scholarly journals Immunocytochemical localization of Na+,K+-ATPase catalytic polypeptide in mouse choroid plexus.

1986 ◽  
Vol 34 (2) ◽  
pp. 189-195 ◽  
Author(s):  
S A Ernst ◽  
J R Palacios ◽  
G J Siegel
Keyword(s):  
Choroid Plexus ◽  
Epithelial Transport ◽  
Protein A ◽  
Fluid Secretion ◽  
Physiological Data ◽  
Electron Microscopic ◽  
Antibody Technique ◽  
Immunogold Staining ◽  
Thin Sections ◽  
Membrane Surfaces

Na+,K+-ATPase plays a central role in the mechanism of cerebrospinal fluid secretion by the choroid plexus. We have used an antiserum to the 100 KD catalytic polypeptide of the enzyme purified from mouse brain (30) to localize the catalytic unit in mouse choroid plexus at the light and electron microscopic levels. Pre-embedding immunostaining with the peroxidase-conjugated second antibody technique showed that microvillar borders facing the ventricle were intensely reactive. In contrast, basal and lateral plasma membrane surfaces were devoid of activity. Identical localization was obtained with a post-embedding procedure in which protein A-gold was used to stain immunoreactive sites on thin sections of Lowicryl-embedded tissue. For comparison, immunogold staining was shown to be restricted to basolateral membranes of kidney medullary ascending thick limbs. The apical localization of Na+,K+-ATPase in choroid plexus is in striking contrast to the almost exclusive basolateral localization seen in other ion-transporting tissues. The immunocytochemical data are completely consistent with physiological data on choroidal epithelial transport and with light microscopic autoradiographic localization of [3H]-ouabain binding sites.

scholarly journals Immunocytochemical localization of D-amino acid oxidase in the central clear matrix of rat kidney peroxisomes.

1986 ◽  
Vol 34 (12) ◽  
pp. 1709-1718 ◽  
Author(s):  
N Usuda ◽  
S Yokota ◽  
T Hashimoto ◽  
T Nagata
Keyword(s):  
Amino Acid ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Protein A ◽  
Immunoblot Analysis ◽  
Acid Oxidase ◽  
Electron Microscopic ◽  
Amino Acid Oxidase ◽  
Thin Sections ◽  
Gold Particles

Light and electron microscopic localizations of D-amino acid oxidase (DAO) in rat kidney was investigated using immunoenzyme and protein A-gold techniques. The enzyme was purified from rat kidney homogenate and its antibody was raised in rabbits. By Ouchterlony double-diffusion analysis and immunoblot analysis with anti-(rat kidney DAO) immunoglobulin, the antibody was confirmed to be monospecific. The tissue sections (200 micron thick) of fixed rat kidney were embedded in Epon or Lowicryl K4M. Semi-thin sections were stained for DAO by the immunoenzyme technique after removal of epoxy resin for LM, and ultra-thin sections of Lowicryl-embedded material were labeled for DAO by the protein A-gold technique for EM. By LM, fine cytoplasmic granules of proximal tubule were stained exclusively. Among three segments of proximal tubules, and S2 and S3 segments were heavily stained but the S1 segment only weakly so. By EM, gold particles indicating the antigenic sites for DAO were exclusively confined to peroxisomes. Within peroxisomes, the gold particles were localized in the central clear matrix but not in the peripheral tubular substructures. The results indicate that D-amino acid oxidase in rat kidney is present exclusively in peroxisomes in the proximal tubule and that within peroxisomes it is found only in central clear matrix and not in the peripheral tubular substructures.

scholarly journals Electron microscopic localization of chromogranin A in osmium-fixed neuroendocrine cells with a protein A-gold technique.

