bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution of immunoreactive Na+,K+-ATPase in gerbil cochlea.
Authors: Schulte B A , Adams J C
Published: April 2011

The distribution of Na+,K+-ATPase was mapped in cochleas of mature gerbils with normal hearing, using a specific and sensitive immunocytochemical method. Na+,K+-ATPase was abundant in the basolateral plasma membrane of marginal cells in the stria vascularis. Considerable levels of enzyme were also associated with the surfaces of spiral ganglion neurons and their central and peripheral processes. An unexpected finding was the detection of high levels of immunoreactive Na+,K+-ATPase in three different populations of cells lying in the inferior portion of the spiral ligament and at the medial and lateral border of the scala vestibuli just superior to the attachment of Reissner's membrane. Cells in these areas shared the morphological characteristics of cells specialized for active transport but appeared to be nonpolarized, suggesting a uniform distribution of Na+,K+-ATPase over their entire plasmalemma. The presence of these three distinct cell populations in the cochlea of several mammalian species suggests that they play an important role in cochlear function, perhaps that of regulating the cation content of perilymph. The absence of discrete concentrations of Na+,K+-ATPase-rich cells in the perilymphatic connective tissue of the bird cochlea and the mammalian vestibular system suggests further that these cells may be involved with generating and maintaining the high endolymphatic potential unique to the mammalian cochlea.
Autometallography for histochemical visualization of rat incisor polyanions with cuprolinic blue.
Authors: Lormée P , Lécolle S , Septier D , Le Denmat D , Goldberg M
Published: April 2011

Autometallography was applied to semi-thin sections of rat incisors fixed a solution of cuprolinic blue-aldehyde. The resulting reduction of silver ions to metallic silver amplifies the copper sulfide signal of the cationic dye. Silver grains were seen over the cell bodies of ameloblasts and odontoblasts but not over their processes. This was owing to the interaction of cuprolinic blue with the DNA and RNA of these cells. In the extracellular matrix, silver grains were unevenly distributed over the predentin, dentin, and forming enamel. The distal predentin near the mineralization front and a thin band of dentin located near the dentino-enamel junction displayed unexpectedly intense accumulation of silver grains, whereas all other portions of the extracellular matrix exhibited the distribution of glycosaminoglycans expected from previous studies. The present investigation constitutes a new application of autometallography to glycosaminoglycan histochemistry.
Effects of prolonged ethanol exposure on the glial fibrillary acidic protein-containing intermediate filaments of astrocytes in primary culture: a quantitative immunofluorescence and immunogold electron microscopic study.
Authors: Renau-Piqueras J , Zaragoza R , De Paz P , Baguena-Cervellera R , Megias L , Guerri C
Published: April 2011

We investigated the effects of ethanol exposure on the shape of the cell and the morphology of intermediate filaments (IF) of cortical astrocytes in primary culture. The content and distribution of glial fibrillary acidic protein (GFAP), the major component of glial IF, was assessed using an anti-GFAP monoclonal antibody and fluorescence scanning densitometry together with quantitative pre- and post-embedding immunogold electron microscopy. The astrocytes were from 21-day-old fetuses obtained from both control and chronic alcoholic rats and were cultured for 28 days in the absence or presence of ethanol (25 mM). The main findings were: (a) ethanol-exposed astrocytes failed to develop processes or to acquire a filamentous IF distribution pattern; (b) these cells showed less GFAP than astrocytes without alcohol; (c) ethanol interfered with the reorganization of the anti-GFAP binding sites from clustered to random; and (d) astrocytes from alcohol-exposed fetuses cultured in the absence of ethanol also showed these alterations, suggesting initial damage to astrocyte precursor cells. Since the glial filaments play a crucial role in creating a scaffolding that guides neuronal migration, the effect of ethanol on astrocyte IF may possibly be correlated with the mechanisms underlying mental retardation and motor dysfunction which are characteristics of fetal alcohol syndrome.
Freeze-substitution staining of rat growth plate cartilage with alcian blue for electron microscopic study of proteoglycans.
Authors: Maitland M E , Arsenault A L
Published: April 2011

To selectively stain polyanionic macromolecules of growth plate cartilage and to prevent artifacts induced by aqueous fixation, proximal tibial growth plates were excised from rats, slam-frozen, and freeze-substituted in 100% methanol containing the cationic dye Alcian blue. Electron microscopic examination showed the tissue stained with Alcian blue to be comparable in ultrastructural preservation to tissues slam-frozen and freeze-substituted in the absence of Alcian blue. The extracellular matrix exhibited a characteristic staining pattern when stained by this method. The pericellular rim was identified as a band of varying width encircling the chondrocyte and its cell processes. Peripheral to the pericellular rim the heterogeneity of staining within the extracellular matrix increased, taking the form of polymorphic densities. X-ray microanalysis showed that the visual interpretation of electron density was related to the concentration of copper present, and that the concentration of sulfur was variable in the pericellular rim and in the interterritorial matrix. The difficulties associated with aqueous fixation and staining procedures are discussed in contrast to the improved preservation achieved by cryogenic methods.
Synthesis of a more stable osmium ammine electron-dense DNA stain.
Authors: Olins A L , Moyer B A , Kim S H , Allison D P
Published: April 2011

