bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoradiographic investigation of estrogen binding in cultured rat ovarian surface epithelial cells.
Authors: Adams A T , Auersperg N
Published: April 2011

Autoradiographic evidence presented that the cultured rat ovarian surface epithelium exhibits estrogen receptor-like activity. Two autoradiographic techniques were used; one involved live cells that were labeled and freeze-dried, and the other the labeling of ethanol-fixed cells. Autoradiograms were prepared by dipping cells grown on plastic cover slips in liquid nuclear track emulsion. Exposure times were 1 to 4 weeks. Experiments using a pulse-chase technique in live cells and steroid competition tests in fixed and live cells gave evidence for translocation of tritiated estradiol from cytoplasm to nucleus in live cells and for a component of estrogen-specific binding in live and fixed cells. The techniques presented here for the investigation of estrogen receptors in cultured cells require little tissue, are simple, and relatively quick. Reports based on biochemical analyses of tissue homogenates claim the presence of estrogen receptors in human ovarian carcinomas of surface epithelial origin and in rat ovarian surface epithelium. The results of this study add further evidence that the ovarian surface epithelium has estrogen receptor activity and should be considered an estrogen target tissue.
Actin and secretion of surfactant.
Authors: Tsilibary E C , Williams M C
Published: April 2011

Type II cells were examined in situ in order to study the role played by actin filaments in secretion of surfactant. Following beta-adrenergic stimulation, the number of lamellar bodies per cell profile decreased substantially, an indication that exocytosis had occurred. In isoproterenol-stimulated cells decoration of actin by S1 revealed greater numbers of filaments associated with lamellar bodies, free in the cytoplasm, and associated with the cellular apex than in control cells. If cells were treated prior to beta-agonist stimulation with cytochalasin D, a drug which disrupts filamentous actin, the secretory response to the agonist was abolished. Together these two observations suggest that actin filaments participate in the intracellular movement and/or extrusion of lamellar bodies from the cells.
Dehydration and embedding temperatures affect the antigenic specificity of tubulin and immunolabeling by the protein A-colloidal gold technique.
Authors: Armbruster B L , Garavito R M , Kellenberger E
Published: April 2011

Qualitative and quantitative tests were performed to determine whether the temperature at which dehydration and embedding occur affects the antigenic specificity of tubulin and the protein A-gold (pAg) immunolabeling technique. The analysis indicates that low temperature (-35 degrees C) treatment increased the specificity and density of pAg labeled anti-tubulin antibodies to Leishmania tropica subpellicular microtubules as compared to samples prepared at 0 degrees C or 20 degrees C.
Localization of biotinylated gonadotropin releasing hormone on pituitary monolayer cells with avidin-biotin-peroxidase complexes.
Authors: Childs G V , Naor Z , Hazum E , Tibolt R , Westlund K N , Hancock M B
Published: April 2011

The new avidin-biotin-peroxidase complex (ABC) technique was used to localize the [D-Lys6] analog of gonadotropin releasing hormone (GnRH), labeled with biotin, on pituitary monolayer cultures from female rats. Staining was diffuse, or in patches, on the surface of 10-17% of the cells 30 sec-3 min after the addition of 10(-10)-10(-12) M biotin-labeled GnRH. In parallel studies, double stains for gonadotropins showed label on 16.3 +/- 2% of the monolayers. Capping was evident by 3 min after exposure and the stain appeared in dense patches, vesicles, or granules 10-30 min after exposure. The stain was abolished by the addition of a 10- to 100-fold excess of unlabeled [D-Lys6] GnRH. Biotinylated GnRH released luteinizing hormone (LH) and follicle stimulating hormone (FSH) and was either equipotent or 10 times more potent than the unlabeled analog in multiple dose-response tests. The ED50 of the 4 hr release was 0.075 nM for LH and 0.02 nM for FSH. Competitive binding assays showed that the binding affinity of the biotinylated GnRH was within the range found for the unlabeled analog (0.7 nM-IC50). This report describes the localization of biotinylated GnRH on the surfaces of cells exposed to low concentrations of the analog with a technique that requires minimal manipulation of the cells, and is performed in less than one day.
Distribution of glucose-6-phosphatase activity in mice studied by in vitro whole-body autoradiography.
Authors: Watanabe M , Goto H , Matsushima M , Shimono R , Kihara T
Published: April 2011

The distribution of the glucose-6-phosphatase (G6Pase) activities was demonstrated by in vitro whole-body autoradiography. Relatively high activities were observed in the liver, kidney, and small intestine of mice. However, these organs did not exhibit uniform activity throughout. The activity of the liver was found heterogeneous, but that of the duodenum and jejunum were higher than for those of other parts of the intestine. G6Pase activity was higher in the cortex of the kidney than in the medulla. In addition to these observations, it was also found that the skeletal system had high radioactivity. These results were similar to those from biochemical experiments. This method of in vitro whole-body autoradiography would seem to be of value in studying macroscopic distributions of other enzyme activities in organs as well as in whole-body.
Immunohistochemical studies on the gastrointestinal tract using antisera to Met-enkephalin and Met-enkephalin Arg6Phe7.
Authors: Bu'Lock A J , Vaillant C , Dockray G J
Published: April 2011

