Fluorescent and Immuno-Electron Microscopy of Detergent Extracted Cytoskeletal and Associated Antigens

1985 ◽  
Vol 43 ◽  
pp. 582-583
Author(s):  
E.J. Basgall ◽  
M.M. Soong ◽  
W.A.F. Tompkins
Keyword(s):  
Electron Microscopy ◽  
Cultured Cells ◽  
Leukemia Virus ◽  
Protein A ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Simple Method ◽  
Viral Antigens ◽  
Associated Proteins ◽  
Nih 3T3

Triton X-100 detergent extraction has been shown to be a relatively simple method for revealing the internal architecture of cultured cells. This method has proven valuable in studies of cytoskeleton-associated proteins and their functions in regulating cell activities. Exposed cytoskeletal elements can be examined using either transmission or high-resolution scanning electron microscopy. Immunochemical studies at the electron microscopic level have been simplified by the availability of Protein A-colloidal gold as a high affinity marker for immunoglobulins, specifically IgG.Several investigators have reported an association between the cytoskeleton and viral antigens. Evidence has indicated that, in some systems, the cytoskeleton plays a significant role in virus infection. Immunofluorescent studies on canine distemper virus infected, extracted cells have revealed altered cytoskeletal staining patterns, as compared to non-infected controls. In our laboratory, immuno-electronmicroscopy studies of extracted NIH/3T3 cells infected with Maloney-murine leukemia virus have indicated an association between cytoskeletal actin and the viral antigens gp70 and pl5E.

scholarly journals FIXATION OF NEURAL TISSUES FOR ELECTRON MICROSCOPY BY PERFUSION WITH SOLUTIONS OF OSMIUM TETROXIDE

1962 ◽  
Vol 12 (2) ◽  
pp. 385-410 ◽  
Author(s):  
Sanford L. Palay ◽  
S. M. McGee-Russell ◽  
Spencer Gordon ◽  
Mary A. Grillo
Keyword(s):  
Electron Microscopy ◽  
Nervous System ◽  
Osmium Tetroxide ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Vascular Perfusion ◽  
Neural Tissues ◽  
Structural Relations ◽  
Cellular Components

This paper describes in detail a method for obtaining nearly uniform fixation of the nervous system by vascular perfusion with solutions of osmium tetroxide. Criteria are given for evaluating the degree of success achieved in the preservation of all the cellular components of the nervous system. The method permits analysis of the structural relations between cells at the electron microscopic level to an extent that has not been possible heretofore.

Phase-contrast and electron-microscopic observations on a membranous complex in primary spermatocytes of the mouse

1975 ◽  
Vol 18 (1) ◽  
pp. 1-17
Author(s):  
A. Pleshkewych ◽  
L. Levine
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Phase Contrast ◽  
Cultured Cells ◽  
Light And Electron Microscopy ◽  
Cytoplasmic Inclusion ◽  
Electron Microscopic ◽  
Secondary Spermatocyte ◽  
Living Mouse ◽  
Circular Contour

A prominent cytoplasmic inclusion present in living mouse primary spermatocytes has been observed by both light and electron microscopy. It began to form at prometaphase and continued to increase in thickness and length as the cells developed. By metaphase it was a distinct sausage-shaped boundary that enclosed a portion of the cytoplasm between the spindle and the cell membrane. At the end of metaphase, the inclusion reached its maximum length. At telophase, it was divided between the daughter secondaries. The inclusion persisted as a circular contour in the interphase secondary spermatocyte. Electron microscopy of the same cultured cells that were previously observed with light microscopy revealed that the inclusion was a distinctive formation of membranes. It consisted of agranular cisternae and vesicles, and was therefore a membranous complex. Many of the smaller vesicles in the membranous complex resembled those found in the spindle. The cisternae in the membranous complex were identical to the cisternal endoplasmic reticulum of interphase primary spermatocytes. Nevertheless, the organization of vesicles and cisternae into the membranous complex was unique for the primaries in division stages, since such an organization was not present in their interphase stages.

scholarly journals Analytical electron microscopic evaluation of the effects of paraformaldehyde pretreatment on the reaction of glutaraldehyde with biogenic amines.

