme. Some sulphomucins stained with Alcian Blue throughout the pH range studied and some only at the more acid pH levels. At pH1.0 Alcian Blue stained only sulphomucins, thus distinguishing them from sialomucins. Alcian Blue staining combined with the high iron diamine technique has enabled three sulphate groups to be identified: one stained with high iron diamine, the other two did not, and, of the latter, one stained with Alcian Blue at pH 2.6 and1.0, and the other only at pH1.0.     

Alcian Blue staining of cartilage for electron microscopy. Application of the critical electrolyte concentation principle.

The critical electrolyte concentration principle was applied to the Alcian Blue staining of rat epiphyseal cartilage proteoglycans for electron microscopy. The distribution and structure of material in glutaraldehyde-fixed cartilage stained at pH 5.8 without MgCl2 and in the presence of 0.05, 0.4, 0.5, 0.9 and 1.0 M MgCl2 was compared with that produced by simultaneous staining and fixation at neutral pH. Both methods resulted in staining of intracellular material within vacuoles as well as staining of non-collagenous matrix material. The structure and distribution of Alcian Blue-positive matrix material consisted of rounded or polygonal granules which accumulated around cells in the proliferative and hypertrophied zones. A similar pattern of distribution was observed in samples stained in the presence of 0.4 or 0.5 M MgCl2. In these cases, however, the stained material exhibited a ribbon-like configuration and granules were few in number. Increasing the MgCl2 concentration to 1.0 M resulted in a marked reduction of Alcian Blue stained material. No ribbon-like structures were observed, and matrix granules were reduced in both number and size. The decreased staining associated with increased electrolyte concentration lends support to the concept that epiphyseal cartilage matrix granules are composed primarily of chondroitin sulphate, and suggest that this same material is present in vacuoles associated with the Golgi apparatus in chondrocytes of the proliferative and hypertrophying zones.     

Ontogeny of sulphated glycoconjugate-producing cells in the rat fundic gland

Summary The ontogeny of sulphated glycoconjugate-producing cells in the rat fundic gland has been studied using high iron diamine (HID), Alcian Blue (AB) at pH 1.0, high iron diamine in combination with Alcian Blue at pH 2.5 (HID-AB), cationic colloidal gold (CCG) at pH 1.0 under light microscopy and CCG (1.0), HID-thiocarbohydrazide (TCH)-silver proteinate (SP)-physical development (PD) under electron microscopy. From day 19.5 of gestation, sulphated glycoconjugate-producing cells were discernible under both light and electron microscopy. The development of such cells can be classified into four stages: (1) a prenatal period from day 19.5 of gestation extending to 0.5 days after birth; (2) 1 day to 2 weeks after birth; (3) 2 to 4 weeks after birth; and (4) the final period from 4 to 8 weeks after birth. Glycoconjugate-producing cells reached maturity by 4 weeks after birth. Our results indicated that glycoconjugate-producing cells were cells along the wall of foveolar lumen, but not those covering the gastric mucosa surface. Our results also suggested that thetrans totransmost Golgi apparatus lamellae were the sites of sulphation in the developing rat stomach.     

Light and scanning electron microcopy study of the tongue in Rhea americana

Morphological characteristics of the tongue were studied in adult rhea (Rhea americana). The lingual surface and the surface of epithelium-connective tissue interface of rhea tongue were examined macroscopically and by light and scanning electron microscopy. The rhea tongue revealed a triangular aspect, without adjustment of the inferior bill formation, occupying approximately ? of the length of the oral cavity. Lingual papilla-like structures were not observed over the lingual surface. The tongue mucosa was composed of a thick non-keratinized stratified squamous epithelium in the dorsal and ventral part, supported by a connective tissue core. The submucosa contained numerous glands with cytoplasmic granules, and luminal secretion was positive for histochemical reaction to Alcian Blue in pH 2.5 and PAS, and negative to Alcian Blue in pH 0.5. Despite the rudimentary characteristic of the tongue in rhea, our results suggest an important role of tongue secretions in food lubrication and humidification during the swallowing process, based on the enormous quantity of lingual glands in the submucosa and the histochemical characteristics of their secretions.     