Specific DNA staining for electron microscopic observation is simplified by a shorter synthesis of the staining reagent. The new, more reliable reagent, osmium ammine-B, is stable for more than a year, dissolves completely in water, and does not require reoptimization of staining conditions for every batch, yet reproducibly gives strong contrast to DNA-containing structures.
Distribution, ultrastructural localization, and ontogeny of the core protein of a heparan sulfate proteoglycan in human skin and other basement membranes.
Authors: Horiguchi Y , Couchman J R , Ljubimov A V , Yamasaki H , Fine J D
Published: April 2011

A variety of heparan sulfate proteoglycans (HSPG) have been identified on cell surfaces and in basement membrane (BM). To more fully characterize HSPG in human skin BM, we used two monoclonal antibodies (MAb) directed against epitopes of the core protein of a high molecular weight HSPG isolated from murine EHS tumor. Indirect immunofluorescence revealed linear distribution of HSPG within all skin BM, and within BM of all other human organs investigated. In a study of the ontogeny of HSPG in human skin BM, HSPG was detectable as early as 54 gestational days, comparable with other ubiquitous BM components, such as laminin and type IV collagen. Immunoelectron microscopy on adult skin and neonatal foreskin showed staining primarily within the lamina densa (LD) and sub-lamina densa regions of the dermoepidermal junction (DEJ) and vascular BM. In neonatal foreskin, additional staining was noted of basilar cytoplasmic membranes of keratinocytes, endothelial cells, and pericytes. We conclude that the core protein of a high molecular weight HSPG is ubiquitous in human BM, appears in fetal skin on or before 54 days, and is present primarily in the regions of the LD and sub-LD.
Quantitative immunocytochemical localization of Na+,K+-ATPase in rat ciliary epithelial cells.
Authors: Okami T , Yamamoto A , Omori K , Akayama M , Uyama M , Tashiro Y
Published: April 2011

Ultrastructural localization of Na+,K+-ATPase in rat ciliary epithelium was investigated quantitatively by the protein A-gold technique, using an affinity-purified antibody against the alpha-subunit of Na+,K+-ATPase. Immunoblot analysis showed that the antibody bound specifically to the alpha-subunit of Na+,K+-ATPase in the ciliary body. Gold particles were found mainly on the basolateral surfaces of both the pigmented epithelial (PE) and nonpigmented epithelial (NPE) cells with an approximately twofold higher labeling density in the PE cells. A few gold particles were also found on the apical and ciliary channel surfaces of the PE cells, whereas no significant binding was found on the apical surfaces of the NPE cells. The basolateral surfaces of PE and NPE cells are markedly infolded and are much greater in area than the apical surfaces. This means that Na+,K+-ATPase is almost exclusively located on the basolateral surfaces of both the NPE and PE cells. We suggest that the Na+,K+-ATPase of both the NPE and PE cells play an important role in the formation of aqueous humor.
LN-6: a monoclonal antibody to vimentin expressed in non-hematopoietic mesenchymal cells and derived tumors and reactive in B5-fixed, paraffin-embedded tissues.
Authors: Stathopoulos E , Naeve G S , Taylor C R , Epstein A L
Published: April 2011

We generated a monoclonal antibody (MAb), designated LN-6, directed against human vimentin, which retains its immunoreactivity in B5-fixed, paraffin-embedded tissues. Like other anti-vimentin MAb, LN-6 was found to be reactive with a wide spectrum of human sarcomas and normal cells of mesenchymal derivation. However, unlike other similar reagents, LN-6 was unreactive with normal and malignant human lymphoid cells and therefore displays a more restricted immunoreactivity. Because of its ability to stain routinely processed pathological tissues and its marked reactivity with human sarcomas, LN-6 is a unique reagent for the immunohistochemical diagnosis of human cancer.
Immunohistochemical localization of P-glycoprotein in adult human ovary and female genital tract of patients with benign gynecological conditions.
Authors: Finstad C L , Saigo P E , Rubin S C , Federici M G , Provencher D M , Hoskins W J , Lewis J L , Lloyd K O
Published: April 2011

The multidrug-resistance gene, MDR1, encodes a plasma membrane glycoprotein termed P-glycoprotein, which mediates active cellular efflux of certain cytotoxic agents. Two mouse monoclonal antibodies (MAb), C219 and JSB-1, were used to identify P-glycoprotein in frozen tissue from the female genital tract of 14 women with benign gynecological conditions; multiple samples from several sites in the genital tract were available from seven patients. P-glycoprotein was detected in the ovarian surface epithelium in four of 14 cases, in the Fallopian tube in three of five cases, in occasional epithelial cells of the endometrial glands in two of five cases, in some endocervical glandular epithelium in three of five cases, in ectocervical squamous epithelium in one of the two cases, and in luteinized cells of the eight cases in which a corpus luteum was present in the specimen. Positive staining with these two MAb was also observed in some endothelial cells in the cortex of the ovary and in the stromal tissue of the myometrium, endometrium, and endocervix. These studies suggest that, if epithelial ovarian cancers are derived from the surface epithelial cells of the ovary, a small proportion of the cancers might be expected to retain the phenotype found in non-cancerous cells and to express P-glycoprotein.
Immunocytochemical identification of myoepithelial cells in normal and neoplastic rat mammary glands with the lectins Griffonia simplicifolia-1 and pokeweed mitogen.
Authors: Hughes C M , Rudland P S
Published: April 2011

