bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucokinase in B-cell-depleted islets of Langerhans.
Authors: Bedoya F J , Oberholtzer J C , Matschinsky F M
Published: April 2011

Glucose phosphorylation was studied in B-cell-enriched or in B-cell-depleted pancreatic islets from normal or streptozotocin-diabetic rats, respectively, using quantitative histochemical procedures. The data indicate that B-cell-enriched preparations from normal animals and whole islets from normals, diabetics, and insulin-treated diabetic animals have comparable glucokinase activities. Average maximum velocities were (mmol/kg dry tissue/hr) 134.1 +/- 7.3 for whole islets and 125.6 +/- 10.7 for the B-cell-enriched preparations from normal rats, 143.1 +/- 13.6 for B-cell-depleted islets from diabetic rats, and 124.4 +/- 10.7 for B-cell-depleted islets from insulin-treated diabetic animals. The Kmax for glucose of the enzyme in islets from untreated diabetic rats was 16 mM, comparable to the Kmax found for glucokinase from normal rat islets. Mannoheptulose, previously shown to be a competitive inhibitor of glucokinase from liver and normal islets, also inhibited glucokinase in B-cell-depleted islets from diabetic rats. The data indicate that glucokinase is not selectively located in the B-cell, as was previously assumed, but is also found in A- and/or D-cells of diabetic rats. This observation raises significant questions about the functional role of islet glucokinase under control and diabetic conditions.
Immunocytochemical studies of desmin and vimentin in pericapillary cells of chicken.
Authors: Fujimoto T , Singer S J
Published: April 2011

The composition of intermediate filaments in pericytes was examined by immunofluorescent and immunoelectron microscopic labeling of frozen sections of various chicken microvascular beds in situ. Pericytes in capillaries of cardiac muscle, exocrine pancreas, and kidney (peritubular capillary) were found to contain both desmin and vimentin. In some capillaries where pericytes do not exist, cells apposed to endothelial cells--the Ito cell in the hepatic sinusoid and the reticular cell in the splenic sinusoid--were shown to contain both of the intermediate filament proteins. In contrast, podocytes and mesangial cells around renal glomerular capillaries contained only vimentin. The presence of desmin supports the hypothesis that pericytes may have a contractile apparatus similar to that of vascular smooth muscle cells. Our results also revealed that even in microvascular beds where pericytes are not found, cells having both desmin and vimentin exist next to endothelial cells and may assume similar functions to pericytes.
Autoradiographic localization of dihydrotestosterone binding in the major salivary glands and other androgen-responsive organs of the mouse.
Authors: Morrell J I , Gresik E W , Barka T
Published: April 2011

Mouse submandibular glands show an androgen-dependent sexual dimorphism, reflected in higher concentrations in males than in females of bioactive peptides, such as epidermal growth factor (EGF), nerve growth factor, and renin in the cells of the granular convoluted tubules (GCT). Biochemical studies have demonstrated androgen receptors in submandibular gland and other androgen-responsive organs in mouse. We have determined the cellular localization of these receptors using steroid autoradiography. Fifteen adult gonadectomized male mice were injected intravenously with 0.13 microgram or 0.26 microgram [3H]-dihydrotestosterone (SA 135 Ci/mM); some animals were pre-treated with cyclocytidine to stimulate secretion by GCT cells. Animals were killed 15 min, 1, 2, or 3 hr after isotope injection. Steroid autoradiographs were prepared, and some were stained immunocytochemically for EGF. Of the different cell types of submandibular gland, the acinar cells most frequently and intensely concentrated [3H]-DHT; GCT cells also concentrated the hormone, as did a small number of striated duct cells. In the other major salivary glands, the only cells that concentrated the androgen were interlobular striated duct cells in sublingual gland. In prostate, anterior pituitary, and brain a large number of cells concentrated androgen, as has been previously reported. Androgen binding by the GCT cells was a predictable finding, since androgen-induced alterations in composition and form of these cells are well documented. The intense androgen concentration by the acinar cells was an unexpected finding and suggests a hitherto unknown androgen regulation of these cells. An incidental finding was intense concentration of [3H]-DHT in the nuclei of the endothelial cells of the post-capillary venules of the cervical lymph nodes.
Dense HRP filling in pre-fixed brain tissue for light and electron microscopy.
Authors: Kageyama G H , Meyer R L
Published: April 2011

The use of neuroanatomical markers in tissues that have been pre-fixed has been virtually ignored, even though this approach could offer certain advantages over in vivo methods, in terms of convenience of application and choice of markers. We have found that HRP can be used on well-fixed brains of cats and goldfish to fill neurons, dendrites, axons, terminals, glial cells, and glial processes for high-resolution light microscopy and electron microscopy. Best results were obtained using brains that were perfusion-fixed with 2.5% depolymerized paraformaldehyde and 1.5% glutaraldehyde. Two methods of HRP application were used: optically guided injections of microliter quantities into various regions of cat brain, and optic nerve fills in goldfish by attaching an HRP-filled polyethylene tube for periods of 1 day to 2 weeks. HRP applied in these ways to pre-fixed tissue was found to fill neurons or glial cells with solid label in the anterograde and retrograde directions.
A new interpretation of the direct Schiff reaction of elastic connective tissue.
Authors: Rosenquist T H , McCoy J R
Published: April 2011

