Ultrastructure of multiple microtubule initiation sites in mouse neuroblastoma cells

1981 ◽  
Vol 47 (1) ◽  
pp. 1-24
Author(s):  
G.A. Sharp ◽  
M. Osborn ◽  
K. Weber
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Immunofluorescence Microscopy ◽  
Neuroblastoma Cells ◽  
Biological Significance ◽  
Optimal Conditions ◽  
Serial Sectioning ◽  
Mouse Neuroblastoma ◽  
Microtubule Organizing Centres ◽  
Perinuclear Area

Morphologically undifferentiated and differentiated mouse neuroblastoma N115 and N18 cells were examined after serial sectioning by electron microscopy. A sizeable percentage of the cells revealed multiple centrioles, usually clustered together in the perinuclear area with 2 preferential locations, i.e. above and below the largest nuclear diameter. These results indicate that the multiple microtubule-organizing centres previously visualized by immunofluorescence microscopy with tubulin antibody in neuroblastoma cells recovering from Colcemid poisoning are most likely in majority related to multiple centrioles. This interpretation is further strengthened by experiments in which cells are first recorded in the fluorescence microscope and then after serial sectioning in the electron microscope. The results show that under optimal conditions immunofluorescence microscopy is able to visualize single centrioles. The possible biological significance of the combined electron and immunofluorescence microscopical results is discussed.

scholarly journals Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell

1978 ◽  
Vol 77 (3) ◽  
pp. R27 ◽  
Author(s):  
M Osborn ◽  
RE Webster ◽  
K Weber
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Light Microscope ◽  
Immunofluorescence Microscopy ◽  
Uranyl Acetate ◽  
Tubulin Antibodies ◽  
Ptk2 Cell ◽  
Cytoplasmic Microtubules ◽  
Triton X ◽  
Microtubular Structures

PtK2 cells were grown on gold grids and treated with Triton X-100 in a microtubule stabilizing buffer. The resulting cytoskeletons were fixed with glutaraldehyde and subjected to the indirect immunofluorescence procedure using monospecific tubulin antibodies. Grids were examined first by fluorescence microscopy, and the display of fluorescent cytoplasmic microtubules was recorded. The grids were then stained with uranyl acetate and the display of fibrous structures recorded by electron microscopy. Thus the display of cytoplasmic microtubular structures in the light microscope and the electron microscope can be compared within the same cytoskeleton. The results show a direct correspondence of the fluorescent fibers in the light microscope with uninterrupted fibers of diameter approximately 550 A in the electron microscope. This is the diameter reported for a single microtubule decorated around its circumference by two layers of antibody molecules. Thus under optimal conditions immunofluorescence microscopy can visualize individual microtubules.

scholarly journals Laminin in cultured mouse C1300 neuroblastoma cells: immunocytochemical localization by pre- and postembedding electron microscope procedures.

1983 ◽  
Vol 31 (6) ◽  
pp. 755-764 ◽  
Author(s):  
P Liesi
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Plasma Membranes ◽  
Neuroblastoma Cells ◽  
Immunocytochemical Localization ◽  
Adhesion Protein ◽  
Antibody Method ◽  
Ultrathin Sections ◽  
Intercellular Connections

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.

Development of the quadripolar meiotic apparatus in Funaria spore mother cells: analysis by means of anti-microtubule drug treatments

1989 ◽  
Vol 93 (2) ◽  
pp. 267-277
Author(s):  
C. H. BUSBY ◽  
B. E. GUNNING
Keyword(s):  
Electron Microscopy ◽  
Lipid Droplets ◽  
Immunofluorescence Microscopy ◽  
Video Recording ◽  
Live Cells ◽  
Spindle Poles ◽  
Microtubule Organizing Centres ◽  
Drug Treatments ◽  
Mother Cells ◽  
Dependent Processes