Antisera specific for the C-terminus of Met-enkephalin and two of its variants isolated from adrenal medulla and brain, namely Met-enk Arg6 and Met-enk Arg6Phe7, have been used in immunohistochemical studies of the gastrointestinal tract in rat, mouse and guinea pig. Met-enk and Met-enk Arg6Phe7-like immunoreactivities were found with similar distribution in nerve cell bodies of the myenteric plexus, and in fibers that were particularly dense in the myenteric plexus and circular smooth muscle. The Met-enk Arg6 antiserum showed weak staining of nerve cells and fibers. In rat and mouse, the antiserum to Met-enk Arg6Phe7, but not those to Met-enk or Met-enk Arg6, also stained numerous endocrine-like cells of the antral mucosa. These were identified as gastrin cells by elution and re-staining experiments with C-terminal gastrin antisera. The rat and mouse gastrin cells might conceivably express the enkephalin gene, but fail to process it to yield Met-enkephalin; alternatively, the Met-enk Arg6Phe7-like immunoreactivity in gastrin cells could be due to a cross-reacting peptide that does not belong to the opioid series.
Immunocytochemical localization of cytoskeletal proteins and histone 2B in isolated membrane-depleted nuclei, metaphase chromatin, and whole Chinese hamster ovary cells.
Authors: Armbruster B L , Wunderli H , Turner B M , Raska I , Kellenberger E
Published: April 2011

Immunocytochemical techniques employing protein A-gold labeling were used to locate actin, tubulin, and histone 2B in thin sections of embedded, isolated membrane-depleted nuclei, metaphase chromosomes, and whole Chinese hamster ovary (CHO) cells. Actin and tubulin were detected in significant amounts in the cytoplasm and the nucleus of whole cells. The isolated membrane-depleted nuclei and metaphase chromosomes showed levels of actin and tubulin labeling either comparable to or sometimes lower than the levels of labeling in whole interphase cells. The results suggest that actin and tubulin are normal components of nuclei throughout the cell cycle. A mouse monoclonal antibody to histone 2B gave relatively weak labeling of nuclei and chromosomes in whole cells but intense labeling of isolated nucleoids and chromosomes. An increased concentration of antibody at the periphery of interphase nucleoids was noted.
Metal compound intensification of the electron-density of diaminobenzidine.
Authors: Newman G R , Jasani B , Williams E D
Published: April 2011

Diaminobenzidine (DAB), commonly used in immunocytochemistry as the substrate for peroxidase, has a low electron density. DAB has a known affinity for the salts of some metals and therefore an examination of the ability of six metal compounds (including osmium tetroxide) to increase the electron density associated with DAB deposits has been undertaken. Ultra-thin sections of unosmicated rat pituitary gland, embedded in L. R. White resin, were immunostained by a hapten sandwich immunoperoxidase method, using antibodies to ACTH and TSH. The unintensified electron density of the DAB polymer reaction product on the specific endocrine granules was compared with the electron density resulting from the use of each of the six metal compounds. Lead and silver nitrate gave unsatisfactory results, while phosphotungstic acid and uranyl acetate produced a limited increase in specific electron density under the conditions used. Gold chloride was found to give the highest electron density to the specific endocrine granules, followed closely by osmium tetroxide. Background staining was greater when osmium was used. We conclude that several metal compounds may be used to intensify the electron density of DAB, but of the ones tested, gold chloride, which is safer, more stable, and cheaper than osmium tetroxide, was clearly the best. This approach not only increases the electron density of the DAB reaction product, but allows of the possibility of quantitation using energy dispersive X-ray analysis.
Zinc-aldehyde fixation for light-microscopic immunocytochemistry of nervous tissues.
Authors: Mugnaini E , Dahl A L
Published: April 2011

Two procedures are described for vascular perfusion of the nervous system with a zinc-aldehyde fixative. The procedures, simple and economical, combine the advantages of perfusion fixation with an aldehyde solution and matrix stabilization by a mordating agent, and improve the sensitivity of the peroxidase-antiperoxidase (PAP) method for the immunocytochemical localization of several antigens. Procedure A is intended for the light-microscopic immunostaining of cellular elements containing high concentrations of antigen. Penetration of the immunoreagents is adequate without the use of detergents. Procedure B is particularly advantageous for the light-microscopic immunostaining of cellular elements that contain low concentrations of antigen, and for high-resolution microphotography. With procedure B, the tissue penetration of immunoreagents is more limited than with procedure A; however, neuronal cell bodies and dendrites are more easily penetrated by the immunoreagents than are axons. Neuronal cell bodies and dendrites thus become clearly detectable in the light-microscope, even when they are surrounded by numerous immunoreactive axon terminals, and especially after the blockage of axoplasmic transport by the topical injection of colchicine.
Cytologic characterization of pulmonary alveolar macrophages by enzyme histochemistry in plastic.
Authors: Soufleris A J , Pretlow T P , Bartolucci A A , Pitts A M , MacFadyen A J , Boohaker E A , Pretlow T G
Published: April 2011

