Morphology and glycoconjugate histochemistry of the palpebral glands of the adult newt, Notophthalmus viridescens.

The eyelids of the newt were studied in 10 microns serial paraffin and 1-2 microns plastic sections using standard histological stains and special stains for glycoconjugates. The eyelids contain four different glands. Simple acinar serous and simple acinar mucous glands occur in the skin; unicellular mucous glands occur in the conjunctiva; and convoluted tubular seromucous glands are present in connective tissue beneath the conjunctiva. The first two are identical to cutaneous glands found elsewhere on the head and body. The simple acinar serous glands are surrounded by myoepithelial cells and release their secretion, which is composed largely of proteins with minimal glycoconjugate content, by a holocrine mechanism. The secretory product of the simple acinar mucous glands is composed of neutral glycoconjugates with a minor content of acidic glycoconjugates; the mucin exhibits strong PAS and PAPD staining and weak staining by AB and PAPS methods. The unicellular conjunctival mucous glands secrete both neutral and acidic glycoconjugates as shown by positive reactions with PAS, PAPD, PAPS, and AB methods. Convoluted tubular seromucous glands in the ventral eyelid synthesize both proteins and neutral glycoconjugates. The mucous secretions of the conjunctival glands probably provide lubrication and protection for the cornea.     

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.     

THE REACTIVITY AND STAINING OF TISSUE PROTEINS WITH PHOSPHOTUNGSTIC ACID

After aldehyde-fixation, treatment with phosphotungstic acid (PTA) in aqueous acidic medium was shown to produce an intense electron-opaque stain with minimal distortion of organelles. Mitochondrial matrix, cisternae of the endoplasmic reticulum, and the Z-band of muscle were densely stained, whereas membranes stood out in negative contrast. Staining of glycogen or lipid was not apparent. Under certain conditions the stain density reflected the concentration of protein based on the quantitative reaction of PTA with the positively charged groups, although the stoichiometry of the reaction between PTA and protein varied with the kind of protein. The staining conditions established should provide a base for the use of the method in quantitative electron microscopy, particularly on thin sections.     

Glycoproteins in membranes of secretory granules of the anterior pituitary gland

Summary Rat anterior pituitary glands were examined by electron microscopy after staining with five different histochemical stains. Histochemical reactions were observed in the cell coat, cell membrane and the membrane surrounding the secretory granules in all anterior pituitary cells following staining with phosphotungstic acid (PTA), chromic acid and PTA, the periodic acid-thiosemicarbazide-silver protein method (PA-TSC-SP) of Thiéry, ruthenium red and concanavalin A. The staining was abolished when the sections were preincubated with pronase, neuraminidase or trypsin and subsequently exposed to PTA, chromic acid and PTA or PA-TSC-SP. The possible functional role of the glycoproteins present in the membrane surrounding the secretory granules is considered.     

Cytochemical properties of mitochondria in the gastric parietal cell.

The matrix of some mitochondria in gastric parietal cells of rat and guinea pig evidenced affinity for the high iron diamine method which localizes sulfated complex carbohydrates selectively by light and electron microscopy. Such staining has not been observed elsewhere in the stomach. The high iron diamine reactive mitochondria about equaled in number those which were unreactive, and the two groups were indistinguishable morphologically. The distinction was not apparent either when mitochondria were stained by other cytochemical procedures including dialyzed iron for acidic complex carbohydrates, 3-3' diaminobenzidine-H2O2 at pH 6.0 for cytochrome oxidase, and Kominick's pyroantimonate osmium tetroxide for antimonate precipitable cations. The dialyzed iron method stained acid glycoconjugates in the outer intermembrane space in parietal cell mitochondria. These mitochondria stained more strongly with dialyzed iron than have any others examined heretofore with this method and comprised the only reactive mitochondria in the stomach. Parietal cell mitochondria also stained strongly for cytochrome oxidase but those of other gastric cells failed to evidence this reactivity.     

Ultrastructural localization of acidic glycoconjugates with the low iron diamine method

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.     

Glycoproteins in membranes of secretory granules of the anterior pituitary gland.

Rat anterior pituitary glands were examined by electron microscopy after staining with five different histochemical stains. Histochemical reactions were observed in the cell coat, cell membrane and the membrane surrounding the secretory granules in all anterior pituitary cells following staining with phosphotungstic acid (PTA), chromic acid and PTA, the periodic acid-thiosemicarbazide-silver protein method (PA-TSC-SP) of Thiéry, ruthenium red and concanavalin A. The staining was abolished when the sections were preincubated with pronase, neuraminidase or trypsin and subsequently exposed to PTA, chromic acid and PTA or PA-TSC-SP. The possible functional role of the glycoproteins present in the membrane surrounding the secretory granules is considered.     