1987 ◽  
Vol 35 (7) ◽  
pp. 795-801 ◽  
Author(s):  
S A Hearn
Keyword(s):  
Neuroendocrine Tumors ◽  
Chromogranin A ◽  
Osmium Tetroxide ◽  
Secretory Granules ◽  
Protein A ◽  
Neuroendocrine Cells ◽  
Neurosecretory Granules ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Carotid Body Tumors

An antibody (LK2H10) to chromogranin A has been recommended for use in ultrastructural identification of neuroendocrine secretory granules. Previous studies have demonstrated immunoreactive chromogranin A in specimens prepared for electron microscopy by glutaraldehyde fixation only. In this study, the effect of specimen post-fixation by osmium tetroxide on post-embedding localization of chromogranin A was evaluated. Human tissues from benign endocrine glands, neuroendocrine tumors, and non-neuroendocrine tumors were post-fixed in osmium, embedded in epoxy resin, and the sample thin sections immunolabeled using a protein A-gold technique. Chromogranin A-positive neurosecretory granules were detected in pancreatic islets, adrenal medulla, stomach, ileum, anterior pituitary, and parathyroid. Mid-gut carcinoids, bronchial carcinoids, pheochromocytomas, paragangliomas, carotid body tumors, and thyroid medullary carcinomas contained immunoreactive granules. Cytoplasmic granules in non-neuroendocrine tumors did not react for chromogranin A. Tissues post-fixed in osmium tetroxide had optimally preserved ultrastructural features, and use of this fixative is compatible with postembedding localization of chromogranin A in neurosecretory granules.

Immuno-electron microscopic identification of Methanosarcina spp. in anaerobic digester fluid

1985 ◽  
Vol 31 (9) ◽  
pp. 839-844 ◽  
Author(s):  
Ralph W. Robinson ◽  
Gregory W. Erdos
Keyword(s):  
Protein A ◽  
Methanobacterium Thermoautotrophicum ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Methanosarcina Mazei ◽  
Pure Cultures ◽  
Electron Microscopic Identification ◽  
Mixed Samples ◽  
Heavy Labelling

Whole cell antibodies against Methanosarcina mazei strain S6 were used with protein A – colloidal gold to identify bacteria in thin sections of samples from anaerobic methane producing digesters. It was possible to identify bacteria at the genus level and to show relatedness at the species and strain levels. Heavy labelling was observed on thin sections of the immunogenic strain S6. Lighter labelling was observed with pure cultures of M. mazei strain LYC. Pure cultures of Methanococcus voltae or Methanobacterium thermoautotrophicum did not label. In samples from mesophilic sewage digesters, sarcinal colonies of bacteria similar to Methanosarcina showed heavy labelling per cell while colonies from 55 °C digesters show lighter labelling. A few free cocci, which were released from the sarcinal colonies, also labelled. Labelling was not observed on other bacterial forms in either set of digesters. These studies indicate that a collection of bacterial antibodies can be used to identify and map bacteria in situ in mixed samples.

scholarly journals Applications of immunocolloids in light microscopy. Preparation of protein A-silver and protein A-gold complexes and their application for localization of single and multiple antigens in paraffin sections.

1982 ◽  
Vol 30 (7) ◽  
pp. 691-696 ◽  
Author(s):  
J Roth
Keyword(s):  
Protein A ◽  
Second Step ◽  
Staphylococcal Protein A ◽  
Paraffin Sections ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Antigenic Sites ◽  
Antigen Localization ◽  
Color Mixing ◽  
Electron Microscopic Localization

The protein A-gold (pAg) complex, a useful reagent for electron microscopic localization of antigens in thin sections, is tested for its suitability as second step reagent in light microscopic immunohistochemistry. In addition, the preparation of colloidal silver, its complex formation with staphylococcal protein A and the application of the protein A-silver complex for antigen localization in paraffin sections is reported. The antigens were visualized in a two-step technique with specific antisera in the first incubation step and pAg or pA-silver as a general second step reagent. The pAg complex gives a red coloration of antigenic sites, whereas the pA-silver stained yellow. The contrasting color provided by the two immunocolloids allowed localization of two antigens in the same section. No color mixing occurred, showing that removal of the antibodies of the first staining sequence is unnecessary. Staining is virtually permanent with the light microscopic immunocolloid method. It is concluded that pAg and pA-silver complexes are useful as general second step reagents for the localization of a variety of antigens in paraffin sections.

scholarly journals Immunocytochemical localization of cathepsin B in rat kidney. II. Electron microscopic study using the protein A-gold technique.

