John Crocker is the editor of Molecular Biology in Cellular Pathology, published by Wiley. Paul G. Murray is the editor of Molecular Biology in Cellular Pathology, published by Wiley.

• Preface xiiiPreface to Molecular Biology in Histopathology xvList of Contributors xvii1 Blotting Techniques: Methodology and Applications 1Fiona Watson and C. Simon Herrington1.1 Introduction 11.2 Blotting techniques 11.3 References 152 In-situ Hybridisation in Histopathology 19Gerald Niedobitek and Hermann Herbst2.1 Introduction 192.2 Experimental conditions 202.3 Probes and labels 232.4 Controls and pitfalls 272.5 Double-labelling 292.6 Increasing the sensitivity of ISH 312.7 What we do in our laboratories 332.8 Applications of ISH: examples 352.9 Perspective 392.10 References 403 DNA Flow Cytometry 49M.G. Ormerod3.1 Introduction 493.2 Definitions and terms 493.3 Dye used for DNA analysis 503.4 Sample preparation for DNA analysis 523.5 Analysis of the DNA histogram 533.6 Quality control 533.7 Computer analysis of the DNA histogram 553.8 Multiparametric measurement 573.9 Acknowledgements 593.10 References 594 Interphase Cytogenetics 61Sara A. Dyer and Jonathan J. Waters4.1 Introduction 614.2 Interphase cytogenetics 624.3 Applications 674.4 Conclusion 764.5 References 775 Oncogenes 79Fiona Macdonald5.1 Introduction 795.2 Identification of the oncogenes 795.3 Functions of the proto-oncogenes 805.4 Mechanism of oncogene activation 895.5 Oncogenes in colorectal cancer 915.6 Oncogenes in breast cancer 945.7 Oncogenes in lung cancer 955.8 Oncogenes in haematological malignancies 965.9 Other cancers 995.10 Conclusion 1005.11 References 1006 Molecular and Immunological Aspects of Cell Proliferation 105Karl Baumforth and John Crocker6.1 The cell cycle and its importance in clinical pathology 1056.2 Molecular control of the cell cycle 1086.3 Cell cycle control 1116.4 The cell cycle and cancer 1126.5 Immunocytochemical markers of proliferating cells 1156.6 References 1336.7 Further Reading 1357 Interphase Nucleolar Organiser Regions in Tumour Pathology 137Massimo Derenzini, Davide Treré, Marie-Françoise O’Donohue and Dominique Ploton7.1 Introduction 1377.2 The AgNORs 1387.3 NOR silver-staining 1427.4 Quantitative AgNOR analysis 1457.5 AgNORs as a parameter of the level of cell proliferation 1467.6 Application of the AgNOR technique to tumour pathology 1477.7 What future for AgNORs in tumour pathology? 1517.8 References 1528 Apoptosis and Cell Senescence 153Lee B. Jordan and David J. Harrison8.1 Introduction 1538.2 Apoptosis 1538.3 Cell senescence 1748.4 Summary 1788.5 References 1799 The Polymerase Chain Reaction 193Timothy Diss9.1 Introduction 1939.2 Principles 1949.3 Analysis of products 1979.4 Rt-pcr 1999.5 Quantitative PCR 2009.6 DNA and RNA extraction 2009.7 Correlation of the PCR with morphology 2019.8 Problems 2029.9 Applications 2029.10 Diagnostic applications 2039.11 Infectious diseases 2099.12 Identity 2099.13 The future 2109.14 References 2109.15 Online information 21210 Laser Capture Microdissection: Techniques and Applications in the Molecular Analysis of the Cancer Cell 213Amanda Dutton, Victor Lopes and Paul G. Murray10.1 Introduction 21310.2 The principle of LCM 21410.3 Technical considerations 21610.4 Advantages and disadvantages of LCM 21710.5 Applications of LCM 22210.6 Future perspectives 22910.7 Acknowledgements 22910.8 References 22911 The In-situ Polymerase Chain Reaction 233John J. O’Leary, Cara Martin and Orla Sheils11.1 Introduction 23311.2 Overview of the methodology 23411.3 In-cell PCR technologies 23511.4 In-cell amplification of DNA 23811.5 Detection of amplicons 24211.6 Reaction, tissue and detection controls for use with in-cell DNA PCR assays 24311.7 In-cell RNA amplification 24411.8 Problems encountered with in-cell PCR amplification 24611.9 Amplicon diffusion and back diffusion 24711.10 Future work with in-cell PCR-based assays 24711.11 References 24912 TaqMan® Technology and Real-Time Polymerase Chain Reaction 251John J. O’Leary, Orla Sheils, Cara Martin, and Aoife Crowley12.1 Introduction 25112.2 Probe technologies 25212.3 TaqMan® probe and chemistry (first generation) 25412.4 Second generation TaqMan® probes 25612.5 Hybridisation 