Peroxidase-conjugated Griffonia simplicifolia-1 (GS-1) and pokeweed mitogen (PWM) histochemically stain only the myoepithelial cells and not the epithelial or fibroblastic cells of rat mammary glands preserved in methacarn or glutaraldehyde and embedded in paraffin. This pattern of staining occurs in other rat exocrine glands except the pancreas, but is the reverse of that seen in most lining epithelium. The histochemical binding of GS-1 and PWM to myoepithelial cells is inhibited specifically by D-galactose and by polymers of N-acetylglucosamine, respectively. GS-1 and its subcomponent, GS-1-B4, also bind to extracellular structures similar to those stained by anti-laminin serum. At the ultrastructural level, both conjugated GS-1 and PWM bind to the plasma membrane of the myoepithelial cells, as well as to the adjacent basement membrane. Non-metastasizing rat mammary tumors produced by dimethylbenz[a]anthracene, by derivative epithelial stem-cell lines, and by a transplantable tumor all contain more elongated myoepithelium-like cells as well as cuboidal epithelium-like cells; both cell types are neoplastic. The more elongated myoepithelium-like cells are stained by GS-1 and PWM, whereas the cuboidal epithelium-like cells are unstained. Moderately and strongly metastatic rat mammary tumors produced by epithelial cell lines and by transplantable tumors, respectively, contain no such neoplastic cells that bind either lectin. We suggest that the carbohydrate receptors for GS-1 and PWM are consistent markers for the presence of the myoepithelial cell in normal and tumorous rat mammary glands.
Lipocortin-1 immunoreactivity in the human pituitary gland.
Authors: Johnson M D , Gray M E , Pepinsky R B , Stahlman M T
Published: April 2011

We evaluated the distribution of lipocortin-1 immunoreactivity in 118 immature or mature human hypophyses using the peroxidase-antiperoxidase (PAP) technique with a polyclonal rabbit antiserum against lipocortin-1. Serial sections were evaluated for five pituitary hormones and S-100 protein immunoreactivity to compare their distributions with that of lipocortin-1. Scattered or moderate numbers of cells exhibited lipocortin-1 immunoreactivity in the pars distalis of 89 subjects ranging in age from 27 weeks' gestation to 83 years. Seven immature and seven aged specimens exhibited no immunostaining, while 15 specimens from older individuals exhibited only rare immunostaining. Immunostaining did not appear to co-localize selectively with any specific pituitary hormone, although the distribution of immunoreactivity did overlap that of some corticotrophs and was seen in elongated processes of S-100-containing folliculostellate cells. Lipocortin-1 was also found in epithelial cells lining colloid cysts of the residual pars intermedia in 115 of 118 pituitaries ranging in age from 23 weeks' gestation to 83 years. In many intermediate lobe cysts, lipocortin-1 exhibited a pattern of immunoreactivity that partially overlapped the distribution of S-100 protein immunostaining, although the pattern was not identical. Pre-absorption of anti-lipocortin-1 antiserum with lipocortin-1-coupled Sepharose-4B immunoreactivity resulted in loss of immunoreactivity in both lobes. No lipocortin-1 immunoreactivity was seen in the neurohypophysis.
Characterization of the isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes by lectin cytochemistry.
Authors: Yamamoto A , Masaki R , Tashiro Y
Published: April 2011

The isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes were characterized by lectin cytochemistry using concanavalin A (ConA), Ricinus communis agglutinin 120 (RCA-120), and wheat germ agglutinin (WGA). We found that RCA-120, ConA, and WGA bind to these membranes. The distribution of the lectins on the isolation membranes was heterogeneous, mainly found on the rims, which we referred to as the peripheral dilated portion. When the rims fused and thus formed autophagosomes the apparent sites of fusion were strongly labeled by the lectins. After autophagosomes were transformed to autolysosomes by fusion with lysosomes, the limiting membranes became more densely and homogeneously labeled with the lectins. We previously reported that cytochrome P-450 does not exist on the limiting membranes of the autophagosomes. Taken together, these results suggest that the isolation membranes may originate not from endoplasmic reticulum membranes but from some post-Golgi membranes that contain complex type N and/or O-linked oligosaccharide chains.
Modulation of glycogen stores in epithelial cells during airway development in Syrian golden hamsters: a histochemical study comparing concanavalin A binding with the periodic acid-Schiff reaction.
Authors: Ito T , Newkirk C , Strum J M , McDowell E M
Published: April 2011