Quantitative morphometric analyses of macrophages should facilitate a more precise definition of the role of macrophages in neoplastic diseases and inflammatory diseases of lymphoid and hemopoietic tissues. The usefulness of the available phenotypic markers is affected by the extent to which they are expressed by macrophages in tissue sections. For our study of phenotypic markers for macrophages in human tissue sections, we selected lung, since pulmonary alveolar macrophages are more readily identified morphologically in tissue sections than macrophages in most other tissues. In 1-2 micron sections of freshly fixed lung embedded in methacrylate, 89.7% of pulmonary alveolar macrophages had histochemically demonstrable acid phosphatase; 86.2%, nonspecific esterase with naphthol ASD chloroacetate as the substrate; 86.2%, nonspecific esterase with alpha-naphthyl butyrate as the substrate; 80.5%, peroxidase; and 75.8%, iron. With respect to their expression of markers, the observed heterogeneity among pulmonary alveolar macrophages is interesting; this heterogeneity may reflect the degree to which they have been activated, the different periods of time since they arrived in the lung, and differences in their local environments. Except for peroxidase, all examined markers were as well demonstrated when tissues were fixed after storage over liquid nitrogen as when fixation was carried out with fresh tissue. Acid phosphatase and nonspecific esterase with the chloroactetate substrate gave bright colors that would facilitate morphometric analyses. The storage of tissue over liquid nitrogen will be equally satisfactory for the characterization of macrophages with histochemical markers and monoclonal antibodies.
Calcium Transport Mechanism in Molting Crayfish Revealed by Microanalysis
Authors: Mizuhira Vinci , Ueno Masaki
Published: April 2011

Crayfish provide a good model in which to study the transport mechanism of Ca ions. During the molting stage, decalcified Ca ions are transferred into the blood and accumulate in the gastrolith epithelium, after which a gastrolith is formed on the surface of the epithelium. The gastrolith is dissolved in the stomach after molting, and the Ca is reabsorbed and redistributed throughout the newly formed exoskeleton. We studied the mechanism of Ca transport by cytochemical precipitation of Ca ions and by electron microanalysis, including X-ray microanalysis (EDX) and electron energy-loss spectroscopy (EELS), with a computer. In EDX analysis, the fine precipitates of K-antimonate in the gastrolith mitochondria clearly defined Ca with antimony; we also observed a large amount of Ca-oxalate in the mitochondria, and Ca-K X-ray pulses were clearly defined. Ca-K X-rays were also detected from fresh freeze-substituted mitochondria. Finally, we succeeded in taking a Ca-L EELS image from the mitochondria of fresh freeze-substituted thin sections. Only a very small amount of Ca was detected from the cell membrane and other organelles. Ca-adenosine triphosphatase (ATPase) and Mg-ATPase activity was also very clearly demonstrated in the mitochondria. These enzymes may play an important role in Ca metabolism.
Distribution of Glycoconjugates in the Kidney Studied by Use of Labeled Lectins
Authors: Murata F. , Tsuyama S. , Suzuki S. , Hamada H. , Ozawa M. , Muramatsu T.
Published: April 2011

Distribution of glycoconjugates in different areas of the rat kidney was studied by light and electron microscopy using six different horseradish peroxidase-labeled lectins. Glomeruli and brush borders of the proximal tubules reacted differently to these lectins, which indicated differences in the carbohydrate compositions of those regions. The ascending limb of Henle's loop (ALH) had strong binding sites for peanut agglutinin (PNA) and soybean agglutinin (SBA). Dolichos biflorus agglutinin (DBA) did not stain the cells of ALH but did stain those of distal convoluted tubules (DCT). DBA is a good marker for distinguishing ALH from DCT. DBA, PNA, and SBA were also good markers of the collecting duct. Ricinus communis agglutinin (RCA-1) and wheat germ agglutinin (WGA) diffusely stained the various components of different parts of the kidney.
Peroxisomes (Microbodies) in Human Liver: Cytochemical and Quantitative Studies of 85 Biopsies
Authors: Roels Frank , Pauwels Marina , Cornelis Alfons , Kerckaert Ingrid , Spek Peter Van Der , Goovaerts Gerda , Versieck Jacques , Goldfischer Sidney
Published: April 2011

The number, intracellular distribution, and staining characteristics of human hepatocellular peroxisomes that had been made visible by cytochemical staining for catalase were evaluated in biopsies from 75 patients with hepatic, inflammatory, or malignant disease and ten normal individuals. Intensity of staining was found to be proportional to enzymatic activity by microspectrophotometry. Scanning transmission electron microscopy (STEM) image analysis demonstrated an inverse relationship between peroxisomal size and contrast. Peroxisomes were more abundant, and often concentrated in a perinuclear configuration in cholestatic and cirrhotic livers. Decreased peroxisomal staining was common in cholestasis, cirrhosis, hepatitis, and in almost all patients with malignancies, both with and without hepatic metastases.
Cellular Synthesis of Macromolecular Glycogen and Its Deposition
Authors: Takeuchi Tadao
Published: April 2011

The largest macromolecules of glycogen synthesized histochemically in tissue cells were spherical branching bodies composed of several large branches with many smaller branches, presumably containing a Meyer structure of α-1,4-1,6-glucosidic linkages. The synthesized macromolecules of glycogen were variable in size and structure, and were closely related to the activities of enzymes, Phosphorylase, branching glycosyltransferase, and glycogen synthetase. The glycogen molecules appeared to be individually free in the cytoplasmic matrices. It is possible that they are synthesized there, and not related to the membrane system.
Immunohistochemistry of Gastric Carcinomas and Associated Diseases: Novel Distribution of Carcinoembryonic Antigen and Secretory Component on the Surface of Gastric Cancer Cells
Authors: Nagura Hiroshi , Tsutsumi Yutaka , Shioda Yoshinobu , Watanabe Keiichi
Published: April 2011

