scholarly journals THE PROPERTIES OF SPECIFIC STAINS FOR ELECTRON MICROSCOPY PREPARED BY THE CONJUGATION OF ANTIBODY MOLECULES WITH FERRITIN

1961 ◽  
Vol 9 (3) ◽  
pp. 519-537 ◽  
Author(s):  
S. J. Singer ◽  
Anita F. Schick
Keyword(s):  
Electron Scattering ◽  
Specific Binding ◽  
Electron Microscopic Examination ◽  
Resolving Power ◽  
Electron Microscopic ◽  
Binding Properties ◽  
Homologous Antigen ◽  
Recent Developments ◽  
Antibody Conjugates ◽  
Antigen Antibody

In order to take full advantage of recent developments in the electron microscopic examination of cellular ultrastructure and composition, it is necessary to develop specific electron stains capable of identifying and localizing a wide variety of macromolecular components of cells. To this end, antibody conjugates have been prepared by chemically coupling the highly electron-scattering ferritin molecule to antibody. Antigen-antibody precipitations with these ferritin-antibody conjugates have demonstrated that under the appropriate conditions they retain the specific binding properties of the antibody from which they are prepared. An electron microscopic study has been made of aggregates of tobacco mosaic virus and its ferritin-conjugated antibody. The aggregates were prepared in solution and then sprayed onto specimen screens. The electron micrographs reveal that the conjugate specifically attached to, and delineated, the virus rods. The chemistry, structure, and resolving power of the ferritin-antibody conjugates, the specificity of their reactions with homologous antigen, and the nature of the problems to be faced in application of these conjugates to the study of the internal antigens of cells are discussed.

scholarly journals Short time bleaching of fluorescein isothiocyanate. A possible parameter for the specific binding of conjugates in immunofluorescence.

1980 ◽  
Vol 28 (9) ◽  
pp. 1029-1031 ◽  
Author(s):  
K Schauenstein ◽  
G Böck ◽  
G Wick
Keyword(s):  
Specific Binding ◽  
Fluorescein Isothiocyanate ◽  
Direct Immunofluorescence ◽  
Homologous Antigen ◽  
Nonspecific Staining ◽  
First Results ◽  
Antibody Conjugates ◽  
Specificity Of Binding ◽  
Short Time ◽  
Kinetics Of

The fluorescence kinetics of fluorescein isothiocyanate (FITC)-antibody conjugates during short (0.01 sec) laser excitations were analyzed for possible information about the immunological specificity of binding to antigenic substrates in direct immunofluorescence assays with antigen-coated Sephadex beads. First results of these investigations suggest marked differences in the bleaching characteristics of specifically and nonspecifically bound FITC conjugates: FITC-anti-bovine serum albumin (BSA) and FITC-anti-rabbit immunoglobulin (Ig) were found to fade significantly more slowly when bound to the respective homologous antigen (anti-BSA to BSA, and anti-rabbit Ig to rabbit Ig) as compared to nonspecifically adherent anti-BSA to rabbit Ig, and anti-rabbit Ig to BSA. Possible implications of these data for discrimination of nonspecific staining in immunofluorescence are discussed.

scholarly journals CELL SURFACE IMMUNOGLOBULIN

1974 ◽  
Vol 139 (4) ◽  
pp. 862-876 ◽  
Author(s):  
Ellen S. Vitetta ◽  
Inge Grundke-Iqbal ◽  
Kathryn V. Holmes ◽  
Jonathan W. Uhr
Keyword(s):  
Cell Surface ◽  
Half Life ◽  
Plasma Cells ◽  
Electron Microscopic Examination ◽  
Lymphoid Cells ◽  
Electron Microscopic ◽  
Igg Synthesis ◽  
Antigen Antibody ◽  
And Control ◽  
Kinetics Of

