Autoradiography and Immunocytochemistry

1st Edition

Practical Methods in Electron Microscopy

Editors: Audrey. M. Glauert
Authors: M.A. Williams
Hardcover ISBN: 9780720406375
eBook ISBN: 9781483293875
Imprint: North Holland
Published Date: 1st January 1980
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Table of Contents

Author's Preface


Chapter 1. Introduction

1.1 The Demand for Autoradiography and Immunocytochemistry

1.2 The Most Advantageous Use of the Techniques

Chapter 2. Immunocytochemistry at the EM Level: Preparation of Labelled Antibodies

2.1 Production and Purification of Antibodies

2.1.1 Selection of Antigens

2.1.2 Purified Specific Antibodies

2.1.3 Partial Isolation of Immunoglobulins

2.1.4 Isolation of Immunoglobulins by Affinity Chromatography Using Protein A

2.2 Ferritin-Labelled Antibodies

2.2.1 Purification of Commercial Ferritin

2.2.2 Preparation of Ferritin-Immunoglobulin Conjugates

2.2.3 Proof of Conjugation

2.3 Purification of Ferritin-Antibody Conjugates

2.3.1 Methods for Purifying Ferritin-Antibody Conjugates

2.3.2 Assessment of the Purity of Conjugates

2.3.3 Dual-Labelling with Fluorescein and Ferritin

2.4 Enzyme-Labelled Antibodies

2.4.1 Preparation of Enzyme-Labelled Antibodies

2.4.2 Proof of Conjugation of Antibodies to Peroxidase

2.5 Heavy Atom Labelling of Antibodies

2.6 Immuno-Electron Microscopy Using Non-Conjugated Electron-Dense Markers

2.6.1 Hybrid Antibodies

2.6.2 Conditions of Storage of Enzyme-Antibody Conjugates, PAP Complex and Hybrid Antibodies

2.7 Lectins as Staining Reagents for Immuno-Electron Microscopy

2.7.1 Preparation of Ferritin-Conjugated Lectins

Chapter 3. Immunocytochemistry at the EM Level: Staining Antigens with Electron-Dense Reagents

3.1 Fixation of Tissues, Cells and Cell Fragments

3.2 General Remarks on 'Staining' with Antibodies


Autoradiography and Immunocytochemistry sets out in detail the preparation of autoradiographs and methods of preparing and use of immunological staining reagents.

This book begins with an introduction to the demand for autoradiography and immunocytochemistry and their most advantageous use, followed by a discussion on the immunocytochemistry at the electron microscopical (EM) level in relation to the preparation of labeled antibodies.

Other topics include the general remarks on “staining” with antibodies; applying Fer-Ab conjugates to ultrathin sections; and controls in immunoferritin staining. The multistep antibody staining methods with unmodified proteins; control observations in staining experiments using lectins; and pattern analysis in EM immunocytochemistry are also covered. This text concludes with a description of the preparation of electron microscope autoradiographs, including the radioisotopes in EM autoradiography, preparation of radioactively-labeled tissues and cells, and methods of applying nuclear emulsions.

This publication serves as a detailed laboratory guide for researchers and workers to successfully conduct electron microscope techniques.


© North Holland 1977
North Holland
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About the Editors

Audrey. M. Glauert Editor

Affiliations and Expertise

Clare Hall, University of Cambridge, UK

About the Authors

M.A. Williams Author