Toxoplasma gondii: A morphometric analysis of the wall and epithelial cells of pigs intestine

The aim of this study was to perform a morphometric analysis of the different layers of the jejunal wall and epithelial cells of pigs with toxoplasmosis. Experiments were conducted using 10, 88-day-old crossbred (Pietran × Wessex) pigs divided into two groups: control (n = 5) and experimental (n = 5). The experimental group consisted of animals inoculated orally with 5000 sporulated oocysts of a genotype III strain of Toxoplasma gondii. At 30 and 60 days following inoculation, the animals were anaesthetised for jejunal biopsy. The intestinal segments were processed routinely for histology. Transverse cuts (4 μm thick) were stained with haematoxylin and eosin (HE), Periodic Acid Schiff (PAS), Alcian Blue (AB), pH 2.5, and Alcian Blue (AB), pH 1.0. We observed hypertrophy of the jejunal wall, increased enterocyte height, and a decreased number of intraepithelial lymphocytes in the infected animals. There were no changes in the number of goblet cells.     

Immunocytohistochemical characterization of pituitary cells of the bluefin tuna, Thunnus thynnus L

In this paper we report the first complete mapping of the pituitary in a tuna species. The various different adenohypophysis cell types of the bluefin tuna, Thunnus thynnus L. have been identified and located using different antisera against mammalian and piscine hormones and various histochemical techniques: PAS, Alcian Blue pH 2.5 and lectins -ConA and WGA(Neutral and Acidic Glycoproteins); Bromophenol Blue (Proteins) and Tioglycollate-Ferric-Ferricianide-FeIII (-S-S- groups). Prolactin (PRL) and adrenocorticotrophic (ACTH) cells were located in the rostral pars distalis (RPD) of the pituitary, while the proximal pars distalis (PPD) displayed gonadotrophic (GTH), thyrotrophic (TSH), somatotrophic (GH) and also a few PRL cells. Moreover, somatolactin (SL) and melanotrophic (MSH) cells were identified inside the pars intermedia (PI). Interestingly, some SL-immunoreactive fibers were also detected in the neurohypophysis. Some GTH cells were also located on the exterior surface of the PI. Glycoproteins containing mannose (Man) and/or glucose (Glc); N-acetyl-glucosamine (GlcNAc) and/or sialic acid sugar residues, as well as -S-S- groups, were observed in GTH, TSH and SL cells. The Bromophenol Blue technique stained amphiphilic SL, acidophilic GH cells and weakly ACTH cells. GH and ACTH cells were unreactive to PAS, Alcian Blue, Tioglycollate-Ferric-Ferricianide-FeIII and lectin (Con A and WGA) techniques. Finally, PAS reaction was positive in amphiphilic SL cells, which were PbH unreactive, while MSH and ACTH cells were stained with PbH technique.     

Ultrastructure of the absorptive cell glycocalyx in hyperplastic colonic polyps after staining with Alcian Blue and high iron diamine

Summary The glycocalyx of absorptive cells in large intestinal hyperplastic polyp was characterized histochemically at the electron microscope level by the use of the Alcian Blue pH2.5 and high iron diamine techniques with the aim of comparing their ability in preserving the fine reticular network of the structure. Both the reagents stained glycocalyx, indicating the presence of sulphated acidic glycoconjugates. However, they showed different degrees of condensation of the reactive sites. Alcian Blue preserved its filamentous appearance better.     

The cytochemistry of glycoconjugates in the zona pellucida of murine ovarian oocytes and two-cell embryos

Summary Changes in glycoconjugates of the zona pellucida induced by maturation, ovulation and fertilization of mouse oocytes have been studied by means of light microscopic methods of cytochemistry. These methods consisted of periodic acid-Schiff (PAS), Alcian Blue pH 1.0 and pH 2.5, and peroxidase-labelled lectin diaminobenzidine (PO—LT—DAB) procedures in combination with the digestion technique with neuraminidase. According to the results obtained, glycoconjugates of the zona pellucida of fertilized eggs contained a smaller amount of sulphate groupings than that in ovarian oocytes, whereas their reactions for sialic acid and fucose residues were significantly stronger in intensity in the former, as compared with those in the latter. The cytophysiological significance of such cytochemical changes in glycoconjugates of the zona pellucida has been discussed with special reference to its functional alterations following maturation, ovulation and fertilization.     

Effects of Streptomyces hyaluronidase digestion on the histochemical reactions of proteoglycans in cartilage compared with its effects on certain non-cartilaginous tissues

Summary To test the value ofStreptomyces hyaluronidase in carbohydrate histochemistry, the effects of digestion with the enzyme on the staining of cartilage and non-cartilaginous tissues by Alcian Blue (AB) pH 1.0, AB pH 2.5, high iron diamine, low iron diamine, aldehyde fuchsin, dialysed iron-ferrocyanide and AB pH 2.5-periodic acid-Schiff were studied by light microscopy. The results obtained lead to the conclusion that theStreptomyces enzyme releases not only hyaluronic acid but also chondroitin sulphates and keratan sulphates in cartilage. Since hyaluronic acid is known to be linked to chondroitin sulphate proteoglycans, the enzyme is of limited value in localizing hyaluronic acid in cartilage. However, it is useful in localizing hyaluronic acid in most non-cartilaginous tissues.     