Lymphoid cells from the spleen, lymph nodes, and thoracic duct of axenic and control mice were incubated with [3H]tyrosine and synthesis and secretion of protein and Ig studied. It was found that only IgM was synthesized by cells from axenic mice whereas cells from control mice also synthesized IgG. Splenocytes from both axenic and control mice had 8S IgM on their surface. Radiolabeled splenocytes from axenic mice were incubated to determine the kinetics of release of 125I-labeled cell surface IgM and [3H]tyrosine-labeled IgM. Cell surface IgM was shed as 8S with an initial half-life of release of 5–8 h whereas [3H]tyrosine-labeled Ig was secreted as 19S with an initial half-life of 2–3 h. These findings suggest that two independent pathways are involved. It is suggested that small lymphocytes shed 8S IgM and plasma cells secrete 19S IgM. It was observed that lymphoid cells from axenic mice synthesize a higher proportion of IgM relative to total protein. Electron microscopic examination of splenocytes from such mice revealed a markedly higher proportion of plasma cells and a paucity of lymphoblasts compared to controls. It was suggested, therefore, that axenic mice lack a population of stimulated T cells which can induce a switch from IgM to IgG synthesis and which is capable of suppressing IgM synthesis. Lymphoid cells from axenic mice synthesize and secrete less protein that coprecipitates with antigen-antibody complexes.

scholarly journals ALLERGIC INFLAMMATION

1963 ◽  
Vol 118 (4) ◽  
pp. 557-564 ◽  
Author(s):  
Henry Z. Movat ◽  
Neil V. P. Fernando ◽  
Tsuneo Uriuhara ◽  
William J. Weiser
Keyword(s):  
Fine Structure ◽  
Microscopic Examination ◽  
Allergic Inflammation ◽  
Electron Microscopic Examination ◽  
Collagen Fibrils ◽  
Electron Microscopic ◽  
Ultrastructural Evidence ◽  
Antigen Antibody

The fine structure of collagen fibrils at sites of antigen-antibody interaction is described. Following injection of antigen (BSA or ferritin) into the center of the cornea of hyperimmune rabbits, an acidophilic ring of precipitate forms at the periphery of the cornea, where antigen and antibody interact in optimal proportions. The precipitate is soon removed by infiltrating polymorphs, but persists longer in leukopenic animals. Electron microscopic examination of the cornea showed no alteration of the collagen fibrils in the area of antigen-antibody precipitation or in the remaining cornea. Contrary to many claims there was no swelling, fragmentation, or disintegration of the fibrils and they had a normal periodicity. Polymorphs infiltrated the precipitates and phagocytosed the antigen-antibody complexes. There was some ultrastructural evidence that degradation of the complexes took place in the polymorphs.

Immunochemical and ultrastructural mapping of the gelatin-binding and cell-attachment regions of human plasma fibronectin with monoclonal antibodies

1986 ◽  
Vol 81 (1) ◽  
pp. 125-141
Author(s):  
D.L. Hasty ◽  
H.S. Courtney ◽  
W.A. Simpson ◽  
J.A. McDonald ◽  
E.H. Beachey
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Quaternary Structure ◽  
Cell Attachment ◽  
Chinese Hamster ◽  
Electron Microscopic Examination ◽  
Ultrastructural Level ◽  
Plasma Fibronectin ◽  
Electron Microscopic ◽  
Antigen Antibody

Monoclonal antibodies against fibronectin were used to locate the gelatin-binding and cell-attachment regions of plasma fibronectin at an ultrastructural level. A total of 23 hybridomas were generated using mice immunized with either intact fibronectin or a 40 000 Mr gelatin-binding fibronectin fragment. One of these antibodies (D9b) strongly inhibited the interaction of radio-labelled fibronectin with gelatin. Another antibody (IB10) inhibited the attachment of Chinese hamster ovary (CHO) cells to a fibronectin substratum by 99%. Both of these antibodies were purified by affinity chromatography on columns of fibronectin-Sepharose and were then incubated with soluble fibronectin to form antigen-antibody complexes. The complexes were separated from free antibody on a column of Sephadex G-200 and were prepared for electron-microscopic examination by spraying on mica discs and rotary shadowing with platinum. As determined by this method, the fibronectin molecules measured 124 +/− 1.7 nm in length. Monoclonal antibody IB10 was visualized as a globular projection 40 +/− 1.4 nm from one end of the fibronectin filament. Monoclonal antibody D9b, on the other hand, was visualized as a globular projection at or near one or both ends of the molecule. These data provide the first morphological localization of the gelatin-binding and cell-attachment regions of fibronectin and indicate that further studies using monoclonal antibodies directed toward other epitopes should shed light not only on function but also on the tertiary and quaternary structure of the fibronectin molecule.