Carcinoembryonic antigen (CEA) and secretory component (SC) were localized by peroxidase-labeled antibody immunocytochemistry in normal and abnormal human gastric mucosa. In normal epithelium, both glycoproteins were absent or only faintly present, but in intestinal metaplasia and carcinoma both were prominently present. CEA and SC on the surfaces of metaplastic epithelial cells were polarized. That is, CEA was expressed only on the microvillous surface and SC was expressed only on the basolateral surface. In gastric cancer, CEA and SC were distributed over the entire surface of the neoplastic cells. Thus, deviations from the normal differentiation and maturation of gastric epithelial cells were accompanied by abnormalities in surface expression of CEA and SC. These observations, together with compatible observations previously made in colonic neoplasia (DJ Ahnen, PK Nakane, and WR Brown, Cancer 49:2077, 1982), suggest that loss of polarity of surface membrane components is a characteristic of neoplastic epithelial cells.
The Microcomputer-based Color Image Analyzer and Its Application to Histochemistry
Authors: Fujita Setsuya
Published: April 2011

For quantification of histochemical reactions, color image analysis, as well as cytophotometry, is important. However, the extremely large amount of information, usually more than one million bits per image, involved in one microscopic picture of a colored histochemical specimen, when digitalized, has inhibited attempts to develop a more convenient system of color image analysis. Recent advances in microcomputer technology have now made it possible to build, at a reasonable cost, an intelligent color terminal equipped with array-processing hardware, a sufficiently large video random-access memory to represent 4096 colors in each pixel, and packages of machine language subroutines called “macro-commands” in the form of read-only memory that can process color images within a few seconds. This intelligent terminal can easily be controlled by a small personal computer via an IEEE-488 interface bus. The digitalized color image can be stored in and retrieved from a floppy disk. As the macro-commands in this system are controlled by BASIC program through CALLS with parameters, it is highly flexible and adapts readily to the varied needs of histochemists. Reported on herein will be the instrumentation of the microcomputer-based color image analyzer and a few examples of applications of this system to histochemistry: color intensification of cytochemical reactions, quantification of enzyme (e.g., horseradish peroxidase) reaction products, periodicacid-Schiff staining, collagen fibers visualized by Azan-Mallory stain, etc. Possible future applications of this color image analyzer will also be discussed.
Ultrastructural immunolocalization of kallikrein in apical granules of striated duct cells of cat submandibular gland.
Authors: Schachter M , Wheeler G D , Matthews R W , Peret M W , Moriwaki C
Published: April 2011

Recent studies on the localization of the serine protease salivary kallikrein have led to the conclusion that it has a ductal localization and to the possibility that it is located there in small "secretory" granules. Until now, the latter inference has been based entirely on circumstantial evidence. In the present study, however, direct evidence from immunolocalization studies at the ultrastructural level establishes the localization of this enzyme in the apical duct granules in the cat submandibular gland. These granules stained only with immune sera to cat submandibular gland kallikrein and were the only subcellular structures that did so. They showed the "studded" appearance characteristic of the electron-dense aggregates of peroxidase-antiperoxidase seen in this type of immune reaction.
Immunogold staining method for the light microscopic detection of leukocyte cell surface antigens with monoclonal antibodies: its application to the enumeration of lymphocyte subpopulations.
Authors: De Waele M , De Mey J , Moeremans M , De Brabander M , Van Camp B
Published: April 2011

Colloidal gold was used as a marker for the light microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies. Suspensions of peripheral blood leukocytes were first incubated with monoclonal mouse antibodies and then with colloidal gold-labeled goat anti-mouse antibodies. The cells were fixed and cytocentrifuge preparations or smears were made. Granulocytes and monocytes were then labeled by the cytochemical staining of their endogenous peroxidase activity. Lymphocytes reacting with the monoclonal antibody had numerous dark granules around the surface membrane. With electron microscopy, these granules appeared as patches of gold particles. This immunogold staining method proved to be a reliable tool for the enumeration of T-lymphocyte subpopulations in peripheral blood. The results were almost identical to those obtained with immunofluorescence microscopy. The procedure can also be applied on small volumes of capillary blood. This constitutes a good microtechnique for the determination of lymphocyte subsets in children.
Intestinal vitamin D-induced calcium-binding protein: time-course of immunocytological localization following 1,25-dihydroxyvitamin D3.
Authors: Taylor A N
Published: April 2011

The vitamin D-induced calcium-binding protein (CaBP) was localized in histological sections of chick duodenum using the peroxidase-antiperoxidase immunocytochemical technique. The time-course of appearance of CaBP in rachitic chicks was investigated from 0 to 120 hr after stimulation by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). CaBP was not routinely detected at 0 hr after 1,25(OH)2D3 administration. CaBP was first noted in some, but not all, of the samples taken 2 hr following 1,25(OH)2D3 and was detected in all 2 1/2 hr samples. The number of CaBP-containing absorptive cells and the apparent CaBP concentration both increased to a maximum at about 16-24 hr. At later times, as CaBP free cells migrated up the villi, the CaBP-containing cells decreased in number, but even at 120 hr post 1,25(OH)2D3 dose there were significant numbers of CaBP-containing cells present. The relationships between time-course of CaBP location on intestinal villi, enterocyte migration rates, and the time-course of 1,25(OH)2D3 stimulated intestinal calcium transport are discussed.
A novel cytochemical marker for liposome decomposition in lysosomes.
Authors: Ho S C , Huang L
Published: April 2011