25812.6 TaqMan® PCR conditions 25912.7 Standards for quantitative PCR 26012.8 Interpretation of results 26112.9 End-point detection 26212.10 Real-time detection 26312.11 Relative quantitation 26312.12 Reference genes 26412.13 Specific TaqMan® PCR applications 26512.14 References 26813 Gene Expression Analysis Using Microarrays 269Sophie E. Wildsmith and Fiona J. Spence13.1 Introduction 26913.2 Microarray experiments 26913.3 Data analysis 27313.4 Recent examples of microarray applications 28413.5 Conclusions 28413.6 Acknowledgements 28413.7 References 28413.8 Further Reading 28613.9 Useful websites 28614 Comparative Genomic Hybridisation in Pathology 287Marjan M. Weiss, Mario A.J.A. Hermsen, Antoine Snijders, Horst Buerger, Werner Boecker, Ernst J. Kuipers, Paul J. van Diest andGerritA.Meijer14.1 Introduction 28714.2 Technique 28914.3 Data analysis 29214.4 Applications 29314.5 Clinical applications 29914.6 Screening for chromosomal abnormalities in fetal and neonatal genomes 29914.7 Future perspectives 30014.8 Acknowledgements 30114.9 References 30115 DNA Sequencing and the Human Genome Project 307Philip Bennett15.1 Introduction 30715.2 DNA sequencing: the basics 30815.3 Applications of DNA sequencing 31815.4 The Human Genome Project 32015.5 References 32715.6 Further Reading 32715.7 Useful websites 32816 Monoclonal Antibodies: The Generation and Application of ‘Tools of the Trade’ Within Biomedical Science 329Paul N. Nelson, S. Jane Astley and Philip Warren16.1 Introduction 32916.2 Antibodies and antigens 33116.3 Polyclonal antibodies 33216.4 Monoclonal antibody development 33316.5 Monoclonal antibody variants 33816.6 Monoclonal antibody applications 34116.7 Therapy 34516.8 Specific applications 34616.9 Conclusions 34716.10 Acknowledgements 34716.11 References 34717 Proteomics 351Kathryn Lilley, Azam Razzaq and Michael J. Deery17.1 Introduction 35117.2 Definitions and applications 35217.3 Stages in proteome analysis 35217.4 Future directions 36817.5 References 368Index 371

John Crocker is the editor of Molecular Biology in Cellular Pathology, published by Wiley. Paul G. Murray is the editor of Molecular Biology in Cellular Pathology, published by Wiley.

• Preface xiiiPreface to Molecular Biology in Histopathology xvList of Contributors xvii1 Blotting Techniques: Methodology and Applications 1Fiona Watson and C. Simon Herrington1.1 Introduction 11.2 Blotting techniques 11.3 References 152 In-situ Hybridisation in Histopathology 19Gerald Niedobitek and Hermann Herbst2.1 Introduction 192.2 Experimental conditions 202.3 Probes and labels 232.4 Controls and pitfalls 272.5 Double-labelling 292.6 Increasing the sensitivity of ISH 312.7 What we do in our laboratories 332.8 Applications of ISH: examples 352.9 Perspective 392.10 References 403 DNA Flow Cytometry 49M.G. Ormerod3.1 Introduction 493.2 Definitions and terms 493.3 Dye used for DNA analysis 503.4 Sample preparation for DNA analysis 523.5 Analysis of the DNA histogram 533.6 Quality control 533.7 Computer analysis of the DNA histogram 553.8 Multiparametric measurement 573.9 Acknowledgements 593.10 References 594 Interphase Cytogenetics 61Sara A. Dyer and Jonathan J. Waters4.1 Introduction 614.2 Interphase cytogenetics 624.3 Applications 674.4 Conclusion 764.5 References 775 Oncogenes 79Fiona Macdonald5.1 Introduction 795.2 Identification of the oncogenes 795.3 Functions of the proto-oncogenes 805.4 Mechanism of oncogene activation 895.5 Oncogenes in colorectal cancer 915.6 Oncogenes in breast cancer 945.7 Oncogenes in lung cancer 955.8 Oncogenes in haematological malignancies 965.9 Other cancers 995.10 Conclusion 1005.11 References 1006 Molecular and Immunological Aspects of Cell Proliferation 105Karl Baumforth and John Crocker6.1 The cell cycle and its importance in clinical pathology 1056.2 Molecular control of the cell cycle 1086.3 Cell cycle control 1116.4 The cell cycle and cancer 1126.5 Immunocytochemical markers of proliferating cells 1156.6 References 1336.7 Further Reading 1357 Interphase Nucleolar Organiser Regions in Tumour Pathology 137Massimo Derenzini, Davide Treré, Marie-Françoise O’Donohue and Dominique Ploton7.1 Introduction 1377.2 The AgNORs 1387.3 NOR silver-staining 1427.4 Quantitative AgNOR analysis 1457.5 AgNORs as a parameter of the level of cell proliferation 1467.6 Application of the AgNOR technique to tumour pathology 1477.7 What future for AgNORs in tumour pathology? 