Contributors ................................................... ix
Preface ........................................................ xi
Acknowledgements ............................................... xi
Abbreviations ................................................. xii
Color section ................................................ xiii
Chapter 1. Introduction ......................................... 1
Simon Renshaw
Chapter 2. Antibodies for immunochemistry
Catherine Onley
1. Introduction ................................................. 3
1.1. Antibodies are a family of glycoproteins with
a central role in the adaptive immune response .......... 3
1.2. Isotypic, allotypic, and idiotypic variations
contribute to the diverse repertoire of antibody
structures .............................................. 6
1.3. The structures of antibodies are optimized for
antigen binding and effector functions .................. 8
1.4. Multiple factors contribute to the antibody-antigen
interaction, including antibody affinity, avidity,
and specificity ........................................ 10
2. Methods and approaches ...................................... 11
2.1. The relationship between antibody affinity, avidity,
and specificity ........................................ 11
2.2. Antibody production .................................... 14
2.3. Recommended protocols .................................. 27
3. References .................................................. 31
Chapter 3. The selection of reporter labels
Sarah Mardle
1. Introduction ................................................ 33
2. Methods and approaches ...................................... 34
2.1. Enzymatic/chromogenic detection ........................ 34
2.2. Fluorescence detection ................................. 37
2.3. Blocking unwanted background signals ................... 40
2.4. Chromogen development .................................. 40
2.5. Mounting media and slide storage ....................... 41
2.6. Storage of enzyme- and fluorochrome-conjugated
reagents ............................................... 41
2.7. Enzymatic or fluorescent? .............................. 41
3. References .................................................. 44
Chapter 4. Immunochemical staining techniques
Simon Renshaw
1. Introduction ................................................ 45
1.1. Paraffin-embedded sections ............................. 45
1.2. Frozen tissue sections ................................. 46
1.3. Free-floating sections ................................. 46
1.4. Cytological specimens .................................. 47
1.5. Reproducible and accurate results ...................... 47
2. Methods and approaches ...................................... 47
2.1. Specimen fixation ...................................... 47
2.2. Processing ............................................. 56
2.3. Specimen storage ....................................... 58
2.4. Decalcification ........................................ 58
2.5. Antigen retrieval ...................................... 59
2.6. Counterstaining following immunochemical staining ...... 61
2.7. Mounting following immunochemical staining ............. 64
2.8. Slide storage following immunochemical staining ........ 67
2.9. Tissue microajrays ..................................... 67
2.10.Recommended protocols .................................. 68
3. Troubleshooting ............................................. 95
4. References .................................................. 95
Chapter 5. Multiple immunochemical staining
Ian William Jones and Adam Westmacott
1. Introduction ................................................ 97
1.1. Choosing an appropriate method ......................... 97
1.2. Experimental design .................................... 98
1.3. Appropriate controls .................................. 101
1.4. Multiple staining using same-species primary
antibodies ............................................ 102
2. Methods and approaches ..................................... 107
2.1. Labels for light microscopy techniques ................ 107
2.2. Labels for fluorescence and confocal microscopy
techniques ............................................ 109
2.3. Labels for electron microscopy techniques ............. 109
2.4. Recommended protocols ................................. 110
3. Troubleshooting ............................................ 124
4. References ................................................. 126
Chapter 6. Confocal microscopy and immunochemistry
Matthew Cuttle
1. Introduction ............................................... 127
1.1. Do you need to use confocal microscopy? ............... 128
1.2. How does the confocal microscope work? ................ 129
2. Methods and approaches ..................................... 131
2.1. Selecting fluorescent dyes ............................ 131
2.2. Setting up the light path on the microscope ........... 132
2.3. Choosing the right lens ............................... 133
2.4. How to set the digital image settings ................. 134
2.5. Getting ready to scan ................................. 135
2.6. Optimizing the image collection settings .............. 136
2.7. Optimizing z stack image collection ................... 139
2.8. The advantages of multiphoton confocal microscopy ..... 140
2.9. Advanced techniques for imaging with multiple
fluorochromes ......................................... 141
2.10.Enhancing the final image ............................. 145
3. References ................................................. 148
Chapter 7. Ultrastructural immunochemistry
Jeremy N. Skepper and Janet M. Powell
1. Introduction ............................................... 149
1.1. Fixation and its effect on antigen-antibody binding ... 150
1.2. Controls .............................................. 153
1.3. Why do we need to use electron microscopy? ............ 156
1.4. Quantification ........................................ 156
2. Methods and approaches ..................................... 157
2.1. Epoxy resin sections .................................. 158
2.2. The acrylic resins London Resin (LR) White and Gold ... 159
2.3. Freeze substitution and low-temperature embedding
in Lowicryl HM20 ...................................... 162
2.4. Ultrathin thawed cryosections ......................... 163
2.5. Recommended protocols ................................. 166
3. References ................................................. 172
Chapter 8. Image capture, analysis, and quantification
Jiahua Wu, Anthony Warford, and David
Tannahill
1. Introduction ............................................... 175
2. Methods and approaches ..................................... 176
2.1. Image capture ......................................... 176
2.2. Image analysis and quantification ..................... 183
2.3. Image data handling ................................... 198
3. References ................................................. 202
Chapter 9. Quality assurance in immunochemistry
Peter Jackson
1. Introduction ............................................... 205
2. Methods and approaches ..................................... 206
2.1. Fixation and tissue processing ........................ 206
2.2. Microtomy ............................................. 209
2.3. Decalcification ....................................... 211
2.4. Antigen retrieval ..................................... 211
2.5. Immunochemical methodologies .......................... 216
2.6. Controls .............................................. 218
2.7. Microscopic interpretation ............................ 221
2.8. Background staining ................................... 222
3. Troubleshooting ............................................ 230
4. References ................................................. 237
Chapter 10.Automated immunochemistry
Emanuel Schenck I.
1. Introduction ............................................... 239
1.1. Defining the needs .................................... 240
Methods and approaches ..................................... 241
2.1. Overview of automated platforms for immunochemical
staining .............................................. 241
2.2. System contrasts ...................................... 241
2.3. Other special features ................................ 246
2.4. System running costs .................................. 246
2.5. System failure safeguards ............................. 246
References ................................................. 248
Appendix ........................................................ 1
Recipes ....................................................... 249
Appendix ........................................................ 2
List of suppliers ............................................. 253
Index ......................................................... 261
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