The endocytosis of large unilamellar liposomes composed of phosphatidylcholine by the cultured Chinese hamster V-79 cells is demonstrated with electron microscopy cytochemistry. A novel cytochemical marker, 5-Br,4-Cl,3-indolylphosphate (BCIP) is used. This marker is a soluble and colorless substrate for the lysosomal acid phosphatase and can be readily entrapped in liposomes. The product of the enzymatic reaction, 5-Br,4-Cl,3-hydroxy indole, rapidly self-condenses and becomes an insoluble derivative of indigo blue. In thin section transmission electron microscopy, the condensed product appears as electron-dense deposits in the lysosomes. Since the electron-dense deposit only appears when the endocytosed liposomes are delivered to the lysosomes as the result of phagosome-lysosome fusion, this marker provides a unique cytochemical means to reveal those liposomes that are lysosomotropic and are actually decomposed within the lysosomes. No electron-dense deposits are found in the liposome-treated cells in the presence of chloroquine, or a combination of NaN3 and deoxyglucose. As a comparison, we have also used horseradish peroxidase entrapped in liposomes to confirm the endocytic uptake of liposomes. Using a radioactive marker, 125I-labeled lysozyme, entrapped in liposomes, it is shown that about 20-30% of liposome uptake by V-79 cells is due to endocytosis.
Cytochemical profile of immunoregulatory T-lymphocyte subsets defined by monoclonal antibodies.
Authors: De Waele M , De Mey J , Moeremans M , Smet L , Broodtaerts L , Van Camp B
Published: April 2011

Immunogold staining in combination with enzyme cytochemistry was used to determine the cytochemical profile of the immunoregulatory T-lymphocyte subpopulations defined by the monoclonal antibodies OKT3, OKT4, OKT8, and OKM1 in normal peripheral blood. Leukocyte suspensions were first incubated with the monoclonal mouse antibodies and then with colloidal gold-labeled goat antimouse antibodies. The cells were fixed and cytocentrifuge preparations were made. Cytochemical reactions for the detection of peroxidase, acid alpha-naphthyl acetate esterase, acid phosphatase, and beta-glucuronidase were performed on these preparations. Under light microscopy, lymphocytes reacting with the monoclonal antibodies had numerous dark granules around their surface membrane. In the cytoplasm the intracellular enzymatic activities were stained. The T-lymphocytes were characterized by a dot-like activity for the three enzymes. No significant difference could be found between the cytochemical profile of the T-helper (OKT4 positive) and T-cytotoxic suppressor cell populations (OKT8 positive). A few cells with lymphocyte morphology reacted with the OKM1 monoclonal antibody. Their cytochemical characteristics were different from those of mature T-cells (OKT3 population) or mature monocytes. From the comparison of their cytochemical characteristics, we can conclude that there is little correlation between the immunoregulatory T-lymphocyte subsets defined by these monoclonal antibodies and those defined by Fc receptors.
Localization of keratin proteins in the human epidermis by a postembedding immunoperoxidase technique.
Authors: Warhol M J , Pinkus G S , Banks-Schlegel S P
Published: April 2011

Individual keratin species were localized ultrastructurally to the tonofilaments and tonofibrils of the human epider-misusing a postembedding immunoperoxidase method. Low molecular weight keratin (45 kD) was localized primarily to the stratum basale and displayed a fine cytoplasmic staining of individual tonofilaments and some tonofibrillar staining. Higher molecular weight keratins (55 and 63 kD) were found predominantly in the suprabasal, differentiated squamous epithelial cells, staining tonofibrils with a web-like pattern. The technique utilized is suitable for studying the distribution of keratin proteins in normal and disease states.
Electron microscopic localization of adenosine triphosphate (ATP)-hydrolyzing activity in isolated matrix vesicles and reconstituted vesicles from calf cartilage.
Authors: Kanabe S , Hsu H H , Cecil R N , Anderson H C
Published: April 2011

The presence and distribution of adenosine triphosphatase (ATPase) activity in isolated matrix vesicles and reconstituted vesicles from fetal calf epiphyseal growth plate cartilage was studied by electron microscopic cytochemical methods to determine whether phosphatase activity would be found concentrated on the inside or the outside of matrix vesicle membranes or on both sides, and whether reconstitution of vesicles from deoxycholate-solubilized substituents would lead to the reassembly of membranes with ATPase incorporated. ATPase activity was observed on both the outer and inner surfaces of the investing membranes of isolated matrix vesicles and reconstituted vesicles. A transmembrane location of ATPase could indicate phosphate transfer across the vesicle membrane. Orthophosphate released by phosphatase activity within the protected microenvironment of the matrix vesicle could combine with membrane- or lipid-bound calcium, known to be present in vesicles, to form the first hydroxyapatite mineral during calcification.
Corticotropin (ACTH) and the N-terminal fragment of pro-opiomelanocortin are located in the same granules in cells of rat pituitary gland.
Authors: Cantin M , Ballak M , Lu C L , Seidah N G , Chrétien M
Published: April 2011

