Autoradiography and Immunocytochemistry (eBook)

Front Cover 1
Autoradiography and Immunocytochemistry 4
Copyright Page 5
Table of Contents 18
Dedication 10
Author's preface 12
Acknowledgements 16
Chapter 1. Introduction 22
1.1 The demand for autoradiography and immunocytochemistry 23
1.2 The most advantageous use of the techniques 24
Chapter 2. Immunocytochemistry at the EM level: preparation of labelled antibodies 26
2.1 Production and purification of antibodies 28
2.2 Ferritin-labelled antibodies 33
2.3 Purification of ferritin–antibody conjugates 40
2.4 Enzyme-labelled antibodies 45
2.5 Heavy atom labelling of antibodies 50
2.6 Immuno-electron microscopy using non-conjugated electron-dense markers 50
2.7 Lectins as staining reagents for immuno-electron microscopy 54
Chapter 3. Immunocytochemistry at the EM level: staining antigen with electron-dense reagents 62
3.1 Fixation of tissues, cells and cell fragments 62
3.2 General remarks on 'staining' with antibodies 64
3.3 Staining with ferritin-labelled antibodies (Fer–Ab) 65
3.4 Controls in immunoferritin staining 78
3.5 Staining with antibody dually-labelled with fluorescein and ferritin 79
3.6 Enhancement of the size and electron opacity of ferritin by heavy metal staining 80
3.7 Methods of applying enzyme-labelled antibodies 80
3.8 Controls when staining with enzyme-labelled antibodies 88
3.9 Multistep antibody staining methods with unmodified proteins 89
3.10 The use of lectins in demonstrating antigenic determinant s or other relevant saccharides 90
3.11 Quantitative studies using immuno-electron microscopy 91
Chapter 4. Preparation of electron microscope autoradiographs 98
4.1 Radioisotopes in EM autoradiography 100
4.2 Radiochemicals 104
4.3 Route of administration of the radiochemical 110
4.4 Dose of radiochemical 111
4.5 Toxicity of radioisotopes 116
4.6 Preparation of radioactively-labelled tissues and cells 117
4.7 Choice of nuclear emulsion 128
4.8 Methods of applying nuclear emulsions 130
4.9 Exposure 145
4.10 Development 146
4.11 Recovery of specimens from their supports 155
4.12 Post-developmental staining and removal of gelatin 158
4.13 Preparation of micrographs from EM autoradiographs 159
4.14 Control observations 161
4.15 Efficiency in EM autoradiography 167
4.16 Resolution in EM autoradiographs 169
4.17 Applications of EM autoradiography 171
Appendix 1. Chemicals, reagents and methods mostly to do with immunocytochemistry 188
Appendix 2. Materials and methods for autoradiography 202
Appendix 3. List of suppliers 220
Subject index 230