A direct Schiff reaction of elastic tissues has been known for many years, but the nature of the native aldehyde-rich components has not been clear. In this study, chicken, quail, and rat embryos and adult rat lung, aorta, and kidney were fixed in methacarn or in a formalin solution, embedded in paraffin, and sections of 8-10 micron obtained. Rehydrated sections were incubated for various periods in solutions of the enzymes chondroitinase ABC, clostripain, collagenase, elastase, heparatinase, hyaluronidase, subtilisin Carlsberg ("protease"), or trypsin, and in solutions of phosphomolybdic acid or sodium borohydride. After incubation, sections were placed, without prior oxidation, in Schiff's reagent, and were ultimately observed and photographed in transmitted light or with blue or green epifluorescence. A Schiff-positive substance was found, always and exclusively, in elastic tissues of the vasculature and lungs, which was hydrolyzed by the proteolytic enzymes to an extent that ranged from complete loss of Schiff reaction in minutes (trypsin) to no loss of Schiff reaction in 22 hr (clostripain). The Schiff-reactive protein preceded the time of appearance of elastin in the early embryos. We conclude that the aldehyde-rich protein responsible for this reaction is a harbinger of elastogenesis in vivo and speculate that it may represent the elastic microfibril or a component thereof.
Genetic differences in the histochemically defined structure of oligosaccharides in mice.
Authors: Spicer S S , Erlandsen S L , Wilson A C , Hammer M F , Hennigar R A , Schulte B A
Published: April 2011

A wide range of tissues from three interfertile species of mice and an interspecific hybrid was examined with lectins conjugated to peroxidase to localize specifically glycoconjugates containing terminal alpha-N-acetylgalactosamine, alpha-galactose, and alpha-fucose, and the terminal disaccharide galactose-(beta 1----3)-N-acetylgalactosamine. This battery of lectins disclosed marked heterogeneity of glycoconjugates in different histological sites in a given animal and even between cells in a presumably homogeneous cell population within an organ. No variation with any lectin was observed between individuals of two closely related inbred strains of Mus domesticus at any specific histological or cytological site. In contrast, littermates of an outbred strain of Mus castaneus differed in binding of certain lectins at various sites, attesting to a genetic basis for individual variation. Hybrids between castaneus and domesticus mice also showed individual variation. Moreover, extensive differences between the mouse species were demonstrable with every lectin in glycoconjugates of stored secretions, Golgi cisternae, and apical or basolateral plasmalemma in many cell types. Totaling the differences in tabulated staining intensities for each possible species pair gave a measure of the overall extent of difference at 53 histological sites. According to this measure, the three species are about equally divergent from one another. Some differences between species appeared to depend on histological rather than histochemical variation, as, for example, a greater abundance of granular duct cells in the sublingual and submandibular glands in Mus hortulanus. Other differences were apparently derived from pathological change, as exemplified by casts and lymphoid infiltrates in kidney and structurally atypical submandibular gland lobules in Mus castaneus, and possibly by infiltrating cells in intestinal lamina propria and epithelium in Mus castaneus and hortulanus.
Localization of phospholipid-rich zones in rat gastric mucosa: possible origin of a protective hydrophobic luminal lining.
Authors: Kao Y C , Lichtenberger L M
Published: April 2011

Mammalian gastric mucosa is unusually hydrophobic or nonwettable, which may be an essential biophysical characteristic of the gastric mucosal barrier. Since this property may be attributable to an adsorbed layer of surface-active phospholipids (SAPL), we investigated the distribution of SAPL in rat oxyntic mucosa. Ferric hematoxylin (FH) and iodoplatinate (IP), selective histochemical stains for phospholipids (as confirmed by spot tests), were used to detect SAPL in frozen sections and aldehyde-fixed tissue, respectively. Using FH staining in conjunction with extraction procedures that either solvate or preserve SAPL, we determined that positive reactivity was the greatest in the apical third of the oxyntic mucosa between the glandular neck region and the surface. IP reactivity appeared to parallel the FH staining pattern. Mucous cells, especially the surface epithelial cells, were heavily stained. Electron microscopic examination revealed that these cells contain inclusion bodies associated with various subcellular organelles, e.g., nuclear envelope, endoplasmic reticulum, Golgi apparatus and its vesicles, and mucous secretory granules. Vesicles and myelin figures, which resembled those found in lung surfactant, were observed extracellularly in close association with the surface mucous cells. Our findings suggest that mucous cells are actively involved in synthesis and storage of SAPL, which may be an essential component of the stomach's protective hydrophobic lining.
Preservation of thrombomodulin antigen on vascular and extravascular surfaces.
Authors: Boffa M C , Burke B , Haudenschild C C
Published: April 2011

The protein C anticoagulant system is mediated by thrombin and is highly accelerated by thrombomodulin. We studied the distribution of thrombomodulin antigen (TM Ag) in the rabbit using an affinity-purified antibody raised in a goat against rabbit thrombomodulin. The preservation of TM Ag was highly dependent on immediate fixation of the surface on which it is located. TM Ag was found on the endothelium of the entire vasculature, whereas it was absent from all connective tissue, smooth and striated muscle, secretory epithelia, cartilage, bone, neural tissue, and all parenchyma examined. A new finding was the presence of TM Ag on nonvascular surfaces of body cavities (the mesothelia of pleura, pericardium, and peritoneum, the synovial membrane, and the arachnoid enveloping the central nervous system). By use of a functional assay, TM activity was recovered in buffered saline/detergent solution which was either injected into the intraperitoneal cavity of rabbits in vivo or incubated with the surface of the arachnoid in vitro. These findings extend the importance of anticoagulant mechanisms to the systems of slowly circulating fluids, in which they might be required for maintenance of the flow, and to mesothelial cavities, in which they could be necessary for preventing adherence between the surfaces, in conditions associated with pathological exudation.
Histological distribution of the 35-KD protein substrate of the epidermal growth factor receptor/kinase in thymus.
Authors: Fava R A , Piltch A S
Published: April 2011