Histomorphological and histochemical characteristics of the intestine of the Senegal sole, Solea senegalensis.

The Senegal sole, Solea senegalensis has been exploited extensively in aquaculture from different countries; at present an intensive production of larvae and adults is being achieved with some nutritional problems. Since this species displays very different life styles and feeding habits at different stages of its life history (larvae, metamorphosis, adults), and because digestive mucins are implicated in different physiological processes including: increase of digestive efficiency, promotion of macromolecules-absorption, buffering of intestinal fluid, prevention of proteolytic damage to the epithelium and defence against bacteria, etc., we studied the histomorphological aspects, as well as the histochemical distribution of carbohydrates, (PAS, Alcian Blue), proteins (Bromophenol Blue), lipids (Oil Red O, Black Sudan B) and glycoproteins (Horseradish peroxidase-conjugated lectins) in the intestinal epithelium of adult Solea senegalensis specimens. Our data are compared with those obtained in larvae and adults of this and other fish species. Primary and secondary folds, microvilli of the intestinal enterocytes, as well as mucous or goblet cells were observed with a scanning electron microscope. Enterocytes and mucocytes of the intestine of adult Solea senegalensis were characterized by a rich supply of sugar and oligosaccharides. Carbohydrates (glycogen and mucins), proteins and lipids were present in cytoplasm and microvilli--brush border--of the enterocytes, which contain GalNAc, GlcNAc, Man, Glc and sialic acid-N-acetyl-D-galactosamine glycoconjugates. Intestinal mucous cells were strongly or weakly stained with Alcian Blue (pH 2.5 and 1). PAS reactivity was intense in numerous goblet cells, but some cells were PAS unreactive or weakly stained. Some goblet cells were positive for Bromophenol Blue but numerous cells were unstained; thus many proteins and possibly lipids may be conjugated with sugars. A similar reactivity to WGA and to neuraminidase-WGA was identified in some intestinal goblet, which were Con A unreactive, indicating the absence of Man and/or Glc and NANA glycoconjugates. GalNAc residues were only scarcely present in glycoproteins of some goblet cells.     

Histochemical reactions of gastrointestinal mucosubstances with orcein, high iron diamine and Alcian blue after prior oxidation of tissue sections.

The histochemical orcein reaction (orc) for mucosubstances in tissue samples from the human gastrointestinal tract was compared with PAS, high iron diamine (HID) and Alcian blue reactions at pH 1.0 or 2.5 (AB 1 and AB 2.5). Orc, HID and AB 1 reactions were performed also with prior oxidation of the tissue sections with potassium permanganate or performic acid (ox-orc, ox-HID and ox-AB reactions, respectively). Orc reaction stained mucosubstances similarly to HID and AB 1; only the brush border and goblet cells in the colon were stained. The reactions of the mucosubstances obtained with ox-orc differed from those with PAS, HID, AB 1 or AB 2.5 but were similar to those with ox-HID or ox-AB; the mucosubstances in the brush border and the goblet cells in the colon and small bowel and in the foveolar epithelium of the stomach were strongly stained. Pyloric and cardiac glands were stained faintly with ox-orc but not with ox-HID or ox-AB. Brunner's glands were negative with ox-orc, ox-HID and ox-AB reactions. It was assumed that the orc reaction stains, like HID or AB 1, sulphate groups in epithelial mucosubstances, and that sulphonic acid residues, resulting from oxidation of disulphide groups in the protein core of mucus glycoproteins, are responsible for the ox-orc as well as for the ox-HID and ox-AB reactions.     

Donnan potentials from the A- and I-bands of glycerinated and chemically skinned muscles, relaxed and in rigor.

Using a combination of microelectrode measurements and high-power microscopy we have demonstrated that different Donnan potentials can be recorded from the A- and I-bands of glycerinated and chemically skinned muscles in rigor, so that the A-band fixed charge concentration exceeds the I-band fixed charge concentration in the rigor condition. In relaxation the two potentials, and therefore the two charge concentrations, are equal in the two bands. X-ray data are presented for relaxed and rigor rat semitendinosus muscle, chemically skinned, and actin and myosin filament charges are calculated under a variety of conditions. Our conclusions are that (a) the fixed (protein) charge is different in the A- and I-bands of striated muscle in the rigor state; (b) the fixed charges are equal in the A- and I-bands of relaxed muscle; (c) the largest charge change between relaxation and rigor is on the thick filament. This occurs whether or not the myosin heads are cross-linked to the thin filaments. (d) Possibly an event on the myosin molecule, the binding of ATP (or certain other ligands) causes a disseminated change that modifies the ion-binding capacity of the myosin rods, or part of them.     