The localization of corticotropin (ACTH) and of the N-terminal fragment (NTF) of pro-opiomelanocortin were assessed by light and electron microscope immunochemistry. Using the unlabeled technique of Sternberger at the light microscope level, a strongly positive reaction for both NTF and ACTH antisera was observed in all secretory cells of the pars intermedia. In the pars distalis, immunostaining with ACTH antiserum was localized in stellate cells dispersed throughout the lobe. As observed in serial, consecutive sections, the NTF antiserum stained exactly the same cells. At the electron microscope level, using the protein A-gold technique, all the secretory granules of the cells of the pars intermedia showed a positive reaction with both antisera. In the pars distalis, after exposure to either of the antisera, gold particles were found over secretory granules of typical stellate corticotrophs. Making use of the possibility of reacting both faces of a fine section with gold particles of different sizes, it was found that the same secretory granules in all the cells of the pars intermedia and in corticotrophs of the pars distalis contained particles of both sizes. These results indicate that ACTH and NTF are contained in the same secretory granules and released together in the circulation where NTF is found in high amounts.
Lectin binding sites in kidney. A comparative study of 14 animal species.
Authors: Holthöfer H
Published: April 2011

Six fluorochrome-coupled lectins with different sugar specificities were used to stain frozen tissue sections of kidneys from 14 animal species including mammals, avians, reptiles, and fresh water fish. Each lectin seemed to have a species-, but not strain-, specific binding pattern. Some lectins, however, bound to the same parts of the nephron in all animals studied. Wheat germ agglutinin (WGA) bound prominently to glomeruli in all kidneys. Dolichos biflorus agglutinin (DBA) seemed to bind to only a group of distal tubules in most animals, whereas either proximal or distal tubules were revealed with soybean (SBA) and peanut (PNA) agglutinins. Heterogeneity of basement membranes in different nephron parts was seen in the binding of some lectins. Ulex europeus agglutinin (UEA I), binding specifically to endothelial cells in human tissues, did not react with the endothelium of any other species, but SBA and PNA seemed to prominently stain vascular endothelia of cow and hen vessels, respectively. These results show a species-specific compartmentalization of saccharides to certain parts of the nephron, while there appears to be some common features in saccharide distribution between different animal species as well.
The development, using poly(Hg-U) in a model system, of a new method to visualize cytochemical hybridization in fluorescence microscopy.
Authors: Bauman J G , Wiegant J , van Duijn P
Published: April 2011

The development, using a model system, of a new method for the detection of cytochemical (in situ) hybrids is described. The method is based on the mercuration of nucleic acids with mercuric acetate. To facilitate hybridization, the acetate ligand is replaced by the CN- ion. In the hybrids formed, the CN- is exchanged for trinitrophenyl(TNP)-glutathione. The TNP-glutathione is subsequently detected by indirect immunofluorescence using anti-TNP antibodies. The feasibility of the approach was investigated using Sepharose- or Sephadex-bound poly(A) and mercurated poly(U). Poly(Hg-U) did hybridize with poly(A)-Sepharose, provided that the acetate ligand was replaced with CN-. The TNP-glutathione hapten thus synthesized bound effectively to mercury-Sepharose but not to amino-Sepharose when the reaction was performed in the dark. Furthermore, binding of TNP-glutathione to Sephadex-bound poly(A) . poly(HG-U) hybrids was detectable with indirect immunofluorescence using anti-TNP antibodies. The fluorescence intensity measured was dependent on the amount of poly(Hg-U) present and on the dilution of the antibody. Nonspecific binding was very low. Calibration of the number of fluorescein molecules found after the complete reaction was performed with fluorescein isothiocyanate-labeled poly(U). It was determined that one fluorochrome molecule per two nucleotides had been obtained, in close agreement with the theoretically expected number. The sensitivity of the method, when applicable to microscopic preparations, is comparable to in situ hybridization with 3H-labeled nucleic acids with a specific activity of 4 x 10(8) dpm/micrograms (two 3H-isotopes per nucleotide) and an exposure time of 1 day. Extension of the method to the cRNA-DNA system and its application to microscopic preparations is under investigation.
Ultrastructural architecture of proteoglycans in the glomerular basement membrane. A cytochemical approach.
Authors: Reale E , Luciano L , Kühn K W
Published: April 2011

Rat kidneys were perfused with fixative solutions containing either a) a polycationic dye (Alcian blue 8 GX, Astra blue 6 GLL, cuprolinic blue, ruthenium red), b) a monocationic dye (safranine 0), or c) Alcian blue in the presence of a 0.3 M MgCl2 concentration. Whereas solutions of a revealed the glomerular basement membrane proteoglycans as particles or threads 60 nm apart and arranged in a reticular pattern, solutions of b and c demonstrated new morphological aspects of these molecules. They appeared as tiny filamentous structures, about 100 to 160 nm long, ordered in a network-like pattern with a mesh of about 60-nm width. The filaments displayed lateral branches about 20 nm apart and about 25 nm long, projecting within the meshes. We suggest that the filamentous structures are the protein core, and the branches are the glycosaminoglycans of proteoglycan molecules. Because of this arrangement the negatively charged sites of the glomerular basement membrane would lie closer to each other than previously assumed.
Localization of alkaline phosphatase and adenosine triphosphatase in the mammalian pancreas.
Authors: Githens S
Published: April 2011

The light microscopic histochemical localization of alkaline phosphatase (APase) and adenosine triphosphatase (ATPase) in the mammalian pancreas is reviewed. Capillary endothelial cells usually exhibit both enzymes. ATPase is usually present in endocrine and acinar cells and absent in duct cells. APase is often found in islet cells but is almost always absent in exocrine cells. These enzymes should therefore be used with caution as markers for specific cells and organelles of the pancreas, and for monitoring diseases that might lead to the release of these enzymes from the pancreas.
A comparison of direct and indirect techniques using ferritin-conjugated ligands for the localization of concanavalin A binding sites on isolated hepatocyte plasma membranes.
Authors: Briggman J V , Widnell C C
Published: April 2011