Rat thymus has been identified as a tissue comparatively enriched in a 35-KD substrate of the epidermal growth factor receptor/kinase (lipocortin-1) (J Biol Chem 261:13784, 1986). A polyclonal antiserum prepared against the 35-KD protein was used to determine histological distribution of the protein in thymus. Frozen sections of rat thymus were examined after indirect labeling of the 35-KD protein with a rhodamine conjugate of secondary antibody. The antigen was localized primarily in the reticular network of the thymic epithelium, with no detectable labeling of resident thymocytes. Immunoblotting (Western blots) of cytosol extracts also demonstrated that thymocytes did not contain detectable amounts of the antigen. Cultured thymic epithelial cells (TEC), however, contained an abundance of two immunologically related protein bands with molecular weights similar but not identical to the antigen from the parental cell line (human A-431 carcinoma). Paraffin sections of rat and human thymus were subjected to an immunoperoxidase staining procedure, and it was observed that Hassall's corpuscles (keratinized epithelial cells) and other cortical and medullary TECs were intensely stained. The demonstration that the antigen is primarily associated with TEC in thymus, in conjunction with its distribution in other tissues, will aid in deducing its physiological role.
Microphotometric quantitation of the reaction product of several indirect immunoperoxidase methods demonstrating monoclonal antibody binding to antigens immobilized on nitrocellulose.
Authors: Nibbering P H , van Furth R
Published: April 2011

The nitrocellulose model and microphotometry were used to investigate whether in immunoperoxidase cytochemical methods the amount of final reaction product reflects the amount of cell surface antigen. The results obtained with four cytochemical peroxidase methods, i.e., those using diaminobenzidine/H2O2 (DAB/H2O)2, DAB/H2O2/COCl2, DAB/H2O2/imidazole, and silver intensification of the DAB end product, were compared first. The quantitative DAB/H2O2/imidazole method proved to be the most sensitive and was selected for further studies. Cell surface antigens prepared by solubilization of peritoneal macrophages with octyl-beta-D-glucopyranoside were immobilized on nitrocellulose. Monoclonal antibody binding to these cell antigens was detected by peroxidase immunocytochemistry. Comparison of the sensitivity of the indirect immunoperoxidase and the biotin-(strept)avidin immunoperoxidase methods on the basis of the highest detectable dilution of a cell lysate showed that these methods were equally sensitive. A linear relationship between the absorbance of the peroxidase reaction product and the amount of cell lysate immobilized on nitrocellulose was found for all three indirect immunoperoxidase methods. This proves that the amount of final immunocytochemical peroxidase reaction product is proportional to the amount of antigen in cell lysates. However, the relative expression of antigens in intact cells differs from that in cell lysates. Therefore, the present method to solubilize cells and immobilize cell antigens cannot be used to quantitate the antigen content of cells.
Reactivation of inhibited bone acid phosphatase and its significance in bone histomorphometry.
Authors: Liu C C , Sherrard D J , Maloney N A , Howard G A
Published: April 2011

Despite biochemical demonstration of acid phosphatase (AcP) activation or reactivation in bone, few attempts have been made to show similar effects histochemically. Bones from growing rats, when fixed in 4% buffered formaldehyde at room temperature and demineralized in 5% formic acid, exhibited expected inactivation of AcP. The inhibited AcP, however, was reactivated by pre-incubation of sections for 1 hr at 37 degrees C in the following buffers: 0.2 M Tris, 0.2 M glycine, 0.2 M NaHCO3, or 0.1 M borax, as well as in alkaline water, but not in 0.2 M Na2HPO4 (all at pH 9). The reactivation was (a) site-specific (e.g., osteoclasts, osteoblasts, osteocytes, and cement lines), (b) temperature- and pH-dependent, (c) unaffected by OH- or SH--binding agents or by an alkaline phosphatase inhibitor, and (d) inhibited completely by 10 mM Na2HPO4. The reactivation process, much simplified and/or more effective than with the methods previously reported, was observed in all 83 human biopsy bones embedded in methyl methacrylate and in human bones stored in cold buffered formaldehyde for 7 months. This study demonstrates a unique method for reactivating and thus localizing the inhibited AcP in bones, and suggests possible applications in bone histomorphometry.
Absolute quantitative autoradiography of low concentrations of [125I]-labeled proteins in arterial tissue.
Authors: Schnitzer J J , Morrel E M , Colton C K , Smith K A , Stemerman M B
Published: April 2011