Preparatory Procedures for Electron Microscopic Analysis at the Molecular Level of Resolution

1978 ◽  
Vol 36 (3) ◽  
pp. 516-520
Author(s):  
F.J. Sjostrand
Keyword(s):  
Electron Microscope ◽  
Molecular Level ◽  
Microscopic Analysis ◽  
Resolving Power ◽  
Cellular Structures ◽  
Electron Microscopic ◽  
Systematic Analysis ◽  
Technical Developments ◽  
Thin Sectioning ◽  
Obvious Reason

In the 1940's and 1950's electron microscopy conferences were attended with everybody interested in learning about the latest technical developments for one very obvious reason. There was the electron microscope with its outstanding performance but nobody could make very much use of it because we were lacking proper techniques to prepare biological specimens. The development of the thin sectioning technique with its perfectioning in 1952 changed the situation and systematic analysis of the structure of cells could now be pursued. Since then electron microscopists have in general become satisfied with the level of resolution at which cellular structures can be analyzed when applying this technique. There has been little interest in trying to push the limit of resolution closer to that determined by the resolving power of the electron microscope.

Electron microscopic studies on ultrathin frozen sections of lactating mouse mammary gland

1978 ◽  
Vol 36 (2) ◽  
pp. 46-47
Author(s):  
Jan Zarzycki ◽  
Joseph Szroeder
Keyword(s):  
Mammary Gland ◽  
Protein Denaturation ◽  
Chemical Constituents ◽  
Freeze Substitution ◽  
Electron Microscopic Examination ◽  
Mouse Mammary Gland ◽  
Electron Microscopic ◽  
Preparation Methods ◽  
Microscopic Studies ◽  
Ultrathin Frozen Sections

The mammary gland ultrastructure in various functional states is the object of our investigations. The material prepared for electron microscopic examination by the conventional chemical methods has several limitations, the most important are the protein denaturation processes and the loss of large amounts of chemical constituents from the cells. In relevance to this,one can't be sure about a degree the observed images are adequate to the realy ultrastructure of a living cell. To avoid the disadvantages of the chemical preparation methods,some autors worked out alternative physical methods based on tissue freezing / freeze-drying, freeze-substitution, freeze-eatching techniqs/; actually the technique of cryoultraraicrotomy,i,e.cutting ultrathin sections from deep frozen specimens is assented as a complete alternative method. According to the limitations of the routine plastic embbeding methods we were interested to analize the mammary gland ultrastructure during lactation by the cryoultramicrotomy method.

Transmission Electron Microscopy of Protein Macromolecules

1971 ◽  
Vol 29 ◽  
pp. 424-425
Author(s):  
Henry S. Slayter
Keyword(s):  
Biological Systems ◽  
Metal Vapor ◽  
Resolving Power ◽  
Electron Microscopic ◽  
Chemical Methods ◽  
Molecular Weights ◽  
The Past ◽  
Physical Chemical ◽  
Transmission Electron

Electron microscopic methods have been applied increasingly during the past fifteen years, to problems in structural molecular biology. Used in conjunction with physical chemical methods and/or Fourier methods of analysis, they constitute powerful tools for determining sizes, shapes and modes of aggregation of biopolymers with molecular weights greater than 50, 000. However, the application of the e.m. to the determination of very fine structure approaching the limit of instrumental resolving power in biological systems has not been productive, due to various difficulties such as the destructive effects of dehydration, damage to the specimen by the electron beam, and lack of adequate and specific contrast. One of the most satisfactory methods for contrasting individual macromolecules involves the deposition of heavy metal vapor upon the specimen. We have investigated this process, and present here what we believe to be the more important considerations for optimizing it. Results of the application of these methods to several biological systems including muscle proteins, fibrinogen, ribosomes and chromatin will be discussed.