1982 ◽  
Vol 30 (5) ◽  
pp. 481-486 ◽  
Author(s):  
R E McClung ◽  
J Wood
Keyword(s):  
Electron Microscopy ◽  
Analytical Electron Microscopy ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Spectrographic Analysis ◽  
Electron Microscopic ◽  
X Ray ◽  
Microscopic Evaluation ◽  
Analytical Electron ◽  
Dense Product

Analytical electron microscopy was used to determine the quantitative effects of paraformaldehyde pretreatment on the formation of the biogenic amine-glutaraldehyde-chrome complex. Pretreatment with paraformaldehyde prevented the glutaraldehyde-chrome reaction with norepinephrine in the rat adrenal medulla. In contrast to the effect of paraformaldehyde on norepinephrine, pretreatment did not prevent the chrome reaction in serotonin-containing argentaffin cells of the gut. X-Ray energy spectrographic analysis revealed a significant decrease in chrome content in the paraformaldehyde treated tissue, but sufficient chrome did react to produce an electron-dense product. Thus by treating tissue with paraformaldehyde prior to the glutaraldehyde chrome procedure, serotonergic sites may be differentiated from catecholaminergic areas at the electron microscopic level.

Correlated 3D Light and Electron Microscopy of Large, Complex Structures: Analysis of Transverse Tubules in Heart Failure

1998 ◽  
Vol 4 (S2) ◽  
pp. 440-441
Author(s):  
Maryann E. Martone ◽  
Andrea Thor ◽  
Stephen J. Young ◽  
Mark H. Ellisman.
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
Structural Features ◽  
Electron Microscopic Level ◽  
Specimen Preparation ◽  
Large Sample Size ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron

Light microscopic imaging has experienced a renaissance in the past decade or so, as new techniques for high resolution 3D light microscopy have become readily available. Light microscopic (LM) analysis of cellular details is desirable in many cases because of the flexibility of staining protocols, the ease of specimen preparation and the relatively large sample size that can be obtained compared to electron microscopic (EM) analysis. Despite these advantages, many light microscopic investigations require additional analysis at the electron microscopic level to resolve fine structural features.High voltage electron microscopy allows the use of relatively thick sections compared to conventional EM and provides the basis for excellent new methods to bridge the gap between microanatomical details revealed by LM and EM methods. When combined with electron tomography, investigators can derive accurate 3D data from these thicker specimens. Through the use of correlated light and electron microscopy, 3D reconstructions of large cellular or subcellular structures can be obtained with the confocal microscope,

Electron microscopy of changes in lower extremity muscles in Duchenne muscular dystrophy

1991 ◽  
Vol 81 (6) ◽  
pp. 294-299
Author(s):  
J Atkin ◽  
VW Thompson ◽  
RB Boyd
Keyword(s):  
Electron Microscopy ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Genetic Disease ◽  
Extensor Digitorum Longus ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Dystrophic Mice ◽  
Insight Into

Duchenne muscular dystrophy is a genetic disease with a sex-linked pattern of inheritance. This disease is present at birth, becomes symptomatic during early childhood, leads to inability to walk near the end of the first decade, and usually results in death by the end of the second decade. In this study, the extensor digitorum longus and soleus muscles from genetically dystrophic mice were examined at the electron microscopic level. The authors describe their results and discuss how these findings might provide some insight into one of the mechanisms of fiber necrosis in Duchenne muscular dystrophy.

Electron microscopic analysis of objects in light microscopic sections

1978 ◽  
Vol 36 (2) ◽  
pp. 80-81
Author(s):  
Arvid B. Maunsbach
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Microscopic Analysis ◽  
Electron Microscopic Level ◽  
Semithin Section ◽  
Cover Glass ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Glass Slides ◽  
Ultrathin Sections

Structural studies in experimental biology or in pathology are frequently extended from the light to the electron microscopic level. This is often done by cutting both semithin (about 1 μm) and thin sections from the same tissue block after embedding for electron microscopy. However, in many studies it would be of great value to analyse the same structure both by light and electron microscopy, i.e. to be able to study by electron microscopy an object which is first detected by light microscopy in a semithin section. To achieve this, a method has been developed by which ultrathin sections are cut directly from the semithin section containing the object of interest.Semithin sections, about 1 μ in thickness, are cut from Epon or Vestopal embedded tissue. The sections are placed on ordinary glass slides and stained with toluidine blue. The sections are studied in the light microscope without a cover glass or mounted in water.

Immunologic Localization of Murine Leukemia Virus Antigens in Thin Sections

1981 ◽  
Vol 39 ◽  
pp. 402-403
Author(s):  
Ray A. Weigand ◽  
Carol I. Paquette ◽  
Clifford Longley ◽  
Philip Furmanski
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Intracellular Localization ◽  
Protein A ◽  
Thin Sections ◽  
Virus Particles ◽  
Tumor Virus ◽  
Labelling Procedure ◽  
Nih 3T3 ◽  
Murine Leukemia