Histochemical Detection of Carbohydrates of Blastocystis hominis

The carbohydrates of Blastocystis hominis were detected by histochemical techniques using light and electron microscopy. B. hominis, fixed with various fixatives, followed by treatment with detergents, were stained with periodic acid-Schiff (PAS) or alcian blue (AB). Intense PAS reactions were observed in cells fixed with glutaraldehyde or 1/2 Karnovsky fixative. The cells fixed with other fixatives showed weak or no reactions with PAS staining. Similar results were seen in the case of AB stain. These results indicated that, depending on the fixative used, B. hominis contained PAS- or AB-reactive carbohydrates. At the electron microscopic level, ultrathin sections of B. hominis were stained with periodic acid methenamine silver (PA-MS) or periodic acid thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining techniques. Intense, positive reactions with PA-MS or PA-TCH-SP were observed on the central vacuole, Golgi apparatus, and cytoplasmic vesicles. The filamentous layer showed moderate reactions with PA-MS, whereas in PA-TCH-SP stain, it was stained more densely. The staining intensity of the central vacuole varied from cell to cell. The presence of membrane fusions of the cytoplasmic vesicles with the central vacuole indicated the accumulation of carbohydrates in the central vacuole.     

Glycoconjugate localization with lectin and PA-TCH-SP cytochemistry in rat hypophysis

Glycoconjugates were localized by light microscopy with lectin-peroxidase conjugates and by electron microscopy with the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) sequence in immunocytochemically or morphologically identified cell types in rat pituitary. Lectin histochemistry demonstrated sialic acid and glycoconjugates with N-glycosidically linked oligosaccharides in gonadotrophs, thyrotrophs, and corticotrophs. Galactose penultimate to sialic acid was observed mostly in gonadotrophs. The terminal galactose-N-acetylgalactosamine disaccharide was detected in a few gonadotrophs and in a moderate number of mammotrophs. Fucose was localized in only corticotrophs with two fucose-binding lectins and in thyrotrophs with another. Several different monosaccharides were seen in glycoconjugates in melanotrophs and in Herring bodies. Melanotrophs displayed heterogeneous staining with fucose-binding lectins. A small number of nonsecretory cells were also visualized in the pars distalis by virtue of their glycogen content. PA-TCH-SP staining revealed complex carbohydrates in secretory granules and some Golgi cisternae in all types of hormone-producing cells in the pars distalis except for the somatotrophs. Melanotrophs of pars intermedia exhibited stained secretory granules and irregular dense bodies containing a stained meshwork. Corticotrophs of the pars distalis lacked the latter bodies, although they form the same glycoprotein precursor hormone as melanotrophs. Lectin conjugates and the PA-TCH-SP sequence stained some groups of secretion granules in Herring bodies, possibly representing vasopressin-containing granules as well as other cell types in the pars nervosa.     

MEASUREMENT OF THICKNESS WITHIN SECTIONS BY QUANTITATIVE ELECTRON MICROSCOPY

To apply the method of quantitative electron microscopy to the measurement of mass in thin sections, the thickness of the section at or very near the structure to be studied must be known. Dowex anion exchange resin AG 1 x 2, stained with phosphotungstic acid (PTA) at pH 6.4, was used as a thickness standard which could be embedded and sectioned. The sectioned PTA-Dowex appeared uniformly stained and exhibited suitable electron opacity. The stoichiometry of the reaction between PTA and the Dowex resin was measured by three independent methods based on gravimetric, colorimetric, and nitrogen determinations whose results showed close agreement. From the PTA uptake, the density of the stained spheres was calculated. Mass of a defined area of PTA-Dowex was measured by quantitative electron microscopy, and from this mass and density, the volume and then the thickness were calculated. The values for thickness were compared to those obtained by interference microscopy on the embedding medium alone in the same sections.     

The histochemistry of glycoconjugates in the colonic epithelium of the chicken

In the colonic epithelium of the chicken, glycoconjugates have been studied by means of selected histochemical methods of light and electron microscopy. According to the results obtained, most of the colonic goblet cells contained acidic and neutral glycoconjugates with sulphate and vicinal diol groupings, alpha-D-mannose and alpha-D-glucose residues and sialic acid-galactose dimers. These goblet cells were found to undergo changes in histochemical reactivity during upward migration along the crypts; alpha-D-mannose and alpha-D-glucose residues and terminal sialic acid-galactose dimers increased in amount. The striated border of the colonic columnar cells has, likewise, been found to contain such glycoconjugates as were similar in reactivity to those of the goblet cells. The histophysiological significances of glycoconjugates involved in the chicken colonic epithelium have been discussed with special reference to the functional activities of the carbohydrates.     

Silver methenamine and phosphotungstic acid staining of the acrosome of Mytilus edulis.