1986 ◽  
Vol 34 (7) ◽  
pp. 899-907 ◽  
Author(s):  
S Yokota ◽  
H Tsuji ◽  
K Kato
Keyword(s):  
Cathepsin B ◽  
Rat Kidney ◽  
Protein A ◽  
Proximal Tubules ◽  
Gold Complex ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Gold Particles ◽  
Apical Vesicles ◽  
Collecting Tubules

Thin sections of Lowicryl K4M-embedded materials were labeled with protein A-gold complex. Gold particles representing the antigen sites for cathepsin B were exclusively confined to lysosomes of each segment of the nephron. The heaviest labeling was noted in the lysosomes of the S1 segment of the proximal tubules. Labeling intensity varied considerably with the individual lysosomes. Lysosomes of the other tubular segments, such as the S2 and S3 segments of the proximal tubules, distal convoluted tubules, and collecting tubules were weakly labeled by gold particles. Quantitative analysis of labeling density also confirmed that lysosomes in the S1 segment have the highest labeling density and that approximately 65% of labeling in the whole renal segments, except for the glomerulus, was found in the S1 segment. These results indicate that in rat kidney the lysosomes of the S1 segment are a main location of cathepsin B. Further precise observations on lysosomes of the S1 segment revealed that apical vesicles, tubules, and vacuoles were devoid of gold particles, but when the vacuoles contained fine fibrillar materials, gold labeling was detectable in such vacuoles. As the lysosomal matrix becomes denser, the labeling density is increased. Some small vesicles around the Golgi complex were also labeled. These results indicate that the endocytotic apparatus including the apical vesicles, tubules, and vacuoles contains no cathepsin B. When the vacuoles develop into phagosomes, they acquire this enzyme to digest the absorbed proteins.

scholarly journals Cerebrospinal Fluid Secretion by the Choroid Plexus

2013 ◽  
Vol 93 (4) ◽  
pp. 1847-1892 ◽  
Author(s):  
Helle H. Damkier ◽  
Peter D. Brown ◽  
Jeppe Praetorius
Keyword(s):  
Cerebrospinal Fluid ◽  
Choroid Plexus ◽  
Epithelial Transport ◽  
Cell Monolayer ◽  
Fluid Secretion ◽  
Transport Proteins ◽  
High Rate ◽  
Electrolyte Balance ◽  
Cuboidal Cell ◽  
Cerebrospinal Fluid Secretion

The choroid plexus epithelium is a cuboidal cell monolayer, which produces the majority of the cerebrospinal fluid. The concerted action of a variety of integral membrane proteins mediates the transepithelial movement of solutes and water across the epithelium. Secretion by the choroid plexus is characterized by an extremely high rate and by the unusual cellular polarization of well-known epithelial transport proteins. This review focuses on the specific ion and water transport by the choroid plexus cells, and then attempts to integrate the action of specific transport proteins to formulate a model of cerebrospinal fluid secretion. Significant emphasis is placed on the concept of isotonic fluid transport across epithelia, as there is still surprisingly little consensus on the basic biophysics of this phenomenon. The role of the choroid plexus in the regulation of fluid and electrolyte balance in the central nervous system is discussed, and choroid plexus dysfunctions are described in a very diverse set of clinical conditions such as aging, Alzheimer's disease, brain edema, neoplasms, and hydrocephalus. Although the choroid plexus may only have an indirect influence on the pathogenesis of these conditions, the ability to modify epithelial function may be an important component of future therapies.

The Immuno-Electron Microscopic Localization of the Mo-Fe Protein Component of Nitrogenase in Cells of Azotobacter Vinelandii

1973 ◽  
Vol 31 ◽  
pp. 574-575
Author(s):  
J. T. Stasny ◽  
R. C. Burns ◽  
R. W. F. Hardy
Keyword(s):  
Cellular Localization ◽  
Direct Evidence ◽  
Azotobacter Vinelandii ◽  
Intracellular Localization ◽  
Protein Component ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Fe Protein ◽  
Intracytoplasmic Membranes

Structure-functlon studies of biological N2-fixation have correlated the presence of the enzyme nitrogenase with increased numbers of intracytoplasmic membranes in Azotobacter. However no direct evidence has been provided for the internal cellular localization of any nitrogenase. Recent advances concerned with the crystallizatiorTand the electron microscopic characterization of the Mo-Fe protein component of Azotobacter nitrogenase, prompted the use of this purified protein to obtain antibodies (Ab) to be conjugated to electron dense markers for the intracellular localization of the protein by electron microscopy. The present study describes the use of ferritin conjugated to goat antitMo-Fe protein immunoglobulin (IgG) and the observations following its topical application to thin sections of N2-grown Azotobacter.