Concanavalin A binding sites have been localized on isolated plasma membranes both by a direct technique involving ferritin-concanavalin A and by an indirect technique in which membranes were treated successively with concanavalin A, rabbit anti-concanavalin A, and ferritin-conjugated sheep anti-rabbit F(ab')2. Binding studies showed that, at saturation, less than 25% of the concanavalin A binding sites were accessible to ferritin-concanavalin A. The decreased binding was apparently related to steric factors, since membranes saturated with the conjugated ligand were able to bind additional concanavalin A, and since the conjugated ligand, once bound to the membrane, caused the same inhibition of the membrane-bound enzyme 5'-nucleotidase as concanavalin A. Nonspecific binding sites accounted for 10% of the total binding of ferritin-concanavalin A and were localized mainly on the cytoplasmic side of the membrane, whereas specific sites were on the external side. The indirect technique, which was expected to increase the binding of ferritin-conjugate to the membrane, resulted in the binding of ferritin to less than 15% of the concanavalin A binding sites, and did not decrease the nonspecific binding.
Membrane-integrated proteins at preformed exocytosis sites.
Authors: Vilmart J , Plattner H
Published: April 2011

Parameters tetraurelia cells were used for analyzing the crucial but also controversial question regarding the organization of those sites of the cell membrane that undergo fusion during exocytosis. Paramecia are unique in that they display a "rosette" of from eight to ten MIPs (Membrane-intercalated particles, as seen on freeze-fracture replicas) at each of their numerous preformed exocytosis sites. We analyzed these structures by exposing slightly fixed cells to various enzymes and by subsequent freeze fracturing. We show that "rosette" MIPs are selectively sensitive to proteolytic enzymes. Since "rosettes" are known to be necessary for membrane fusion, this cytochemical result would be in line with the assumption that membrane-integrated proteins may play an active role in regulating exocytotic membrane fusion.
Gastric inhibitory peptide-like immunoreactivity in glucagon and glicentin cells: properties and origin. An immunocytochemical study using several antisera.
Authors: Sjölund K , Ekelund M , Håkanson R , Moody A J , Sundler F
Published: April 2011

Although gastric inhibitory peptide (GIP) has never been detected outside the upper small intestine by immunochemical methods, GIP-like immunoreactivity has been demonstrated by immunocytochemistry in the glucagon/glicentin cells of pancreas, and gut. In the present study several GIP antisera (five polyclonal and one monoclonal) were tested on specimens from pancreas and intestines of several mammalian species, including man. Two of the polyclonal antisera and the monoclonal one stained cells in the upper small intestine only, while the other three also stained cells in the pancreas, ileum, and colon. Monoclonal anti-GIP did not stain GIP cells in man. The immunostaining produced could not be abolished by pretreatment of the antisera with glucagon or glicentin in excess, whereas small amounts of synthetic or natural porcine GIP prevented the immunostaining. Thus, three of the antisera are specific for GIP, while the other three recognize not only GIP but also GIP-like peptides. The results suggest that the glucagon/glicentin cells contain peptides distinct from GIP but sharing an immunodeterminant with GIP. The GIP-like immunoreactivity in the glucagon cells of the rat pancreas was not altered by infusion of GIP or by elimination of the bulk of endogenous GIP by resection of the upper small intestine, indicating that the GIP-like peptide is produced in the glucagon cells rather than accumulated from the circulation. The nature of this GIP-like peptide is unknown. Conceivably, it represents the cryptic portion of the glucagon precursor molecule. In some species a proportion of the GIP cells in the proximal small intestine displayed glicentin-like immunoreactivity as well, emphasizing the relationship between GIP cells on the one hand and glucagon/glicentin cells on the other.
Cytochemical localization of glucose-6-phosphatase activity in cerebral endothelial cells.
Authors: Broadwell R D , Cataldo A M , Salcman M
Published: April 2011

Glucose-6-phosphatase (G6Pase) activity, with glucose-6-phosphate and mannose-6-phosphate as substrates, was examined by cytochemistry in capillary and arteriole endothelial cells of the mouse brain. G6Pase activity was observed ultrastructurally in the lumen of the nuclear envelope and endoplasmic reticulum (ER) of these cells. The reactive ER and nuclear membrane appeared to be in continuity. Nucleoside diphosphatase activity, also a marker for the ER in some cell types, was not seen within the ER of the cerebral microvasculature. The ER of arterioles and capillaries did not bind lead nonspecifically when incubated in a substrate-free medium. Speculation is raised concerning the involvement of G6Pase in glucose metabolism of cerebral endothelial cells and in making blood-borne glucose available to brain parenchyma.
Use of the avidin-biotin-peroxidase complex (ABC) method for the localization of rabbit cathepsin B in cells and tissues.
Authors: Graf F M , Sträuli P
Published: April 2011