The effect of various fixation procedures on the digestability of sialomucins with neuraminidase

Synopsis The histochemical digestability with neuraminidase of sialomucin in mouse sublingual gland was studied in unfixed and formaldehyde vapour-fixed cryostat sections, and in sections prepared from paraffin-embedded material fixed in several alcohol- or formaldehyde-containing fixatives recommended for mucosubstances.The removal of sialic acid residues from sections treated with neuraminidase was followed histochemically with the following staining methods: Azure A pH 3.5, Alcian Blue pH 2.5, Low Iron Diamine and Alcian Blue pH 2.5 followed by periodic acid-Schiff. When Goland's methanolic cyanuric chloride was used as fixative, only a partial loss of tissue basophilia was evident after enzyme incubation, but in tissues fixed in other ways a complete loss of histochemically detectable sialic acid residues was observed.     

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

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

Seasonal variation in glycoconjugates of the pedal glandular system of the rayed Mediterranean limpet,Patella caerulea (Gastropoda: Patellidae)

Glycoconjugates secreted by the pedal system of the rayed limpet, Patella caerulea, were characterised in situ by histochemical and lectin-histochemical methods in individuals collected around the annual cycle, in November, March, and June. Stainings with periodic acid–Schiff (PAS), Alcian blue pH 2.5 (AB pH 2.5), Alcian blue pH 1.0 (AB pH 1.0), high-iron diamine–Alcian blue pH 2.5 and lectin binding assays with 9 lectins (Con A, WGA, succinylated-WGA, PNA, DBA, SBA, AAA, UEA-I, LTA) were performed. Four secreting cell types were observed in the sole, one in the peripheric region, and two in the sidewall. Glycoconjugate composition varied among cell types and also in one and the same cell type throughout the year. β-Elimination followed by PAS and AB pH 2.5 stainings indicated that most saccharidic chains were O-linked to the protein backbone. Secretion by sole and peripheric region was acidic, carboxylated and/or sulfated, whereas that of the sidewall was neutral. Glucosaminylated and 1,4-fucosylated residuals were predominant in the cell types along the year, 1,2-fucosylated residuals being observed only in the sidewall cells in June. Mannosylated and/or glycosylated residuals were observed in all cells mostly in November. Galactosylated/galactosaminylated residuals were present mostly in the sidewall cells and in the sole subepidermal mucocytes in June. Mannosylated and/or glycosylated residuals in November are probably linked to gonad maturation or to higher locomotion and foraging activity, whereas galactosaminylation in the sole cells and 1,2-fucosylation and glucosaminylation in the sidewall cells in June are linked to a prolonged stationary state, increasing water adsorption to counteract dehydration and/or to modulate microbial interactions.     

Histochemical characterization of the mucins of the alimentary tract of the grass snake, Natrix natrix (Colubridae)

Characterization of mucins in the alimentary tract of the grass snake, Natrix natrix was performed by histochemical (PAS, Alcian Blue, pH 2.5 and pH 1.0, sialidase-Alcian Blue, pH 2.5, HID-AB pH 2.5) and lectin-histochemical (WGA, SWGA, PNA, sialidase-PNA, SBA, sialidase-SBA, DBA, sialidase-DBA, ConA, BSI-B4, AAA, UEA-1, LTA) techniques. Oesophageal lining epithelium consisted of ciliated and goblet cells, with no pluricellular glands. Mannosylated sialosulfomucins were observed. Fundic mucosa of stomach presented surface cells producing sialomucins with terminal sialic acid linked to galactose. In gastric glands neck and oxynticopeptic cells were found. Neck cells had sialomucins with mannose, N-acetylglucosamine, galactose, N-acetylgalactosamine and fucose-α-(1,2)-linked residues. Cytoplasm of oxynticopeptic cells showed N-acetylgalactosamine and fucose residues. Secretion of surface cells in pyloric mucosa was similar to that of fundic ones, differing in having fucose. Goblet cells in the small intestine of N. natrix produced sulfo- and sialomucins, with sialic acid linked to galactose and N-acetylgalactosamine residues. Mucins also presented residues of mannose. Goblet cells in the large intestine presented sulfomucins only, with terminal N-acetylgalactosamine, galactose and N-acetylglucosamine. The glycosylation patterns found are probably related to protection against injuries, gastric juice and microorganisms, both pathogenic and decomposers, as well as to dietary adaptations.