We studied glycogen storage in the developing airway epithelium of Syrian golden hamsters from gestational Day 11 to neonatal Day 2 using concanavalin A (ConA) staining as an adjunct approach to the periodic acid-Schiff (PAS) reaction. One hundred and fourteen fetuses and neonates were fixed in 4% formaldehyde-1% glutaraldehyde, 6% mercuric chloride-1% sodium acetate-0.1% glutaraldehyde, and 95% ethanol, embedded in paraffin, and stained with ConA-horseradish peroxidase conjugate as well as with PAS. ConA staining was abolished by alpha-glucosidase digestion or by pre-treatment with periodic acid, demonstrating that ConA bound to glycogen. In tissues fixed with mercury and/or aldehydes, ConA staining was greatly enhanced by pepsin digestion. Airway glycogen stores, revealed by ConA and PAS, fluctuated during development. At first all the undifferentiated epithelial cells contained abundant glycogen. Then, coincident with the appearance of the first endocrine cells, the glycogen stores were depleted. Thereafter, glycogen accumulated in pre-secretory and basal cells until birth, but by 2 days after birth the glycogen stores were again depleted. The initial depletion of glycogen followed by repletion was observed at all levels of the conducting airways; changes in the trachea preceded those in the bronchi and bronchioles by 1 and 2 days, respectively.
Elastase and cathepsin G of human monocytes: heterogeneity and subcellular localization to peroxidase-positive granules.
Authors: Kargi H A , Campbell E J , Kuhn C
Published: April 2011

We used antibodies to human leukocyte ("neutrophil") elastase and cathepsin G to localize the corresponding antigens in human neutrophils, monocytes, and alveolar macrophages by immunohistochemistry. Furthermore, we combined immunogold localization with enzyme histochemistry to localize proteinase antigens and endogenous peroxidase activity in the same sections. As expected, all neutrophils contained both elastase and cathepsin G, and the proteinases localized to granules with peroxidase activity. In contrast, marked heterogeneity in monocyte staining for elastase, cathepsin G, and endogenous peroxidase was found. Sixty percent or more were unstained, while the remainder varied greatly in staining intensity. The elastase and cathepsin G in monocytes were localized by immunoelectron microscopy, combined with histochemistry, to cytoplasmic granules which had peroxidase activity. Alveolar macrophages were unstained. Therefore, a subpopulation of peripheral blood monocytes contains leukocyte elastase and cathepsin G in a cell compartment from which these enzymes may potentially be released into the extracellular space. The occurrence of peroxidase and neutral proteinases in the same granules in monocytes could permit the H2O2-myeloperoxidase-halide system and the neutral proteinases to act in concert in such functions as microbe killing and extracellular proteolysis.
A monoclonal antibody raised against Alzheimer cortex that specifically recognizes a subpopulation of microglial cells.
Authors: Cras P , Gheuens J , Lübke U , Boons J , Vandermeeren M , Van Heuverswijn H , Martin J J
Published: April 2011

A monoclonal antibody (MAb), termed AMC30, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimer's brain. The antigen to which AMC30 is directed was expressed by microglial cells in senile plaques of Alzheimer's disease (AD). Microglia in the parenchyma surrounding brain tumors or infarctions, multinuclear giant cells, perivascular and parenchymal macrophages throughout the brain of AIDS patients were also labeled. Different non-nervous system lesions in which macrophages participate were also stained. Microglial cells in normal areas of the cortex or white matter were not labeled with MAb AMC30. The antigen to which AMC30 is directed was not detected in normal bone marrow, lymph nodes, lung, or spleen monocytes or macrophages. The epitope recognized by MAb AMC30 was present after formalin fixation and paraffin embedding. Our findings suggest that this MAb is directed against an antigen that is specifically expressed in a subpopulation of microglial cells and macrophages reactive to various pathological conditions.
A quantitative histochemical study of D-amino acid oxidase activity in rat liver in relationship with feeding conditions.
Authors: Patel H R , Frederiks W M , Marx F , Best A J , Van Noorden C J
Published: April 2011

The histochemical method for the demonstration of D-amino acid oxidase activity in rat liver, based on the use of cerium ions and the diaminobenzidine-cobalt-hydrogen peroxide procedure, was improved by the application of unfixed cryostat sections and a semipermeable membrane interposed between section and gelled incubation medium. The amount of final reaction product precipitated in a granular form was about four times higher with this technique in comparison with conventional procedures using fixed sections and aqueous incubation media. The specificity of the reaction was proven by the 70% reduction of the amount of final reaction product when incubating in the presence of substrate and D,L-beta-hydroxybutyrate, a specific inhibitor of D-amino acid oxidase activity. Cytophotometric analysis of liver sections revealed that the specific test minus control reaction was linear with incubation time and section thickness. The Km value of the enzyme of 10.3 +/- 2.7 mM, as determined in periportal areas, is about five times the value found with biochemical methods in liver cell homogenates. The enzyme activity in periportal areas is about five times the activity in pericentral areas. Fasting (24 and 48 hr) induced a significant decrease in D-amino acid activity in periportal and pericentral areas. The possible physiological role of the enzyme in liver is discussed.
Localization of Na(+)-dependent active type and erythrocyte/HepG2-type glucose transporters in rat kidney: immunofluorescence and immunogold study.
Authors: Takata K , Kasahara T , Kasahara M , Ezaki O , Hirano H
Published: April 2011