1517.8 References 1528 Apoptosis and Cell Senescence 153Lee B. Jordan and David J. Harrison8.1 Introduction 1538.2 Apoptosis 1538.3 Cell senescence 1748.4 Summary 1788.5 References 1799 The Polymerase Chain Reaction 193Timothy Diss9.1 Introduction 1939.2 Principles 1949.3 Analysis of products 1979.4 Rt-pcr 1999.5 Quantitative PCR 2009.6 DNA and RNA extraction 2009.7 Correlation of the PCR with morphology 2019.8 Problems 2029.9 Applications 2029.10 Diagnostic applications 2039.11 Infectious diseases 2099.12 Identity 2099.13 The future 2109.14 References 2109.15 Online information 21210 Laser Capture Microdissection: Techniques and Applications in the Molecular Analysis of the Cancer Cell 213Amanda Dutton, Victor Lopes and Paul G. Murray10.1 Introduction 21310.2 The principle of LCM 21410.3 Technical considerations 21610.4 Advantages and disadvantages of LCM 21710.5 Applications of LCM 22210.6 Future perspectives 22910.7 Acknowledgements 22910.8 References 22911 The In-situ Polymerase Chain Reaction 233John J. O’Leary, Cara Martin and Orla Sheils11.1 Introduction 23311.2 Overview of the methodology 23411.3 In-cell PCR technologies 23511.4 In-cell amplification of DNA 23811.5 Detection of amplicons 24211.6 Reaction, tissue and detection controls for use with in-cell DNA PCR assays 24311.7 In-cell RNA amplification 24411.8 Problems encountered with in-cell PCR amplification 24611.9 Amplicon diffusion and back diffusion 24711.10 Future work with in-cell PCR-based assays 24711.11 References 24912 TaqMan® Technology and Real-Time Polymerase Chain Reaction 251John J. O’Leary, Orla Sheils, Cara Martin, and Aoife Crowley12.1 Introduction 25112.2 Probe technologies 25212.3 TaqMan® probe and chemistry (first generation) 25412.4 Second generation TaqMan® probes 25612.5 Hybridisation 25812.6 TaqMan® PCR conditions 25912.7 Standards for quantitative PCR 26012.8 Interpretation of results 26112.9 End-point detection 26212.10 Real-time detection 26312.11 Relative quantitation 26312.12 Reference genes 26412.13 Specific TaqMan® PCR applications 26512.14 References 26813 Gene Expression Analysis Using Microarrays 269Sophie E. Wildsmith and Fiona J. Spence13.1 Introduction 26913.2 Microarray experiments 26913.3 Data analysis 27313.4 Recent examples of microarray applications 28413.5 Conclusions 28413.6 Acknowledgements 28413.7 References 28413.8 Further Reading 28613.9 Useful websites 28614 Comparative Genomic Hybridisation in Pathology 287Marjan M. Weiss, Mario A.J.A. Hermsen, Antoine Snijders, Horst Buerger, Werner Boecker, Ernst J. Kuipers, Paul J. van Diest andGerritA.Meijer14.1 Introduction 28714.2 Technique 28914.3 Data analysis 29214.4 Applications 29314.5 Clinical applications 29914.6 Screening for chromosomal abnormalities in fetal and neonatal genomes 29914.7 Future perspectives 30014.8 Acknowledgements 30114.9 References 30115 DNA Sequencing and the Human Genome Project 307Philip Bennett15.1 Introduction 30715.2 DNA sequencing: the basics 30815.3 Applications of DNA sequencing 31815.4 The Human Genome Project 32015.5 References 32715.6 Further Reading 32715.7 Useful websites 32816 Monoclonal Antibodies: The Generation and Application of ‘Tools of the Trade’ Within Biomedical Science 329Paul N. Nelson, S. Jane Astley and Philip Warren16.1 Introduction 32916.2 Antibodies and antigens 33116.3 Polyclonal antibodies 33216.4 Monoclonal antibody development 33316.5 Monoclonal antibody variants 33816.6 Monoclonal antibody applications 34116.7 Therapy 34516.8 Specific applications 34616.9 Conclusions 34716.10 Acknowledgements 34716.11 References 34717 Proteomics 351Kathryn Lilley, Azam Razzaq and Michael J. Deery17.1 Introduction 35117.2 Definitions and applications 35217.3 Stages in proteome analysis 35217.4 Future directions 36817.5 References 368Index 371

John Crocker is the editor of Molecular Biology in Cellular Pathology, published by Wiley. Paul G. Murray is the editor of Molecular Biology in Cellular Pathology, published by Wiley.