We developed a method for absolute quantitative autoradiographic measurement of very low concentrations of [125I]-labeled proteins in arterial tissue using Kodak NTB-2 nuclear emulsion. A precise linear relationship between measured silver grain density and isotope concentration was obtained with uniformly labeled standard sources composed of epoxy-embedded gelatin containing glutaraldehyde-fixed [125I]-albumin. For up to 308-day exposures of 1 micron-thick tissue sections, background grain densities ranged from about two to eight grains/1000 micron 2, and the technique was sensitive to as little as about one grain/1000 micron 2 above background, which correspond to a radioactivity concentration of about 2 x 10(4) cpm/ml. A detailed statistical analysis of variability was performed and the sum of all sources of variation quantified. The half distance for spatial resolution was 1.7 micron. Both visual and automated techniques were employed for quantitative grain density analysis. The method was illustrated by measurement of in vivo transmural [125I]-low-density lipoprotein [( 125I]-LDL) concentration profiles in de-endothelialized rabbit thoracic aortic wall.
Intensification of the immunocytochemical reaction by staining both sides of tissue sections.
Authors: Ciocca D R , Stati A O , Fasoli L C
Published: April 2011

We describe how the efficiency of immunostaining may be increased by staining paraffin sections on both sides. This modification exposes more antigenic binding sites per unit tissue area, as shown by the peroxidase-antiperoxidase and the avidin-biotin-peroxidase complex methods. Using antigen-rich tissue samples, the modified procedure made it possible to use more dilute primary antiserum or to reduce the incubation time of the tissue with the primary antibody. Alternatively, in tissue samples with sparse antigenic sites, the procedure made it possible to visualize and document very weak immunoreactivities.
Separate cell types that express two different forms of somatostatin in anglerfish islets can be immunohistochemically differentiated.
Authors: McDonald J K , Greiner F , Bauer G E , Elde R P , Noe B D
Published: April 2011

The somatostatin-related peptides somatostatin-14 (SS-14) and somatostatin-28 (aSS-28) are synthesized at the C-terminal end of two separate pre-pro-somatostatins in anglerfish pancreatic islets. The purpose of this study was to determine whether these peptides are expressed in the same or different cell types. Antisera R141 and R293, which recognize the central region of SS-14 and the C-terminal region of aSS-28 ([Tyr7,Gly10] SS-14), respectively, were used in an immunohistochemical examination of anglerfish islets. The R293 antiserum-labeled cells were distributed individually or in small clusters. These same cells, as well as a separate set of cells arranged in large clusters, were stained by the R141 antiserum. Pre-absorption of the R141 antiserum with [Tyr7,Gly10] SS-14 eliminated staining by R141 of only those cells also labeled by R293, whereas pre-absorption of R141 with SS-14 prevented all staining. Pre-absorption of R293 with [Tyr7,Gly10] SS-14 eliminated all staining, whereas pre-absorption with SS-14 had no effect on aSS-28-like immunoreactivity. These results suggest the existence of two separate cell types which express either SS-14 or aSS-28. The cells that contained the somatostatin-related peptides were found to be distinct from those cells that contained insulin, glucagon, or anglerfish peptide Y. However, the cells stained by the R293 antiserum were distributed in close association with glucagon-containing cells. The implications of the existence of separate cell types which express SS-14 or aSS-28 are discussed with regard to processing of the biosynthetic precursors to these peptides.
Differential immunolabeling for electron microscopy of diverse peptidergic neurons.
Authors: Maegawa M , Hisano S , Tsuruo Y , Katoh S , Nakanishi J , Chikamori-Aoyama M , Daikoku S
Published: April 2011

We describe a simple and reliable method for differential immunolabeling of pre- and post-synaptic signal peptides at the ultrastructural level. Hypothalamic tissues of rats, including the suprachiasmatic nucleus, were cut on a Vibratome. Visualization of the immunolabeling of somatostatin (SRIH) and vasoactive intestinal polypeptide (VIP) was performed with avidin-biotin-peroxidase-diaminobenzidine (DAB). The end product of the DAB to VIP was further silver-intensified in a physical processing using silver nitrate, and the silver grains were finally substituted for gold. DAB-labeled SRIH fibers synapse on gold-labeled VIP perikarya and dendrites in the suprachiasmatic nucleus.
Localization of 125I-atrial natriuretic factor (ANF)-binding sites in rat renal medulla. A light and electron microscope autoradiographic study.
Authors: Bianchi C , Gutkowska J , Garcia R , Thibault G , Genest J , Cantin M
Published: April 2011

Using light and electron microscope autoradiography in vivo, the localization of 125I-(Arg 101-Tyr 126) atrial natriuretic factor (ANF)-binding sites was studied in the renal medulla of rats. At the light microscopic level, the autoradiographic reaction was mainly distributed in patches in the outer medulla, and followed the tubular architecture in the innermost part of the inner medulla. At the electron microscopic level, binding sites were mainly found in the outer medullary descending vasa recta and inner medullary collecting ducts. These results suggest that, in rats, the renal medulla may participate in the natriuresis and diuresis produced by ANF through vascular and tubular effects; the former by changing medullary blood flow at the level of descending vasa recta and the latter by acting on electrolyte and water transport at the level of collecting ducts.
Prenatal exposure to alcohol alters the Golgi apparatus of newborn rat hepatocytes: a cytochemical study.
Authors: Renau-Piqueras J , Miragall F , Guerri C , Baguena-Cervellera R
Published: April 2011