Glucose is actively taken up from the glomerular filtrate into the tubule cells by the Na(+)-dependent active glucose transporter (GT), and passively crosses the basolateral membrane via facilitated diffusion GT. With the use of antibodies directed against two types of GTs, we show the immunocytochemical localization of the Na(+)-dependent active GT (SGLT1) and the erythrocyte/HepG2-type facilitated diffusion GT (GLUT1). For light microscopic observation, frozen sections were stained by the rhodamine labeling method. Counterstaining with fluorescein-phalloidin and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) was employed to facilitate cell type identification. Immunogold staining was carried out on ultra-thin frozen sections for electron microscopy. The antibody to SGLT1 reacted with a 77 KD protein in immunoblotting of a kidney lysate. By immunocytochemistry, SGLT1 was localized in the microvillous plasma membrane in the apical brush borders of the cells of all three proximal tubule segments (S1, S2, and S3). The antibodies to GLUT1, a member of the facilitated diffusion GT family, were raised against human erythrocyte GT or synthetic oligopeptides derived from HepG2 GT, which reacted with a 48 KD protein in immunoblotting of the kidney lysate. GLUT1 was found at the basolateral plasma membranes of S3 proximal tubule cells, cells of the thick limb of Henle's loop, and collecting duct cells. Combined with known physiological data, our findings suggest that SGLT1 in the apical plasma membrane of the proximal tubule cells is responsible for the Na(+)-dependent active reabsorption of glucose from the glomerular filtrate. GLUT1 in the basolateral plasma membrane of S3 cells may transport reabsorbed glucose to the blood vessels. GLUT1 in the basolateral plasma membranes of cells of the thick limb of Henle's loop and of the collecting duct, on the other hand, may nourish these metabolically active cells by facilitating the diffusion of extracellular glucose provided from blood through the basolateral side of the cells.
Application of the immunofluorescence technique and confocal laser scanning microscopy for studying the distribution of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptor on rat luteal cells.
Authors: Lakkakorpi J T , Rajaniemi H J
Published: April 2011

We used confocal scanning microscopy to study the semi-quantitative distribution of luteinizing hormone/chorionic gonadotropin (LH/CG) receptors on rat luteal cells at both the two- and the three-dimensional level. The receptors were visualized in 6-microns sections of pseudopregnant rat ovaries using polyclonal rabbit antiserum to hCG-affinity-purified LH/CG receptor in conjunction with rhodamine-conjugated anti-rabbit immunoglobulins. Twenty to 30 optical sections were taken at different focal planes from representative luteal cells with a confocal laser scanning microscope and then processed digitally to two- and three-dimensional pseudocolored images. Distinct differences in fluorescence intensity could be demonstrated at both the two- and the three-dimensional level on the luteal cell surfaces, suggesting an uneven distribution of the LH/CG receptors on the cell membranes. This probably results in the compartmentalization and polarization of luteal cell function.
Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.
Authors: Shi S R , Key M E , Kalra K L
Published: April 2011

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.
Immunocytochemical detection of atrial natriuretic peptide in periarterial myocardiocytes of cardiac ventricles of adult normotensive rat.
Authors: Gu J
Published: April 2011

The cardiac hormone atrial natriuretic peptide (ANP) is known to be produced by the atrial myocardiocytes. In the ventricles, the concentrations of ANP are very low and are often undetectable by immunocytochemistry. Apart from recent reports of ANP in the various Purkinje cell types, the histological distribution of the small amounts of ANP in the ventricular myocardium has not been well established. In this study, the distribution of ANP immunoreactivity in the ventricles of normotensive adult rats was examined with a modified version of an immunostaining procedure. ANP immunoreactivity was present, in addition to typical myocardial cells, in the myocardiocytes surrounding the arteries and arterioles, particularly at their bifurcations. The specificity of the immunostaining was confirmed with pre-absorption controls. Such a distribution indicates that in addition to being an endocrine hormone ANP may have a paracrine function. The periarterial myocardiocytes may be able to sense the pressure and/or volume changes of the surrounded blood vessels and to release ANP, which may in turn affect these blood vessels and thereby regulate coronary perfusion. The physiological and pathological significance of periarterial ANP distribution warrants further investigation.
IDENTIFICATION OF CYTOPLASMIC BASOPHILIA (RIBONUCLEIC ACID) BY FLUORESCENCE MICROSCOPY
Authors: VON BERTALANFFY LUDWIG , BICKIS IVAR
Published: April 2011

1. The fluorochrome, acridin orange (AO), allows identification of basophilic cytoplasmic inclusions in the supravital state.

2. The fluorescence picture of hepatic cells after treatment with AO shows the hyaloplasm faint gray-green; flake-like or granular structures in the cytoplasm, bright red; the nuclei, with chromatin structures, green; the central part of the nucleoli red, their periphery green.

3. The red-fluorescent cytoplasmic inclusions correspond to those observed with the toluidine-blue technique, and shown, by the RNase test, to consist mainly of RNA. The same is demonstrated by the changes of AO fluorescence after application of RNase to fresh or fixed tissues.

4. The fluorescence picture given by other cell elements and tissues in the rat liver is described.

5. Changes in the AO fluorescence of liver tissue with varying pH, dye concentration, staining time, action of salts, prolonged U.V. illumination, fixation, as well as the fluorescence of some biologically important substances (RNA, DNA, heparin, albumin, glycogen), treated with AO, are indicated.