In order to examine the temporal sequence of intracellular events which culminates in the release of infectious virus particles from the cell surface, we have developed immunoferritin techniques to localize viral antigens in thin sections. We previously described the intracellular localization of murine mammary tumor virus p28 protein in thin sections using an unlabelled antibody procedure with ferritin-antiferritin. We now describe the labelling of murine leukemia virus (MuLV) in thin sections using anti-MuLV and Protein A-ferritin.Cultures of F-MuLV in NIH/3T3 cells were grown to 90% confluence, fixed, dehydrated, and embedded in Epon as before. Thin sections were picked up on nickel grids, incubated in 20% H2O2 for 20 min, rinsed in PBS, and subjected to the labelling procedure. The labelling protocol included: preincubation in 5% BSA in PBS, rinse in 1% BSA in PBS, 3 hour incubation in rabbit anti-MuLV, rinse in 1% BSA in PBS, incubation in apoferritin, incubation in Protein A-ferritin (Zymed Labs., Burlingame, CA), rinses in 1% BSA in PBS and PBS, and incubation in 2% glutaraldehyde in PBS.

Techniques Involved in the Acquisition of Serial Sections of the Atrioventricular Node for Light and Electron Microscopy

1968 ◽  
Vol 26 ◽  
pp. 226-227
Author(s):  
Sheila S. Emmett ◽  
J. C. Thaemert
Keyword(s):  
Electron Microscopy ◽  
Osmium Tetroxide ◽  
Light And Electron Microscopy ◽  
Atrioventricular Node ◽  
Electron Microscopic Level ◽  
Serial Sections ◽  
Electron Microscopic ◽  
Trapezoidal Shape ◽  
Old Mice

The acquisition of serial sections of the atrioventricular node for light and electron microscopy is a formidable task. Ordinary techniques are not adequate if the best possible results are to be achieved at the electron microscopic level. The techniques outlined below have proven to be valuable in locating and determining the position of the AV node.Whole hearts of 2-week old mice were fixed, in situ, by perfusion with 1% phosphate-buffered osmium tetroxide. The hearts were removed from the animals, sectioned transversely into 3 slices approximately equal in thickness, dehydrated in graded concentrations of ethanol and embedded in Epon 812. The block faces were trimmed to a trapezoidal shape ranging in size from 0.75 x 1 mm to 4 x 5 mm. Serial sections approximately 2 microns in thickness were cut with glass knives on a Porter-Blum MT-2 Ultramicrotome. While floating on a drop of water on the knife, each section was stretched with 1 drop of a 1:1, xylene in chloroform mixture applied directly to the section. The sections were picked up individually with a brush, transferred to a glass slide and oven dried for several hours prior to staining.

Use of antibody and lectin-colloidal gold conjugates to detect surface and extracellular glycoproteins produced in vivo by the Lyme disease spirochete Borrelia bergdorferi

1990 ◽  
Vol 48 (3) ◽  
pp. 934-935
Author(s):  
David W. Dorward ◽  
Claude F. Garon
Keyword(s):  
Electron Microscopy ◽  
Lyme Disease ◽  
Colloidal Gold ◽  
Clinical Symptoms ◽  
Protein A ◽  
Rabbit Serum ◽  
Extracellular Material ◽  
Electron Microscopic ◽  
Extracellular Glycoproteins

Despite the often serious pathological ramifications of Lyme disease, Borrelia burgdorferi, the causative agent, is rarely demonstrated in nor isolated from naturally-infected mammalian hosts. This dilemma has led to suggestions that a limited number of spirochetes expel extracellular bioproducts that may contribute to the clinical symptoms. Currently, no mammalian toxins have been attributed to B. burgdorferi, however, the production of extracellular vesicles by this bacterium was recently described. In order to assay the possible presence of such extracellular material in infected hosts, we developed a sensitive electron microscopic assay to detect and characterize B. burgdorferi products in biological samples.Polyclonal rabbit serum was raised against purified B. burgdorferi vesicles and against an electrophoretically-purified 83 kilodalton (kDa) vesicle-associated protein. After purification of IgG from the sera, anti-vesicle F(ab’)2 fragments were produced and used to “activate” parlodion-coated grids. Such grids were incubated with cultured spirochetes, or with fluids and tissues from infected and non-infected mammals and ticks. Captured antigens were then labeled with anti-83 kDa IgG and Protein A-colloidal gold conjugates, and/or lectin-colloidal gold conjugates, then examined and characterized by electron microscopy.

Demonstration of Doublecortin Protein, a Neurogenesis Marker, at the Electron Microscopic Level

2017 ◽  
Vol 7 (1) ◽  
pp. 6-10
Author(s):  
Ozlem Tugce Cilingir Kaya ◽  
Cynthia Moore ◽  
Charles Meshul ◽  
Serap Sirvanci
Keyword(s):  
Protein A ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic
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