The effect of prenatal exposure to ethanol on the Golgi apparatus of newborn rat hepatocytes has been studied cytochemically using several trans-Golgi markers (thiamine pyrophosphatase, uridine diphosphatase, inosine diphosphatase, acid phosphatase, and 5'-nucleotidase) as well as a cis-side marker (osmium impregnation). The amount of cerium phosphate formed in the cytochemical reactions was roughly quantitated by stereologic methods. The Golgi apparatus of about 40% of the hepatocytes appeared disorganized after alcohol treatment, and in the other 60%, the electron density of reaction product deposits for all phosphatases investigated was decreased. 5'-Nucleotidase was completely absent in cisternae of Golgi apparatus of treated cells. In control cells impregnated with osmium tetroxide, reduced osmium compounds were observed in most Golgi cisternae and in nearby vesicles. In contrast, only small vesicles appeared positive in treated hepatocytes. These results suggest that prenatal alcohol exposure alters some Golgi functions. Thus, the decrease in nucleoside diphosphatase and 5'-nucleotidase cytochemical activities after ethanol exposure strongly suggests that this treatment could affect glycosylation in the Golgi apparatus of newborn rat hepatocytes.
Polymyxin B binding sites in Escherichia coli as revealed by polymyxin B-gold labeling.
Authors: Morioka H , Tachibana M , Machino M , Suganuma A
Published: April 2011

A complex of polymyxin B, bovine serum albumin, and colloidal gold was prepared and used for the ultrastructural localization of polymyxin B binding sites on thin sections of Epon-embedded Escherichia coli cells. Gold particles were found on the outer membrane of E. coli, which is consistent with reported biochemical findings. We concluded that gold labeling with polymyxin B is useful in localizing the binding sites of polymyxin.
Comparison of the intracellular pathways of transferrin recycling and vesicular stomatitis virus membrane glycoprotein exocytosis by ultrastructural double-label cytochemistry.
Authors: Hedman K , Goldenthal K L , Rutherford A V , Pastan I , Willingham M C
Published: April 2011

Transferrin is taken up by receptor-mediated endocytosis into intracellular vesicles and tubules, and then recycles rapidly to the plasma membrane (diacytosis). We applied double-label cytochemistry to study whether the recycling structures containing transferrin fuse with the intracellular membranous structures that deliver newly synthesized membrane glycoproteins from the ER to the plasma membrane (exocytosis) or whether they remain independent. KB and Vero cells were infected with the temperature-sensitive transport mutant 0-45 of vesicular stomatitis virus (VSV). Temperature-regulated exocytosis of membrane glycoprotein "G" occurred simultaneously with diacytosis of transferrin. The exocytic "G" protein, as detected by immunoperoxidase electron microscopy, passed through the cisternal Golgi stacks and vacuolar, tubular, vesicular, and pit-like structures of the Golgi system. A transferrin-ferritin conjugate used in ultrastructural double-label experiments was detected in diacytic vesicles and tubules that accumulated in the proximal (trans-reticular) Golgi area of the cell. The ferritin-labeled vesicles/tubules were often close to and intermixed with the VSV-"G" containing membranous structures, but in most cases at early times (15-20 min) the transferrin and VSV-"G" containing vesicular structures remained distinct. At later times (30-45 min), the two labels were occasionally found in the same structures. These results indicate that rapid recycling of endocytosed materials and exocytosis of membrane glycoproteins to the cell surface usually occur in distinct vesicles, possibly along the same general morphologic exit pathway.
Regional differences in quantities of histochemically detectable mucosubstances in nasal, paranasal, and nasopharyngeal epithelium of the bonnet monkey.
Authors: Harkema J R , Plopper C G , Hyde D M , St George J A
Published: April 2011

Inhaled irritants induce secretory cell hyperplasia in nasal epithelium of animals. To characterize this response histochemically it is first important to know the histochemical character and distribution of epithelial mucosubstance in the normal nasal cavity. An automated image analyzing method was used to detect and quantitate acidic, neutral, and sulfated mucosubstances in the epithelium lining the nasal and paranasal airways of eight bonnet monkeys. Tissue sections 2 micron thick from defined regions of these airways were stained with either alcian blue/periodic acid-Schiff to demonstrate acid and neutral mucosubstances or high iron diamine to demonstrate sulfated mucins. Respiratory epithelium covering maxilloturbinates had the largest volume of stainable mucosubstance per unit surface area of basal lamina, whereas the maxillary sinus epithelium had the least. There was a general anteroposterior increase in the quantity of total epithelial mucosubstance along the septal and lateral walls of the nasal cavity, and there was more acidic than neutral mucosubstance in the posterior nasal airway than in the anterior. Epithelial mucosubstance in the maxillary sinus was predominantly neutral. Therefore, we conclude that there are substantial regional quantitative differences in stainable mucosubstances in the primate nasal epithelium which must be considered when examining nasal mucosa for irritant-induced changes in epithelial mucins.
Direct visualization of IgM antibodies bound to tissue antigens using a monoclonal anti-type III collagen IgM as a model system.
Authors: Keene D R , Sakai L Y , Burgeson R E , Bächinger H P
Published: April 2011

A mouse monoclonal IgM antibody directed against human Type III collagen was utilized to immunolocalize Type III collagen by transmission and scanning electron microscopy without the use of an electron-dense conjugate. Because bound IgM can be directly visualized, primary or secondary antibody conjugates, such as ferritin, HRP, colloidal gold, etc., are unnecessary in this method. Immunolocalization to Type III collagen in the matrix of human skin and to fibrils formed in vitro using only IgM antibody reveals uninterrupted IgM binding which exactly matches the banding period of the collagen fibrils. In contrast, colloidal gold-conjugated secondary antibody complexes directed against primary IgM binding sites reveal less precise labeling. The data suggest that direct visualization of primary monoclonal IgM antibodies may be useful in a wide variety of highly specific ultrastructural immunolocalization studies without requiring the use of electron-dense conjugates.
Immunolocalization of glyceraldehyde-3-phosphate dehydrogenase, hexokinase, and carboxypeptidase Y in yeast cells at the ultrastructural level.
Authors: van Tuinen E , Riezman H
Published: April 2011