6. The advantages and limitations of the method, and the theories of AO staining are discussed.

7. As compared to conventional histochemical methods, the applicability to living or supravital tissues of the fluorochrome technique decreases errors due to fixation, dehydration, embedding and related procedures, and the low concentrations applied in fluorochrome staining further safeguard against histochemical artifacts.
STUDIES IN HISTOCHEMISTRY: XLIII. THE DESIGN AND USE OF IMPROVED APPARATUS FOR THE PREPARATION AND FREEZING-DRYING OF FRESH-FROZEN SECTIONS OF TISSUE
Authors: GRUNBAUM BENJAMIN W. , GEARY JOHN R. , GLICK DAVID
Published: April 2011

The design and use of three pieces of apparatus which facilitate and speed sampling of fresh frozen tissue, sectioning, and subsequent handling of sections for freezing-drying or other procedures were described. These are:

A tissue borer that produces cylinders of tissue of an accurately determined diameter with minimum distortion, and provides a mounting support for microtoming that permits even thin samples to be sectioned without the contamination of a supporting medium.

An alignment device that is used to set the borer with its tissue in the most suitable position in the head of the microtome.

A section holder for freeze-drying and handling cut sections conveniently without damage to them.

Tests have shown that the use of this equipment mechanizes the procedure to the point where subjective factors are largely reduced and accurate sectioning with little variability can be achieved with relative ease.
Co-localization of an endocytic marker and acid phosphatase in a tubular/reticular compartment in macrophages.
Authors: Luo Z R , Robinson J M
Published: April 2011

Cultured resident murine macrophages are incubated in the continuous presence of the fluorescent endocytic marker Lucifer Yellow and a phorbol ester that activates protein kinase C. Under these steady-state labeling conditions the fluorescent tracer was, for the most part, in a tubular/reticular compartment. Enzyme cytochemical localization of acid phosphatase in the same cells showed essentially a one-to-one correlation between the Lucifer Yellow- and acid phosphatase-containing compartments. Procedures for epifluorescence observation and subsequent enzyme cytochemical examination of the same whole mount cells are described. In addition, chemical fixation methods for the preservation of this labile tubular/reticular compartment are presented.
Application of an optimized immunostaining technique to evaluate the heterogeneous secretory response from porcine somatotropes by cell blotting.
Authors: Dobado-Berrios P M , Ruiz-Navarro A , Torronteras R , Gracia-Navarro F
Published: April 2011

The objective of the present study was to quantify the absolute hormone release from individual porcine pituitary cells incubated on polyvinylidene difluoride (PVDF) transfer membranes (cell-blot assay). After immunoperoxidase staining, growth hormone (GH) release from isolated somatotrope cells appeared like a colored zone of secretion surrounding the cell. Optical densities of these secretion zones were quantitated by computerized image analysis and translated into picograms by means of an appropriate standard curve. As a prior step, the staining method and the optimal immunocytochemical conditions were selected by applying purified porcine growth hormone (pGH) to the transfer membranes. The avidin-biotin-peroxidase nickel-intensified (ABC-Ni) method produced a better resolution than the peroxide-anti-peroxidase (PAP) method, although both techniques were similar with regard to background, sensitivity, and range of quantitation. The amount of GH released from single porcine somatotropes was highly heterogeneous, although the cells were treated under the same conditions. Moreover, this fact was consistent with the stimulation of the average release of GH by GH-releasing factor (GHRF) but not by GHRF+somatostatin (SRIF). Our results confirm the availability of the recently developed cell-blot assay and support the concept of functional heterogeneity in anterior pituitary cell populations.
Developmental expression of a 53 KD dentin sialoprotein in rat tooth organs.
Authors: D'Souza R N , Bronckers A L , Happonen R P , Doga D A , Farach-Carson M C , Butler W T
Published: April 2011

Rat dentin contains a major sialic acid-rich glycoprotein, DSP, with an overall composition similar to that of bone sialoproteins but whose biological role in dentinogenesis is unknown. Using polyclonal affinity-purified antibodies to rat DSP and four immunohistochemical methods of detection, we studied the cell and tissue localization of DSP and the time course of its appearance during odontoblast differentiation. DSP first appeared within young odontoblasts concomitant with early secretion of pre-dentin matrix and before the onset of mineralization but was absent in pre-odontoblasts. DSP immunostaining also localized within secretory odontoblasts and was intense in odontoblastic processes. Early pre-dentin stained positive for DSP, in contrast to more mature pre-dentin, where immunoreactivity was less intense and more restricted to odontoblastic processes. In the zone of mineralized dentin matrix, a moderate and uniform staining pattern was evident. Intense immunostaining was also seen within the cells and matrix of dental pulp during dentinogenesis. Other cells and tissues within the tooth organ and those surrounding it were non-reactive. These findings suggest that DSP is developmentally expressed in cells of the odontoblastic lineage and may be a biochemical marker of odontoblastic activity.
Expression of alpha-lactalbumin, alpha-S1-casein, and lactoferrin genes is heterogeneous in sheep and cattle mammary tissue.
Authors: Molenaar A J , Davis S R , Wilkins R J
Published: April 2011