Visualization of rabbit cathepsin B was achieved utilizing monospecific sheep antibodies and the avidin-biotin-peroxidase complex (ABC) method. This technique was applied to stain 1) paraffin sections of the liver, 2) fixed fibroblasts from tissue culture, and 3) fixed mesenteries. Cathepsin B was found to be localized within cells of the lining of the liver sinusoids (most probably Kupffer cells), in perinuclear granules of cultured fibroblasts, and within histiocytes of the mesentery. The results demonstrate that the method permits precise and highly sensitive localization of cathepsin B within cells and tissues. Compared to fluorescent staining of cathepsin B, the ABC method has the advantage that routine paraffin sections can be stained, and that all the orthodox histological staining procedures can still be carried out.
Retardation of fading and enhancement of intensity of immunofluorescence by p-phenylenediamine.
Authors: Platt J L , Michael A F
Published: April 2011

The effects of p-phenylenediamine (PD) on the extinction and intensity of fluorescence and on antigen binding in immunofluorescence microscopy was investigated. Visual and photometric comparison of tissue sections reacted sequentially with polyclonal or monoclonal antisera and appropriate fluorochromes and mounted with and without PD indicated that PD both prolonged and intensified fluorescence without evident effect on antibody binding.
Factors affecting the immunoperoxidase demonstration of intracellular immunoglobulins and J chain from cytocentrifuged cell smears.
Authors: Reitamo S , Käyhkö K , Konttinen Y T , Bergroth V
Published: April 2011

Immunohistochemical methods were used to study 1) the optimum fixation conditions for the preservation of human J chain and immunoglobulin (Ig) immunoreactivity and 2) the relation of J chain synthesis by plasmablasts and plasma cells to Ig synthesis in cell smears of cultured human peripheral blood lymphocytes stimulated with pokeweed mitogen (PWM). J chain was demonstrated using the indirect immunoperoxidase method, and intracellular Ig was demonstrated with the unlabeled antibody--enzyme method. In the sequential double staining procedure, J chain was demonstrated using the indirect immunoperoxidase method followed by the demonstration of Ig with the direct immunofluorescence method. Optimum preservation of J chain immunoreactivity was obtained with fixation in neutral buffered formalin at 22 degrees C for 5 min followed by immediate immunoperoxidase staining. False negative results were seen when the slides were stained 2 weeks after fixation. In PWM-stimulated smears, J chain appeared on day three, simultaneously with or after the onset of Ig synthesis. In double stained smears most IgG-positive cells also showed immunoreactivity for J chain from the third day on.
Laminin in cultured mouse C1300 neuroblastoma cells: immunocytochemical localization by pre- and postembedding electron microscope procedures.
Authors: Liesi P
Published: April 2011

Laminin was localized in cultured mouse C1300 neuroblastoma cells by applying the peroxidase-antiperoxidase technique in preembedding electron microscopy. The results were compared to those obtained by indirect immunofluorescence and by the colloidal gold second antibody method on Epon-embedded ultrathin sections. Laminin was found in the cell membranes and within the rough endoplasmic reticulum as well as in intracytoplasmic vacuoles. Plasma membranes of the neuroblastoma cells showed a patchy localization of laminin that was apparently involved in cell-to-substrate attachment and in gap junction-like intercellular connections. Under normal conditions, the Golgi cisternae contained no laminin. Pretreatment of cells with micromolar concentrations of monensin, however, lead to an accumulation of laminin within the Golgi cisternae. These results support a role for laminin as an adhesion protein in cultured neuroblastoma cells and indicate that laminin is transported through the Golgi complex.
Pericellular matrix of growth plate chondrocytes: a study using postfixation with osmium-ferrocyanide.
Authors: Farnum C E , Wilsman N J
Published: April 2011

The pericellular matrix surrounding chondrocytes from all zones of epiphyseal growth plate cartilage, as well as from articular, tracheal, and auricular cartilage, was examined using a number of variations of osmium ferrocyanide postfixation of aldehyde-fixed tissues. Comparisons were made with other fixative techniques, including ruthenium red, safranin O, and lanthanum nitrate, all of which have previously been reported to stabilize a variety of lacunar matrix components. An electron-dense material was preserved uniquely by osmium-ferrocyanide in the lacunar matrix of mid and late zone hypertrophying chondrocytes and was absent from all other zones of the growth plate as well as from the other types of cartilage examined. Because of its highly restricted distribution, this electron-dense material is hypothesized to represent a pericellular matrix component involved with either matrix calcification or metaphyseal capillary penetration. Several hypotheses are presented as to its specific composition.
Dye interactions. A basis for specific detection and histochemistry of polysaccharides.
Authors: Wood P J , Fulcher R G
Published: April 2011

Specific histochemical staining can be related to specific interactions of dyes and polysaccharides in solution. Specificity evidently depends upon polysaccharide conformation and the need for a close "fit" between polysaccharide and ligand. As a consequence, microscopic identification of cereal beta-(1 leads to 4)(1 leads to 3)-D-glucan and beta-(1 leads to 3)-D-glucan is possible, and is demonstrated.
Immunogold-silver staining: new method of immunostaining with enhanced sensitivity.
Authors: Holgate C S , Jackson P , Cowen P N , Bird C C
Published: April 2011

A new method for demonstrating antigens in paraffin sections of formol sublimate-fixed tissue is described that utilizes an "indirect" immunohistological technique employing immunoglobulin adsorbed to colloidal gold as the secondary antiserum. The gold particles introduced to antigenic sites are revealed by a silver precipitation reaction. This technique, the immunogold-silver staining method, is of much enhanced sensitivity (up to 200-fold) as compared with standard immunoperoxidase and immunogold staining methods. The results have been confirmed in a study of immunoglobulins in reactive human tonsil. The use of this new method for double immunolabeling is also described.