We have developed a simple and effective method to embed whole yeast cells in Lowicryl resins with excellent ultrastructural and antigenic preservation. Using affinity-purified antibodies eluted from electrophoretically separated proteins transferred to nitrocellulose, we have shown by immunoelectron microscopy that two glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and hexokinase, are present in the cytoplasm and the nucleus. Carboxypeptidase Y is localized in the yeast vacuole. These results agree with earlier localization studies based on subcellular fractionation.
Studies with anti-bromodeoxyuridine antibodies: II. Simultaneous immunocytochemical detection of antigen expression and DNA synthesis by in vivo labeling of mouse intestinal mucosa.
Authors: Schutte B , Reynders M M , Bosman F T , Blijham G H
Published: April 2011

We developed a rapid and convenient immunocytochemical method for simultaneous detection of antigen expression and S-phase cells by means of anti-bromodeoxyuridine (BrdUrd) antibodies. Immunocytochemical detection of BrdUrd after in vivo administration in mice was compared with autoradiography using [3H]-BrdUrd. Both the sensitivity and specificity of the technique were high. For the dual peroxidase staining technique, DAB color modification by cobalt ions was used. We showed that antigen localization was not affected by the BrdUrd staining protocol. The technique we describe here can be performed on frozen or paraffin-embedded tissue and on cytocentrifuge preparations for analysis of the cytokinetics of phenotypically defined cells in heterogeneous populations.
Ultrastructural localization of a breast tumor-associated antigen.
Authors: Major P , Lavallée M , Minassian H , Kovac P , Wang N S
Published: April 2011

We used a monoclonal antibody to a high molecular weight glycoprotein of the milk fat globule membrane to study at the ultrastructural level the sites of accumulation of immunoreactive material in breast tumors. By light microscopy, the antigen was found only on the luminal membrane of normal resting or lactating breast cells. In tumors, antigen could be found in the cytoplasm. We used the avidin-biotin-peroxidase complex immunoperoxidase technique to localize the site of cytoplasmic accumulation of immunoreactive material in breast tumors. This technique showed that antigen is found on the membrane of cytoplasmic vesicles. Some of these vesicles were collapsed and gave rise to what appeared to be clumps of free cytoplasmic immunoreactive material. We have also documented that in some tumors the entire cytoplasmic membrane bears antigen, whereas in other tumors only areas of cytoplasmic membrane that form microscopic lumina express antigen.
Postnatal development of growth hormone and prolactin cells in male and female rat pituitary. An immunocytochemical light and electron microscopic study.
Authors: Smets G , Velkeniers B , Finne E , Baldys A , Gepts W , Vanhaelst L
Published: April 2011

Localization and ultrastructural maturation of prolactin (PRL) and growth hormone (GH) cells were studied in pituitaries from neonatal, immature (4-6 weeks old), and adult rats (2-3 months old) by light and electron microscopic immunocytochemistry. The distribution pattern of these cells did not change with age. Both cell types were concentrated laterodorsally, with PRL cells adjacent to the intermediate lobe and GH cells nearer the center of the pars distalis. Labeling density of the immunogold reaction was highest for both hormones in immature rats. In neonatal and immature rats, one PRL cell type with granules 200 nm in diameter was present. In adult rats, two types of PRL cells were present: one containing polymorphous granules measuring about 500 nm (prevalent in female rats), the other with spherical granules about 200 nm (prevalent in male rats). No changes were detected in GH cells during maturation.
Ultrastructural cytochemical and autoradiographic demonstration of nonspecific esterase(s) in guinea pig basophils.
Authors: Dvorak A M , Monahan-Earley R A , Dvorak H F , Galli S J
Published: April 2011

We used ultrastructural autoradiographic and cytochemical methods to localize esterase activities in unstimulated guinea pig basophils and in basophils undergoing degranulation or recovery from degranulation. We used tritium-labeled diisopropylfluorophosphate (DFP) as a probe for serine enzymes and localized this probe by ultrastructural autoradiography to cytoplasmic granules of immature or mature unstimulated basophils, as well as to granules released by degranulating basophils. Ultrastructural cytochemistry using alpha naphthyl acetate (ANA) as substrate localized nonspecific esterase activity to extruded granules, either within the interiors of degranulation sacs or within granules completely separated from degranulating basophils. Extruded granules retained their esterase activity for as long as 24 hr after antigen-induced degranulation. The plasma membranes of unstimulated or degranulating basophils, as well as of basophils recovering from degranulation, displayed prominent cell surface ANA esterase ectoenzyme activity. Lipid bodies, organelles present in the cytoplasm of both control and recovering basophils, were also alpha naphthyl acetate esterase (ANAE)-positive. Thus, cytochemical and autoradiographic techniques localized esterase and/or [3H]-DFP-binding activities to cytoplasmic granules, lipid bodies, and cell surface of basophils, and these enzyme activities persisted during both degranulation and recovery from degranulation.
Lectin-labeled membrane is transferred to the Golgi complex in mouse pituitary cells in vivo.
Authors: Balin B J , Broadwell R D
Published: April 2011