We used 35S-labeled cRNA probes to localize the sites of alpha-lactalbumin, alpha-S1-casein, and lactoferrin mRNA synthesis in sheep and forcibly weaned cattle mammary tissue. Expression of alpha-lactalbumin was absent in three of four "virgin" glands studied, present in some alveoli of "pregnant" glands but not in others, despite a similar histological appearance. In the early lactating gland, expression was high in those alveoli with few fat globules in their cells and lumen and was absent in alveoli with abundant fat globules. These observations suggest either that alpha-lactalbumin gene expression is linked to the long-term secretory activity of cells and falls once cells are resting or regressing, or that there are cyclical variations in expression, or that in the lactating gland some groups of epithelial cells are synthesizing alpha-lactalbumin and some are synthesizing fat. Expression patterns of alpha-S1-casein were similar to those of alpha-lactalbumin. Lactoferrin, in contrast, was expressed almost exclusively in the "fatty alveoli" of both species. Our results show that dramatic variations in milk gene expression can occur throughout the mammary gland of sheep and cattle and that at no stage of pregnancy, lactation, or involution can the gland be considered metabolically homogeneous.
Procedures for specific detection of silver-stained nucleolar proteins on western blots.
Authors: Hozák P , Roussel P , Hernandez-Verdun D
Published: April 2011

Nucleolar organizer region (NOR)-silver staining of the chromosomes and nucleoli is a method that enables the detection of proteins associated with the ribosomal genes. We adapted the most commonly used cytochemical NOR-silver staining techniques to Western-blotted proteins of HeLa cells, mimicking the silver staining of cells in situ, and testing several parameters that may influence the in situ reaction. Two of these techniques, both one-step methods with colloidal developers, were standardized to obtain reproducible results. The specificity of NOR staining is documented by: (a) only a few bands are revealed among the many proteins detected by total proteins staining on gels or blots; two major groups of bands are found around 100 KD and 40 KD that could correspond at least in part to nucleolin and B23 nucleolar proteins; (b) the silver staining of bands was not the result of the high relative protein concentrations; and (c) the same number of NOR-silver-stained bands was observed across a large range of protein concentrations. The reaction appeared to be specific for a subset of nucleolar proteins, because the same bands were observed with the use of nucleolar, nuclear, or total cell protein extracts, and the silver grains observed in electron microscopy were clearly confined to the nucleolar fibrillar centers and dense fibrillar component. The efficiency of the reaction was not modified by any of the tested fixative pre-treatments except that involving methanol. The presented standardization of NOR-silver staining on Western blots allows the characterization of the Ag-NOR proteins and their specific regions responsible for silver staining of the nucleolus.
The expression of desmosomal and corneodesmosomal antigens shows specific variations during the terminal differentiation of epidermis and hair follicle epithelia.
Authors: Mils V , Vincent C , Croute F , Serre G
Published: April 2011

Using five monoclonal antibodies (MAb), we studied by indirect immunofluorescence the desmosomes and a junctional structure specific to cornified layers, the corneodesmosome, in normal and plantar epidermis and in the various sheaths of the anagen hair follicle. The monoclonal antibodies DP1&2.2-15, PG5.1, and DG3.10, specific for desmoplakins I/II, plakoglobin, and desmoglein I, respectively, were used to study the desmosome antigens, and G36-19 and G20-21 to study the corneodesmosome antigens. The distribution and sequence of expression of the five antigens allowed the nine epithelial differentiation pathways studied to be merged into four distinct families: non-plantar epidermis, characterized by the absence of desmosome and corneodesmosome antigens in the stratum corneum; the outer root sheath of the hair follicle, which behaves like the viable layers of the epidermis with regard to the desmosome antigens but does not express the corneodesmosome antigens; plantar epidermis and the three components of the inner root sheath in which the corneodesmosome antigens are present up to the desquamating layer; and the three components of the hair shaft, which are characterized by the absence of expression of both the desmosome and the corneodesmosome antigens in its mature portion.
Superiority of in situ hybridization over immunolabeling for detecting DNA on Lowicryl sections: a study on adenovirus-infected cells.
Authors: Puvion-Dutilleul F , Pichard E
Published: April 2011

We investigated the intranuclear distribution of viral genomes in adenovirus Type 5-infected HeLa cells on ultra-thin sections of Lowicryl K4M-embedded material by immunolabeling of DNA and in situ hybridization with a viral DNA probe. Monoclonal AC-30-10, raised against double- and single-stranded DNA (dsDNA, ssDNA), and HB2 antibodies, raised against only the dsDNA, were used. Both antibodies intensely labeled the condensed host chromatin and the virus-induced substructures containing inactive encapsidated and non-encapsidated viral genomes. The antibodies labeled only slightly those substructures containing replicating and transcribing viral genomes. Viral ssDNA, which is accumulated within well-delineated compact fibrillar structures, was not revealed by AC-30-10 even after elimination of the proteins of the section. In situ hybridization detected all of these types of viral DNA, depending on the protocol used. Therefore, the sensitivity of in situ hybridization for detecting DNA appears superior to immunolabeling with specific antibodies.
Localization of amidating enzymes (PAM) in rat gastrointestinal tract.
Authors: Martínez A , Burrell M A , Kuijk M , Montuenga L M , Treston A , Cuttitta F , Polak J M
Published: April 2011