Microtubule-dependent processes in Funaria hygrometrica spore mother cells (SMCs) were analysed by monitoring the effects of colchicine and oryzalin on pre-meiotic and meiotic events. The techniques used were electron microscopy, immunofluorescence microscopy of microtubules (MTs)and continuous video recording of events in treated and recovering live cells sampled at various stages of sporogenesis. Inferences drawn from previous work that the SMC plastids serve as MT-organizing centres were confirmed in so far as MT recovery in MT-depleted cells starts at the tips of the plastids. The MTs that emanate from these regions are required for positioning the plastids in the tetrahedral conformation, which defines the meiotic poles, for positioning lipid droplets in clusters at these poles and for positioning and holding the nucleus in the tetrahedral cage. If released, the nucleus can be moved by a non-MT system. Other phenomena not controlled by MTs are plastid elongation, maintenance of the tetrahedral conformation-when the MTs are absent (during divisions or as a result of drug treatment) and (probably) development of the organelle band that spans the cell between divisions I and II. In cells treated during division, when there is nonuclear envelope, the pattern of MT recovery is different: the plastids are inactive as microtubule-organizing centres (MTOCs) but MTs reappear among the chromosomes. Spindles capable of transporting chromosomes regenerate. However, the importance of interactions between nucleus and plastids is highlighted by cases in which treatmenthas resulted in: (1) movement of the nucleus out of the quadripolar plastid cage; and (2) loss of the MTs at plastid tips that normally contribute to the spindle poles; in such cases quadripolarity is lost even though functional spindles return. Plastid MTOC activity returns when the nuclear envelope isin place, i.e. in interkinesis and after telophase II.

Fluoronanogold as a Probe for High Resolution Correlation Between ImmunoFluorescence and Electron Microscopy

1999 ◽  
Vol 5 (S2) ◽  
pp. 476-477
Author(s):  
T. Takizawa ◽  
J. M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
High Resolution ◽  
Immunofluorescence Microscopy ◽  
Human Neutrophils ◽  
Secondary Antibody ◽  
Marker Protein ◽  
Fab Fragment ◽  
Ultrathin Cryosections

[Introduction] Immunocytochemical labeling of cryosections, especially immunofluorescence microscopy using semi-thin (0.5-μm) cryosections, has been a powerful technique for detection of cellular antigens in situ and has been widely employed in cell and molecular biology studies. In many cases, immunofluorescence provides sufficient resolution and sensitivity to answer the question being addressed. However, in certain cases the increased resolution of the electron microscope using ultrathin (90-nm) cryosections may be required to define more precisely the localization of specific molecules. Recently, a unique fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), has been developed for use as a secondary antibody in immunocytochemical applications. It consists of a Fab' fragment of an antibody to which a 1.4-nm gold particle and fluorochromes are conjugated. FNG permits correlative microscopic observation of a sample stained in a single labeling procedure by multiple optical imaging. Recently, we have shown FNG immunocytochemistry on ultrathin cryosections to be valuable for high-resolution correlation of immunofluorescence and immunoelectron microscopy. In the present study, we have examined the utility of FNG as a secondary antibody for immunolabeling of myeloperoxidase (a marker protein for the azurophillic granules) in ultrathin cryosectioned human neutrophils.[Materials and Methods] Purified human neutrophils were fixed with paraformaldehyde, embedded in gelatin, infiltrated with sucrose, cut as ultrathin cryosections, and then collected on formvar film-coated nickel EM grids as described previously. Grids containing ultrathin cryosections were incubated with antimyeloperoxidase and then incubated with FNG.

scholarly journals THE EFFECTS OF HALOTHANE ON CULTURED MOUSE NEUROBLASTOMA CELLS

1974 ◽  
Vol 63 (2) ◽  
pp. 531-540 ◽  
Author(s):  
Robert E. Hinkley ◽  
Alvin G. Telser
Keyword(s):  
Electron Microscopy ◽  
Cell Differentiation ◽  
Gas Phase ◽  
Culture Medium ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Short Term ◽  
Neurite Extension ◽  
Mouse Neuroblastoma ◽  
Subcortical Regions

Mouse neuroblastoma cells (clone NB2a) were cultured in the presence of 0.3–2.1% halothane in the gas phase for up to 72 h. Halothane inhibited neurite extension dose dependently and virtually abolished microspike formation even at the lowest concentration tested. These effects were completely reversible. Electron microscopy demonstrated that microfilaments measuring 40–80 Å in diameter are the only fibrous organelles visible within microspikes. When the cells were exposed to halothane, no microfilamentous complexes could be identified in any cells and the subcortical regions of neurites often appeared devoid of individual microfilaments. Microtubules were still present in neurites after exposure to halothane concentrations at which microfilaments disappeared. However, at concentrations above 1.0%, microtubules gradually appeared to decrease in number. Short-term experiments showed that existing neurites and microspikes rapidly retracted when suddenly exposed to culture medium equilibrated with 1.0% halothane and quickly reformed when the halothane was removed. The inhibition of neuroblastoma cell differentiation by halothane appears to be mediated by disruption of 40–80 Å diameter microfilaments.

scholarly journals Immunolocalisation of PrPSc in scrapie-infected N2a mouse neuroblastoma cells by light and electron microscopy

2009 ◽  
Vol 88 (1) ◽  
pp. 45-63 ◽  
Author(s):  
Nathalie M. Veith ◽  
Helmut Plattner ◽  
Claudia A.O. Stuermer ◽  
Walter J. Schulz-Schaeffer ◽  
Alexander Bürkle
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Neuroblastoma Cells ◽  
Mouse Neuroblastoma

scholarly journals Studies on the organization and localization of actin and myosin in neurons.