Labeling of the Golgi complex with the lectin conjugate wheat germ agglutinin-horseradish peroxidase (WGA-HRP), which binds to cell surface membrane and enters cells by adsorptive endocytosis, was analyzed in secretory cells of the anterior, intermediate, and posterior lobes of mouse pituitary gland in vivo. WGA-HRP was administered intravenously or by ventriculo-cisternal perfusion to control and salt-stressed mice; post-injection survival times were 30 min-24 hr. Peroxidase reaction product was identified within the extracellular clefts of anterior and posterior pituitary lobes through 24 hr but was absent in intermediate lobe. Endocytic vesicles, spherical endosomes, tubules, dense and multivesicular bodies, the trans-most saccule of the Golgi complex, and dense-core secretory granules attached or unattached to the trans Golgi saccule were peroxidase-positive in the different types of anterior pituitary cells and in perikarya of supraoptico-neurohypophyseal neurons; endoplasmic reticulum and the cis and intermediate Golgi saccules in the same cell types were consistently devoid of peroxidase reaction product. Dense-core secretory granules derived from cis and intermediate Golgi saccules in salt-stressed supraoptic perikarya likewise failed to exhibit peroxidase reaction product. The results suggest that in secretory cells of anterior and posterior pituitary lobes, WGA-HRP, initially internalized with cell surface membrane, is eventually conveyed to the trans-most Golgi saccule, in which the lectin conjugate and associated membrane are packaged in dense-core secretory granules for export and potential exocytosis of the tracer. Endoplasmic reticulum and the cis and intermediate Golgi saccules appear not to be involved in the endocytic/exocytic pathways of pituitary cells exposed to WGA-HRP.
Quantitative assessment of Na+,K+-ATPase localization by direct and indirect p-nitrophenyl phosphatase methods.
Authors: Firth J A
Published: April 2011

Na+,K+-ATPase histochemistry, using direct (Pb) and indirect (Mg/Sr-Co) p-nitrophenyl phosphatase methods, was assessed by scanning integrative microdensitometry of three classes of tubules in mouse and guinea pig kidneys. The methods yielded similar and appropriate patterns of activity distribution and inhibitor response. The indirect method gave preferable results, in that non-enzymic background was lower and rate of reaction product accumulation was considerably higher.
Localization of adenosine deaminase and adenosine deaminase complexing protein in rabbit brain.
Authors: Schrader W P , West C A , Strominger N L
Published: April 2011

Adenosine deaminase and adenosine deaminase complexing protein have been localized in rabbit brain. Brains fixed in paraformaldehyde or in Clarke's solution were blocked coronally. Blocks from brains fixed in paraformaldehyde were either frozen in liquid nitrogen or embedded in paraffin. Tissue fixed in Clarke's solution was embedded in paraffin. Sections from each block were stained by the peroxidase-antiperoxidase method for adenosine deaminase or complexing protein using affinity-purified goat antibodies. Adenosine deaminase and complexing protein did not co-localize. Adenosine deaminase was detected in oligodendroglia and in endothelial cells lining blood vessels, whereas complexing protein was concentrated in neurons. The subcellular location and appearance of the peroxidase reaction product associated with individual cells was also quite distinctive. The cell bodies of adenosine deaminase-positive oligodendroglia were filled with intense deposits of peroxidase reaction product. In contrast to oligodendroglia, the reaction product associated with most neurons stained for complexing protein was concentrated in granular-appearing cytoplasmic deposits. In some instances, these deposits were clustered about the nuclear membrane. Staining of neurons in the granular layer of cerebellum was an exception. Granule cells were lightly outlined by peroxidase reaction product. Cerebellar islands, also referred to as glomeruli, were stained an intense uniform brown. These results raise the possibility that oligodendroglia and blood vessel endothelia, through the action of adenosine deaminase, might play a role in controlling the concentration of extracellular adenosine in brain. They do not, however, support the suggestion that complexing protein aids in adenosine metabolism by positioning adenosine deaminase on the plasma membrane.
Detection of albumin mRNAs in rat liver by in situ hybridization: usefulness of paraffin embedding and comparison of various fixation procedures.
Authors: Tournier I , Bernuau D , Poliard A , Schoevaert D , Feldmann G
Published: April 2011

Our aim was to define optimal conditions for efficient and reproducible albumin mRNA detection in rat liver by in situ hybridization. We used an albumin-specific [3H]-labeled cDNA probe with a specific activity of 6-8.10(6) cpm/microgram DNA. In situ hybridization is as efficient on paraffin sections as on cryostat sections for detecting albumin mRNAs. Perfusion fixation with a 4% paraformaldehyde solution results in homogeneous RNA retention within tissue blocks, in contrast with immersion fixation, which yields heterogeneous RNA preservation. Comparison of immersion fixation with three different fixatives (paraformaldehyde, ethanol-acetic acid, and Bouin's fixative) shows that the highest level of hybridization signal is obtained with paraformaldehyde. Ethanol-acetic acid and Bouin's fixative appear less efficient for albumin mRNA detection. Loss of mRNAs within liver tissue blocks over time is largely although not completely prevented by paraffin embedding.
Rapid-freezing and malachite green-acrolein-osmium tetroxide freeze-substitution fixation improve visualization of extracellular lipids in rat incisor pre-dentin and dentin.
Authors: Goldberg M , Escaig F
Published: April 2011