The Mytilus acrosome was investigated by histochemical methods combined with electron microscopy, using silver methenamine (SM) and phosphotungstic acid (PTA) staining, as well as some chemical and enzymatic pretreatments followed by the staining. As one of two major components in the Mytilus acrosome, the egg-membrane lysin was conspicuously stainable with PTA and susceptible to pronase digestion. The other component, that occupies the space between the acrosomal membrane and the axially located strand containing lysin, was stained with SM very specifically. This staining property was not affected by pronase digestion or treatment that blocked aldehyde and SH groups.     

Lectin-binding pattern in normal human gastric mucosa

Summary Normal human gastric mucosal cells were examined by light and electron microscopy using lectins as a probe. The ABC method was used with biotinylated lectins for light microscopy and HRP-labeled lectins for electron microscopy. The human gastric mucosal cells revealed specific binding patterns for each lectin by light microscopy. Among the lectins tested, in particular, DBA gave a characteristic pattern. It specifically stained the supranuclear region of surface epithelial cells and the perinuclear region of parietal cells. By electron microscopy, the stacked cisternae and the vesicles of the Golgi apparatus of the surface epithelial cells were positive for the DBA staining. These results show that the DBA-positive supranuclear region observed by light microscopy corresponds to the Golgi apparatus. In the parietal cells, DBA, RCA and ConA bound to the intracellular secretory canaliculi which are invaginations of the cell membrane running around the nucleus in the cytoplasm. Therefore, the tubular perinuclear positive region observed by light microscopy corresponds to the membranes of the intracellular secretory canaliculi. In addition, the ConA reagent stained the endoplasmic reticulum, Golgi apparatus, nuclear envelope, and cell membrane of the parietal cell, which explains the diffuse cytoplasmic staining observed at the light microscopic level with this lectin. Lectins have proved to be very useful for the evaluation of in situ cytochemical aspects of the glycoconjugates characteristic to human gastric mucosal cells.     

Quantification of protein A-gold staining for peroxisomal enzymes by confocal laser scanning microscopy.

The protein A-gold technique has been widely applied for visual localization and quantification of various antigens by electron microscopy. Observation of specimens stained by the protein A-gold technique with conventional light microscopy is difficult because of insufficient sensitivity of the staining. Light microscopic visualization and quantification of the reaction products were attempted employing a confocal laser scanning microscope (CLSM). Liver tissues of normal and peroxisome proliferator-treated rats were fixed and embedded in Lowicryl K4M resin. Ultrathin and thin sections were stained for catalase and a peroxisome-specific beta-oxidation enzyme by the protein A-gold technique. Ultrathin sections were observed by electron microscopy and the labeling density for each enzyme was analyzed with an image analyzer. Thin sections were observed with a CLSM in the reflection mode and the intensity of the light reflection was analyzed under the same conditions for all specimens. A comparison of these two observation procedures was also attempted using liver tissues stained with various concentrations of the antibody for catalase. The intensity of the reflection for each, as observed by CLSM, correlated well with the labeling density observed by electron microscopy. CLSM made it possible to quantify and to directly observe protein A-gold staining at the light microscopic level.(J Histochem Cytochem 47:1343-1349, 1999)     

Lectin-binding pattern in normal human gastric mucosa. A light and electron microscopic study

Normal human gastric mucosal cells were examined by light and electron microscopy using lectins as a probe. The ABC method was used with biotinylated lectins for light microscopy and HRP-labeled lectins for electron microscopy. The human gastric mucosal cells revealed specific binding patterns for each lectin by light microscopy. Among the lectins tested, in particular, DBA gave a characteristic pattern. It specifically stained the supranuclear region of surface epithelial cells and the perinuclear region of parietal cells. By electron microscopy, the stacked cisternae and the vesicles of the Golgi apparatus of the surface epithelial cells were positive for the DBA staining. These results show that the DBA-positive supranuclear region observed by light microscopy corresponds to the Golgi apparatus. In the parietal cells, DBA, RCA and ConA bound to the intracellular secretory canaliculi which are invaginations of the cell membrane running around the nucleus in the cytoplasm. Therefore, the tubular perinuclear positive region observed by light microscopy corresponds to the membranes of the intracellular secretory canaliculi. In addition, the ConA reagent stained the endoplasmic reticulum, Golgi apparatus, nuclear envelope, and cell membrane of the parietal cell, which explains the diffuse cytoplasmic staining observed at the light microscopic level with this lectin. Lectins have proved to be very useful for the evaluation of in situ cytochemical aspects of the glycoconjugates characteristic to human gastric mucosal cells.     

New sensitive light microscopical detection of colloidal gold on ultrathin sections by reflection contrast microscopy. Combination of reflection contrast and electron microscopy in post-embedding immunogold histochemistry.