The High Resolution Appearance of Biological Substrates

1969 ◽  
Vol 27 ◽  
pp. 416-417
Author(s):  
Glen B. Haydon
Keyword(s):  
High Resolution ◽  
Phase Image ◽  
Biological Structure ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Resolution Electron ◽  
Discrete Structures ◽  
Large Phase ◽  
Biological Substrates ◽  
High Resolution Electron Micrographs

High resolution electron microscopic study of negatively stained macromolecules and thin sections of tissue embedded in a variety of media are difficult to interpret because of the superimposed phase image granularity. Although all of the information concerning the biological structure of interest may be present in a defocused electron micrograph, the high contrast of large phase image granules produced by the substrate makes it impossible to distinguish the phase ‘points’ from discrete structures of the same dimensions. Theory predicts the findings; however, it does not allow an appreciation of the actual appearance of the image under various conditions. Therefore, though perhaps trivial, training of the cheapest computer produced by mass labor has been undertaken in order to learn to appreciate the factors which affect the appearance of the background in high resolution electron micrographs.

Determination of the phase structure of polymerblends by electron microscopy

1978 ◽  
Vol 36 (1) ◽  
pp. 492-493
Author(s):  
Dr. G. Kaemof
Keyword(s):  
Screw Extruder ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Single Screw Extruder ◽  
Transmission Electron ◽  
The Matrix ◽  
Twin Screw ◽  
Special Case ◽  
Microscopic Investigations

A mixture of polycarbonate (PC) and styrene-acrylonitrile-copolymer (SAN) represents a very good example for the efficiency of electron microscopic investigations concerning the determination of optimum production procedures for high grade product properties.The following parameters have been varied:components of charge (PC : SAN 50 : 50, 60 : 40, 70 : 30), kind of compounding machine (single screw extruder, twin screw extruder, discontinuous kneader), mass-temperature (lowest and highest possible temperature).The transmission electron microscopic investigations (TEM) were carried out on ultra thin sections, the PC-phase of which was selectively etched by triethylamine.The phase transition (matrix to disperse phase) does not occur - as might be expected - at a PC to SAN ratio of 50 : 50, but at a ratio of 65 : 35. Our results show that the matrix is preferably formed by the components with the lower melting viscosity (in this special case SAN), even at concentrations of less than 50 %.

An Electron Microscopic Study of the Acute Effects of Hypothalamic Releasing Factors on the Anterior Pituitary Gland

1968 ◽  
Vol 26 ◽  
pp. 232-233
Author(s):  
P.W. Coates ◽  
E.A. Ashby ◽  
L. Krulich ◽  
A. Dhariwal ◽  
S. McCann
Keyword(s):  
Anterior Pituitary ◽  
Microscopic Investigation ◽  
Uranyl Acetate ◽  
Electron Microscopic Investigation ◽  
Electron Microscopic ◽  
Tissue Samples ◽  
Thin Sections ◽  
Dense Membrane ◽  
Membrane Bound ◽  
Releasing Factors

The morphologic effects on somatotrophs of crude sheep hypothalamic extract prepared from stalk-median eminence were studied by electron microscopy in conjunction with concurrently run bioassays performed on the same tissue samples taken from young adult male Sherman rats.Groups were divided into uninjected controls and injected experimentals sacrificed at 5', 15', and 30' after injection. Half of each anterior pituitary was prepared for electron microscopic investigation, the other half for bioassay. Fixation using collidine buffered osmium tetroxide was followed by dehydration and embedment in Maraglas. Uranyl acetate and lead citrate were used as stains. Thin sections were examined in a Philips EM 200.Somatotrophs from uninjected controls appeared as described in the literature (Fig. 1). In addition to other components, these cells contained moderate numbers of spherical, electron-dense, membrane-bound granules approximately 350 millicrons in diameter.

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