We studied the distribution of the two enzymes involved in post-translational C-terminal alpha-amidation of regulatory peptides in rat digestive tract, using immunocytochemical methods and in situ hybridization techniques. The enzymes were located in most of the fibers and neurons of the myenteric and submucous plexus throughout the entire digestive tract and in endocrine cells of the stomach and colon. Staining of reverse-face serial sections demonstrated that the enzymes in endocrine cells of the stomach co-localized with gastrin in the bottom of the gastric glands. Some gastrin-immunoreactive cells near the neck of the gland were negative for PAM, suggesting that amidation takes place only in the more mature cells. In the colon all cells immunoreactive for glucagon and GLP1 were also positive for peptidylglycine alpha-hydroxylating monooxygenase (PHM) but not for peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The absence of immunoreactivity for the amidating enzymes in endocrine cells of the small intestine, known to produce C-terminally amidated peptides, suggests the existence of other amidating enzymes.
A simple and reliable method for correlative light and electron microscopic studies.
Authors: DeFelipe J , Fairén A
Published: April 2011

We describe in detail a simple method for flat-embedding that can be subsequently used in correlative light and electron microscopic studies. The method can be applied to any material suitable for electron microscopy and is especially useful for study of the synaptology and ultrastructural characteristics of immunocytochemically or morphologically identified neurons or their processes. We present here an example to show how accurately one can delineate the fine details of a complex axonal arborization impregnated with the Golgi method in the mouse cerebral cortex. Golgi-impregnated sections to be studied at the electron microscopic level are osmicated, dehydrated, infiltrated with Araldite resin, flat-embedded, and identified cells or processes photographed. Serial semi-thin sections (1-2 microns thick) are then cut with an ultramicrotome, examined with the light microscope, and the elements rephotographed. Selected semi-thin sections are then resectioned on the ultramicrotome at 60-70 nm and examined electron microscopically. This method allows the systematic and accurate localization of stained cells and processes throughout the successive steps of the procedure.
Optimized detection of cytoplasmic immunoglobulin and CD3 in benign and malignant lymphoid cells: enhanced sensitivity combined with differential staining of endogenous peroxidases and light microscopic morphology.
Authors: Kranz B R , Thierfelder S
Published: April 2011

A novel immunocytochemical approach to the detection of cytoplasmic antigens, exemplified by immunoglobulin and CD3, was evaluated using benign and 155 malignant lymphohematopoietic cell specimens and conventional techniques for detailed comparison. It relies on (a) electrostatic cell attachment to poly-L-lysine-coated multispot slides, (b) sequential use of glutaraldehyde fixation and detergent permeabilization, and (c) staining of endogenous peroxidases and antigens in contrasting colors. Differential staining instead of inactivation of endogenous peroxidases and avoidance of artifacts incurred in conventional cytocentrifugation, dehydration, and alcohol or acetone fixation uniquely afforded improved precision in cell identification, clear distinction of cytoplasmic from surface antigenic staining, and greatly enhanced sensitivity in antigen detection, all combined with increased performance efficiency achieved by the use of multispot slides. With this method, cytoplasmic immunoglobulin and CD3 were found more widely distributed than has been previously recognized. Thus, almost all malignant cells in all cases of B-cell lymphoma/leukemia (encompassing the major subtypes) and T-ALL, as well as substantial proportions of benign mature B- and T-lymphocytes, stained strongly positive for cIg and cCD3, respectively, whereas myeloid cells were consistently negative. High sensitivity combined with excellent cytomorphology and differential myeloperoxidase staining permitted unequivocal differentiation of minute fractions of malignant cells against a heterogeneous background of benign cells.
Altered collagen expression in human dentin: increased reactivity of type III and presence of type VI in dentinogenesis imperfecta, as revealed by immunoelectron microscopy.
Authors: Waltimo J , Risteli L , Risteli J , Lukinmaa P L
Published: April 2011

We used transmission immunoelectron microscopy and polyclonal antibodies to study the reactivities of Types III and VI collagen in dentin of normal human permanent and primary teeth and in primary teeth from five patients with dentinogenesis imperfecta (DI) associated with osteogenesis imperfecta and occurring as a single trait. In the normal permanent tooth, reactivity of Type III collagen was occasional and, where present, peritubular. Staining of normal primary teeth was less occasional but still rare, whereas the abnormal dentin stained more uniformly. Atypical, non-striated fibrillar structures that also showed Type III collagen reactivity were observed in dentin of two of the three patients with DI as a single trait. Later, these two patients proved to be first cousins. Unlike antibodies to the N-terminal pro-peptide of Type I pro-collagen, antibodies to the C-terminal telopeptide of Type I collagen, used for comparison stained the affected dentin homogeneously. Reactivity of Type VI collagen, not detected in normal teeth, was seen in the dentin of all abnormal teeth, in association with non-fibrillar delicate material. This study also shows that although readily detectable in dentin affected by DI, Type III collagen is a minor constituent of normal human dentin matrix.