1979 ◽  
Vol 80 (2) ◽  
pp. 356-371 ◽  
Author(s):  
E R Kuczmarski ◽  
J L Rosenbaum
Keyword(s):  
Electron Microscope ◽  
Dorsal Root Ganglion ◽  
Actin Filaments ◽  
Dorsal Root ◽  
Neuroblastoma Cells ◽  
Growth Cones ◽  
Nerve Cells ◽  
Stress Fibers ◽  
Mouse Neuroblastoma ◽  
High Concentrations

The organization of actin in mouse neuroblastoma and chicken dorsal root ganglion (DRG) nerve cells was investigated by means of a variety of electron microscope techniques. Microspikes of neuroblastoma cells contained bundles of 7- to 8-nm actin filaments which originated in the interior of the neurite. In the presence of high concentrations of Mg++ ion, filaments in these bundles became highly ordered to form paracrystals. Actin filaments, but not bundles, were observed in growth cones of DRG cells. Actin was localized in the cell body, neurites, and microspikes of both DRG and neuroblastoma nerve cells by fluorescein-labeled S1. Myosin was localized primarily in the neurites of chick DRG nerve cells with fluorescein-labeled anti-brain myosin antibody. This antibody also stained stress fibers in fibroblasts and myoblasts but did not stain muscle myofibrils.

Immune electron microscopy of haemocyanins from two southern african whelks

1978 ◽  
Vol 36 (2) ◽  
pp. 158-159
Author(s):  
Linda M. Stannard ◽  
Margaret Lennon
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Outer Ring ◽  
Immune Electron Microscopy ◽  
Intertidal Zones ◽  
Central Belt ◽  
Cape Peninsula ◽  
Circular Contour

Burnupena cincta and Fusus verruculatus are two whelks which inhabit the intertidal zones of the Cape Peninsula shore. Their respiratory pigments, or haemocyanins, are morphologically similar in structure (Figs. 1 and 2) and appear in the electron microscope as short cylindrical rods about 34 nm in diameter and 36 nm high. Viewed side-on the molecules show regular banding suggesting a structure composed of six equidistant rings of sub-units. Occasionally the particles have the appearance of possessing a central “belt” in the position of the 3rd and 4th rows of sub-units. End-on views of the haemocyanin molecules show a circular contour with a dense outer ring and a less dense inner ring in which 10 definite sub-units may frequently be distinguished. A number of molecules display an extra central inner component which appears either as a diffuse plug or as a discrete ring-shaped core ± 8 nm in diameter.

Electron microscopy and diffraction studies of polyimides

1983 ◽  
Vol 41 ◽  
pp. 28-29
Author(s):  
O.C. de Hodgins ◽  
K. R. Lawless ◽  
R. Anderson
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Structural Features ◽  
Inert Atmosphere ◽  
Polyimide Films ◽  
Resolution Electron ◽  
The Matrix ◽  
High Resolution Electron Microscope ◽  
Diffraction Patterns ◽  
Polymeric Samples

Commercial polyimide films have shown to be homogeneous on a scale of 5 to 200 nm. The observation of Skybond (SKB) 705 and PI5878 was carried out by using a Philips 400, 120 KeV STEM. The objective was to elucidate the structural features of the polymeric samples. The specimens were spun and cured at stepped temperatures in an inert atmosphere and cooled slowly for eight hours. TEM micrographs showed heterogeneities (or nodular structures) generally on a scale of 100 nm for PI5878 and approximately 40 nm for SKB 705, present in large volume fractions of both specimens. See Figures 1 and 2. It is possible that the nodulus observed may be associated with surface effects and the structure of the polymers be regarded as random amorphous arrays. Diffraction patterns of the matrix and the nodular areas showed different amorphous ring patterns in both materials. The specimens were viewed in both bright and dark fields using a high resolution electron microscope which provided magnifications of 100,000X or more on the photographic plates if desired.

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