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Fine Structure Immunocytochemistry

Fine Structure Immunocytochemistry

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1 Introduction to Immunocytochemistry and Historical Background.- 1.1 Approaches to Immunocytochemistry.- 1.2 Criteria for an Electron-Microscopic Immunocytochemical Technique.- 1.3 Problems with the Pre-Embedding Techniques.- References.- 2 Fine Structure Preservation.- 2.1 Introduction.- 2.2 Fine-Structure Evaluation in Practice.- 2.2.1 Fine-Structure Stabilization by Fixation.- 2.2.2 Contrasting.- 2.2.3 Analysis.- 2.2.4 Interpretation.- 2.2.5 Selective Destruction.- 2.3 Summary.- References.- 3 Fixation for Fine Structure Preservation and Immunocytochemistry.- 3.1 Fine Structure Preservation.- 3.1.1 Glutaraldehyde.- 3.1.2 Formaldehyde.- 3.1.3 Effects of Aldehydes on Cell Components Other than Proteins.- 3.1.4 Cross-Linking Agents Other than Glutaraldehyde and Formaldehyde.- 3.2 Factors Affecting the Quality of Fixation for Fine Structure Preservation.- 3.2.1 Concentration and Length of Fixation.- 3.2.2 Temperature of Fixation.- 3.2.3 pH and the Buffer Vehicle.- 3.2.4 Osmolarity and Ionic Strength of the Buffer Vehicle.- 3.2.5 Purity of the Aldehyde Fixative; Storage and Disposal.- 3.2.6 Tissue Type.- 3.2.7 Methods of Fixation.- 3.2.8 Rate of Fixative Penetration.- 3.3 Fixation Artefacts.- 3.4 Effect of Fixatives on Enzyme Activity.- 3.4.1 Effect of Glutaraldehyde on Enzyme Activity.- 3.4.2 Effect of Formaldehyde on Enzyme Activity.- 3.5 Fixation for Immunocytochemistry.- 3.5.1 Effect of Glutaraldehyde on Antigenicity.- 3.5.2 Effect of Formaldehyde on Antigenicity.- 3.5.3 Effect of Acrolein on Antigenicity.- 3.5.4 Effect of Osmium Tetroxide on Antigenicity.- 3.5.5 Effect of Factors Affecting Cross-Linking on Antigenicity.- 3.5.6 Assessing the Effects of Fixation on Antigenicity.- 3.6 A Concluding Remark.- References.- 4 Embedding Media for Section Immunocytochemistry.- 4.1 Resin-Free Sections - Temporary Embedement.- 4.1.1 The PEG Method.- 4.1.2 The Poly-Methylmethacrylate Method.- 4.2 Permanent Embedding Media.- 4.2.1 Water-Miscible Media.- 4.2.2 New Acrylic Resins.- 4.2.3 Epoxy Resins.- 4.3 Freeze Substitution.- 4.3.1 Rapid Freezing.- 4.3.2 Freeze Substitution and Immunolabelling.- References.- Chapters 5 Cryo and Replica Techniques for Immunolabelling.- 5.1 Cryo Sectioning Techniques.- 5.1.1 Historical Perspectives.- 5.1.2 Theory of Freezing.- 5.1.3 The Knife.- 5.1.4 The Microtome.- 5.1.5 Cryo Sectioning Theory.- 5.1.6 The Hydrated Cryo Sectioning Technique.- 5.1.7 Tokuyasu Thawed Cryo-Section Technique.- 5.2 Freeze-Fracture and Replica Labelling Methods.- 5.2.1 Fracture-Label Method.- 5.2.2 Label-Fracture Method.- 5.2.3 Fracture-Flip.- 5.3 Other Replica Techniques for Labelling.- 5.3.1 Surface Replica Methods.- 5.3.2 Replicas of Labelled Sections.- References.- 6 Elementary Immunology.- 6.1 Basic Immunology.- 6.1.1 The Immune Response.- 6.1.2 Cells of the Immune System.- 6.2 The Structure of Antibodies.- 6.3 The Biological Functions of Immunoglobulins.- 6.3.1 lgG.- 6.3.2 lgM.- 6.3.3 IgA.- 6.3.4 lgD and lgE.- 6.3.5 Generation of Antibody Diversity.- 6.4. The Nature of Antigenicity.- 6.4.1 Antigen Size - Carriers and Haptens.- 6.4.2 Antigen Presentation.- 6.4.3 Proteins as Antigens.- 6.4.4 The Interaction of Antibodies with Their Antigens.- 6.5 Practical Aspects of Immunology.- 6.5.1 Preparation of Antibodies.- 6.5.2 Production of Polyclonal Antibodies.- 6.5.3 Monoclonal Antibodies.- 6.5.4 Anti-Peptide Antibodies.- 6.5.5 Use of Molecular Biology Approaches to Express Immunoglobulin Molecules in Cells.- 6.5.6 Purification of Antibodies.- 6.6 Affinity Purification.- 6.7 Characterization of Antibodies.- 6.8 Precipitin Techniques.- 6.9 Immunoblotting.- 6.10 Immunoprecipitation.- 6.11 Immunoassay.- References.- 7 Labelling Reactions for Immunocytochemistry.- 7.1 Historical Perspectives.- 7.2 Labelling in Practice.- 7.2.1 Purity of Antibody: Use of Light Microscopy.- 7.2.2 Class of Antibody; Hybrid Antibodies.- 7.2.3 Antibody Concentrations.- 7.2.4 Time, Temperature and Repeated Applications of Antibody.- 7.2.5 Avoiding Non-
Electron microscopy in the biological sciences can be divided into two disciplines. The first, concerned with high resolution detail of particles or periodic structures, is mostly based on sound theoretical principles of physics. The second, by far the larger discipline, is interested in the information obtainable from thin sections. The theoretical back ground to those groups of techniques for preparing and looking at thin sections is often inexact and "loose", for want of a better word. What should be chemistry is often closer to alchemy. This kind of electron microscopy is often enshrined with mystical recipes, handed down from generation to generation. Admittedly, many of the processes involved, such as those required to embed tissue in epoxy resins, involve multiple interconnected steps, which make it difficult to follow the details of anyone of these steps. If all these steps are shrouded in some mystery, however, can one really trust the final image that emerges on the EM screen? When we present the data in some semi quantitative form is there really no better way to do it than to categorize the parameters with ++, +/-, etc? What happens when one labels the sections with antibodies? Does the whole business necess arily need to be more of an "art" than a "science"? Upon reflecting on these problems in 1981, I had the impression that many of the multi-authored textbooks that existed then (and that have appeared since) tended to exacerbate or at least perpetuate this

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    antibody; biology; chemistry; electron microscopy; enzyme; enzymes; ferritin; glycoprotein; membrane; microscopy; molecular biology; protein; proteins; receptor; tissue

Produktdetails

Autor: Gareth Griffiths
ISBN-13: 9783642770975
ISBN: 3642770975
Einband: Book
Seiten: 484
Gewicht: 725 g
Format: 235x155x25 mm
Sprache: Englisch
Autor: Gareth Griffiths
Rezension"a wealth of solid, well-referenced information.The more experienced user will find this is an essential reference text" Australian Journal of Medical Science

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Autor: Gareth Griffiths
ISBN-13:: 9783642770975
ISBN: 3642770975
Erscheinungsjahr: 06.12.2011
Verlag: Springer Berlin Heidelberg
Gewicht: 725g
Seiten: 484
Sprache: Englisch
Auflage Softcover reprint of the original 1st ed. 1993
Sonstiges: Taschenbuch, 235x155x25 mm