One simple post-embedding method for combined light- and electron microscopy is presented. Different types of antigens in normal rat and mouse kidneys as well as in tissues from cases of experimental induced nephritis were stained after Lowicryl K4M embedding by an immunogold (silver) method. The (silver-enhanced) gold particles were visualized by light microscopy, e.g. bright-field (BFM)- and reflection contrast (RCM) microscopy, as well as by electron microscopy. The potentials of RCM visualization in this field were investigated, resulting in the successful detection of colloidal gold (15 nm) particles, or silver enhanced gold particles, on ultrathin sections. Furthermore, an increased detection sensitivity of RCM compared with BFM together with an increase in the sensitivity of the immunostaining by RCM visualization was found. The different ways to use RCM, alone or in combination with bright-field- or phase contrast microscopy for visualization of plastic sections varying in thickness, type of plastic and staining, are discussed.     

Capsular Polysaccharides of <Emphasis Type="Italic">Lactobacillus</Emphasis> spp.: Theoretical and Practical Aspects of Simple Visualization Methods

Lactobacillus strains can synthesize capsular polysaccharides (CPS), which are important substances in the dairy industry—they exhibit many important technological as well as health-promoting properties. Technological advancements have made it possible to detect bacterial capsules using costly and labor-intensive methods, such as serological reactions, molecular genetic techniques, and electron microscopy. Light microscopy, which is the method of interest in this paper, is one of the most widely accessible and cheapest techniques. CPS may be observed under a light microscope after staining bacterial cells and the background with a basic die and an acidic die, respectively (negative–positive staining), with the capsules remaining transparent. The literature offers many polysaccharide staining methods, but due to the considerable structural diversity of CPS and possible dye-capsule interactions, a suitable staining technique should be carefully selected for each strain. The current study showed that not all methods adequately reveal Lactobacillus CPS, with the most effective ones being those proposed by Hiss and Maneval.     

How to stain nucleic acids and proteins in Miller spreads

The spreading technique proposed by Miller and Beatty in 1969 allowed for the first time the visualization at transmission electron microscopy of nucleic acids and chromatin in an isolated and distended conformation. This approach is beneficial since it can reveal many aspects of chromatin organization and function that otherwise can only be indirectly inferred by biochemical methods. The final step of staining chromatin spreads is critical because it can strongly influence the interpretation of the results. We evaluated different staining techniques, and almost all provided a good result. Specifically, well-contrasted micrographs were obtained when staining with H3PW12O40 (phosphotungstic acid, PTA), as originally proposed by Miller and Beatty, and with two alternatives proposed here: uranyl acetate or terbium citrate. Quite a good contrast of the spread DNA could also be achieved using osmium ammine; while no or little contrast of nucleic acids was observed by staining with KMnO₄ (potassium permanganate) and H3PMo12O40 (phosphomolybdic acid, PMA) respectively.Key words: Chromatin spread, transmission electron microscopy, staining techniques     

Lectin-binding pattern on the surface epidermis of Ambystoma tigrinum larvae. A light- and electron-microscopic study

The surface epidermis of Ambystoma tigrinum larvae was examined at the light- and electron-microscope levels using five different lectin conjugates as probes for the detection of sugar residues on the cell membranes. Concanavalin A (Con-A), wheat-germ agglutinin (WGA), Ricinus communis agglutinin I (RCA-I), Dolichos biflorus agglutinin and soybean agglutinin (SBA) conjugates clearly labelled the surface cells, especially their apical surfaces. At electron microscopy, the labelling on plasma membranes was found to exhibit regional differences. Among the lectins tested WGA displayed a particularly characteristic binding pattern. WGA also bound to basolateral cell surfaces, including the tight-junction zone which was also stained by the RCA-I conjugate. The different labelling intensity and staining patterns obtained with the conjugates indicated the polarity of the cell surfaces. It is also assumed that the WGA staining of the basolateral membranes and intercellular spaces reflected transcellular transport, which is facilitated by acidic glycoconjugates. Other functional aspects of the polarized distribution of the lectin conjugates were also correlated with the receptor sites of certain sugar residues.     

Ultrahistochemical studies on the moderately electron dense bodies in teleostean endocardial cells

Summary The staining properties of phosphotungstic acid (PTA) on moderately electron dense bodies (MDB), are studied in the endocardium of Gadus morrhua. In MDB fixed in aldehydes only, and stained with PTA at a low pH (0–1), intensely electron dense material occurs on and beneath the limiting membrane. This latter area displays a declining electron density when stained with PTA solutions in which the pH is raised from 1 to 4. At pH>5 the peripheral matrix appears nearly unstained. Collagen fibres fixed as above, and then stained with PTA at a low pH, appear electron dense. These results suggest that the peripheral matrix of the MDB consists mainly of basic proteins.