Rat incisor tissue sections were fixed by a modified version of the malachite green-aldehyde method (MGA) composed of rapid-freezing, malachite green-acrolein staining, and osmium tetroxide freeze-substitution (Fr.MGAO). In the pre-dentin, a thick, dense network of branched fibrous structures was observed. Cryotechniques allowed visualization of complexes about twice as thick and dense as the aggregates visualized on MGA-treated sections. Pretreatment of rapid-frozen samples with methanol before freeze-substitution fixation and staining prevented staining of the complexes otherwise revealed by the Fr.MGAO method. Electron-dense material stained by this procedure resisted de-mineralization with EDTA, while intramitochondrial granules and dentin crystallites were dissolved. EDTA treatment demonstrated unequal distribution of Fr.MGAO staining in dentin in the form of tiny dots underlining the collagen fibers. These results support the concept that rapid-freezing, followed by staining and freeze-substitution fixation, improves preservation of the phospholipids visualized as extracellular matrix components in pre-dentin and dentin of rat incisors.
Ultrastructural distribution of sulfated glycosaminoglycans in epithelial-mesenchymal interface of developing rat tooth germs.
Authors: Kogaya Y , Furuhashi K
Published: April 2011

We investigated the ultrastructural distribution of sulfated glycosaminoglycans in the epithelial-mesenchymal interface of tooth germs by use of the high-iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining and enzymatic digestion method. At an early stage in odontoblast differentiation, HID-TCH-SP stain deposits were sparsely distributed in the basement membrane and in the intercellular spaces. Subsequently, as formation of the initial predentin matrix began, HID-TCH-SP stain deposits were densely distributed in the interfibrillar spaces and the basement membrane. Testicular hyaluronidase digested most of those in the progenitor pre-dentin, whereas those in the region of basal lamina resisted enzymatic digestion. Testicular hyaluronidase-resistant HID-TCH-SP stain deposits were susceptible to heparitinase, indicating that the sulfated glycosaminoglycan in the basal lamina is heparan sulfate. Furthermore, the heparan sulfate tended to be regularly arranged at the sites of internal and external lamina densa. However, as progenitor pre-dentin matrix formation proceeded, the numbers of stain deposits temporarily increased and their distribution pattern became irregular, finally tending to disappear with the disruption of basal lamina.
Immunoperoxidase detection of myeloid antigens in glycolmethacrylate-embedded human bone marrow.
Authors: Archimbaud E , Islam A , Preisler H D
Published: April 2011

Different methods for fixation and exposure of antigenic determinants were tested for detection of a granulocytic differentiation antigen by the monoclonal antibody L12-2, using an indirect immunoperoxidase method on semi-thin sections of undecalcified, glycolmethacrylate-embedded human bone marrow biopsies. Fixation in Bouin's solution for 3 hr gave a more intense and more homogeneous immunological staining than fixation in absolute methanol, 4% formalin, B5, or Michel's medium, and the morphological detail was excellent. Digestion by pronase or trypsin was required. Coating the glass slides with Alcian blue prevented loss of sections from the slides during the staining procedure. Bouin fixation also made possible detection of two other differentiation antigens expressed in the granulocytic series, using the monoclonal antibodies 1G10 and R1B19. Furthermore, the same technique also permitted detection of factor VIII-RAg in the megakaryocytes, as well as recognition of cells of the erythroid series by use of polyclonal rabbit antisera.
Cerium-based cytochemical method for detection of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity at physiological pH.
Authors: Kobayashi T , Okada T , Seguchi H
Published: April 2011

We have developed a new cytochemical method for detecting the ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase (K-NPPase) activity of the sodium-potassium-activated adenosine triphosphatase (Na-K ATPase) complex. The incubation medium contains p-nitrophenylphosphate (p-NPP) as substrate, cerium chloride as capture agent, Tricine buffer, MgCl2, and KCl. Tricine buffer protected against the medium turbidity caused by non-enzymatic reaction at pH 7.5. Biochemically, the accumulation of p-nitrophenol and phosphate in the reaction precipitate was proportionally related to the enzyme concentration. Ultracytochemically, the reaction products of the K-NPPase activity were localized as fine and uniform electron-dense deposits in the cytoplasmic side of specialized basolateral plasma membranes of cells of kidney distal convoluted tubules, secretory cells of salt gland, and marginal cells of stria vascularis. This method has the advantage of being useful at physiological pH.
Detection of mRNAs present at low concentrations in rat liver by in situ hybridization: application to the study of metabolic regulation and azo dye hepatocarcinogenesis.
Authors: Lamas E , Kahn A , Guillouzo A
Published: April 2011

In situ hybridization on tissue sections was used to detect mRNAs present at low concentrations during metabolic adaptation and azo dye carcinogenesis in rat liver. The method consisted of hybridizing the slices at relatively high stringency with [35S]-labeled single-stranded probes derived from cDNA insert clones into the M13 phage. L-pyruvate kinase mRNA was proved to be present at very low concentrations in hepatocytes of fasted rats and to be relatively abundant in all hepatocytes after 18 hr of refeeding on a carbohydrate-rich diet. Aldolase A mRNA concentrations have been previously shown to increase markedly in liver of 3'-methyl DAB-fed rats, with a maximum at the fourth week. We demonstrate here, using our in situ hybridization technique, that this phenomenon is not due to re-expression of this "fetal marker" in hepatocytes but to its abundancy in proliferating small cells (i.e., so-called oval and transitional cells). Small amounts were also detected in sinusoidal cells. In normal liver, aldolase A mRNAs were detected only in some sinusoidal cells.