A Comparative Study of Fractographic Replicas

1974 ◽  
Vol 32 ◽  
pp. 566-567
Author(s):  
Loren Anderson ◽  
Pat Pizzo ◽  
Glen Haydon
Keyword(s):  
Electron Microscopy ◽  
Electron Microscopic Examination ◽  
Direct Examination ◽  
Electron Microscopic ◽  
Reliable Technique ◽  
Service Failures ◽  
Transmission Electron ◽  
Mode Of Failure ◽  
Scanning Electron Microscopic ◽  
Scanning Electron

Transmission electron microscopy of replicas has long been used to study the fracture surfaces of components which fail in service. Recently, the scanning electron microscope (SEM) has gained popularity because it allows direct examination of the fracture surface. However, the somewhat lower resolution of the SEM coupled with a restriction on the sample size has served to limit the use of this instrument in investigating in-service failures. It is the intent of this paper to show that scanning electron microscopic examination of conventional negative replicas can be a convenient and reliable technique for determining mode of failure.

Novel methods for demonstrating osseointegration of hydroxylapatite-coated titanium hip prostheses

1991 ◽  
Vol 49 ◽  
pp. 34-35 ◽  
Author(s):  
P. Frayssinet ◽  
J. Hanker ◽  
D. Hardy ◽  
B. Giammara
Keyword(s):  
Hip Prosthesis ◽  
Ethanol Concentration ◽  
Bone Matrix ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Hip Prostheses ◽  
Cellular Inclusions ◽  
Microscopic Studies ◽  
Diamond Saw ◽  
Plasma Sprayed

Prostheses implanted in hard tissues cannot be processed for electron microscopic examination or microanalysis in the same way as those in other tissues. For these reasons, we have developed methods allowing light and electron microscopic studies as well as microanalysis of the interface between bone and a metal biomaterial coated by plasma-sprayed hydroxylapatite(HA) ceramic.An HA-coated titanium hip prosthesis (Corail, Landos, France), which had been implanted for two years, was removed after death (unrelated to the orthopaedic problem). After fixation it was dehydrated in solutions of increasing ethanol concentration prior to embedment in polymethylmethacrylate(PMMA). Transverse femur sections were obtained with a diamond saw and the sections then carefully ground to a thickness of 200 microns. Plastic-embedded sections were stained for calcium with a silver methenamine modification of the von Kossa method for calcium staining and coated by carbon. They have been examined by back-scatter SEM on an ISI-SS60 operated at 25 KV. EDAX has been done on cellular inclusions and extracellular bone matrix.

Properties of Isolated Mitochondria of Agaricus Bisporus: Their Relationship to Dormancy and the Germination of Spores

1973 ◽  
Vol 31 ◽  
pp. 458-459
Author(s):  
K. S. McCarty ◽  
R. F. Weave ◽  
L. Kemper ◽  
F. S. Vogel
Keyword(s):  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Microscopic Examination ◽  
Agaricus Bisporus ◽  
Osmotic Shock ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Isolated Mitochondria ◽  
Dna Strands ◽  
Cytoplasmic Ribosomes

During the prodromal stages of sporulation in the Basidiomycete, Agaricus bisporus, mitochondria accumulate in the basidial cells, zygotes, in the gill tissues prior to entry of these mitochondria, together with two haploid nuclei and cytoplasmic ribosomes, into the exospores. The mitochondria contain prominent loci of DNA [Fig. 1]. A modified Kleinschmidt spread technique1 has been used to evaluate the DNA strands from purified whole mitochondria released by osmotic shock, mitochondrial DNA purified on CsCl gradients [density = 1.698 gms/cc], and DNA purified on ethidium bromide CsCl gradients. The DNA appeared as linear strands up to 25 u in length and circular forms 2.2-5.2 u in circumference. In specimens prepared by osmotic shock, many strands of DNA are apparently attached to membrane fragments [Fig. 2]. When mitochondria were ruptured in hypotonic sucrose and then fixed in glutaraldehyde, the ribosomes were released for electron microscopic examination.

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