• Preface xiiiPreface to Molecular Biology in Histopathology xvList of Contributors xvii1 Blotting Techniques: Methodology and Applications 1Fiona Watson and C. Simon Herrington1.1 Introduction 11.2 Blotting techniques 11.3 References 152 In-situ Hybridisation in Histopathology 19Gerald Niedobitek and Hermann Herbst2.1 Introduction 192.2 Experimental conditions 202.3 Probes and labels 232.4 Controls and pitfalls 272.5 Double-labelling 292.6 Increasing the sensitivity of ISH 312.7 What we do in our laboratories 332.8 Applications of ISH: examples 352.9 Perspective 392.10 References 403 DNA Flow Cytometry 49M.G. Ormerod3.1 Introduction 493.2 Definitions and terms 493.3 Dye used for DNA analysis 503.4 Sample preparation for DNA analysis 523.5 Analysis of the DNA histogram 533.6 Quality control 533.7 Computer analysis of the DNA histogram 553.8 Multiparametric measurement 573.9 Acknowledgements 593.10 References 594 Interphase Cytogenetics 61Sara A. Dyer and Jonathan J. Waters4.1 Introduction 614.2 Interphase cytogenetics 624.3 Applications 674.4 Conclusion 764.5 References 775 Oncogenes 79Fiona Macdonald5.1 Introduction 795.2 Identification of the oncogenes 795.3 Functions of the proto-oncogenes 805.4 Mechanism of oncogene activation 895.5 Oncogenes in colorectal cancer 915.6 Oncogenes in breast cancer 945.7 Oncogenes in lung cancer 955.8 Oncogenes in haematological malignancies 965.9 Other cancers 995.10 Conclusion 1005.11 References 1006 Molecular and Immunological Aspects of Cell Proliferation 105Karl Baumforth and John Crocker6.1 The cell cycle and its importance in clinical pathology 1056.2 Molecular control of the cell cycle 1086.3 Cell cycle control 1116.4 The cell cycle and cancer 1126.5 Immunocytochemical markers of proliferating cells 1156.6 References 1336.7 Further Reading 1357 Interphase Nucleolar Organiser Regions in Tumour Pathology 137Massimo Derenzini, Davide Treré, Marie-Françoise O’Donohue and Dominique Ploton7.1 Introduction 1377.2 The AgNORs 1387.3 NOR silver-staining 1427.4 Quantitative AgNOR analysis 1457.5 AgNORs as a parameter of the level of cell proliferation 1467.6 Application of the AgNOR technique to tumour pathology 1477.7 What future for AgNORs in tumour pathology? 1517.8 References 1528 Apoptosis and Cell Senescence 153Lee B. Jordan and David J. Harrison8.1 Introduction 1538.2 Apoptosis 1538.3 Cell senescence 1748.4 Summary 1788.5 References 1799 The Polymerase Chain Reaction 193Timothy Diss9.1 Introduction 1939.2 Principles 1949.3 Analysis of products 1979.4 Rt-pcr 1999.5 Quantitative PCR 2009.6 DNA and RNA extraction 2009.7 Correlation of the PCR with morphology 2019.8 Problems 2029.9 Applications 2029.10 Diagnostic applications 2039.11 Infectious diseases 2099.12 Identity 2099.13 The future 2109.14 References 2109.15 Online information 21210 Laser Capture Microdissection: Techniques and Applications in the Molecular Analysis of the Cancer Cell 213Amanda Dutton, Victor Lopes and Paul G. Murray10.1 Introduction 21310.2 The principle of LCM 21410.3 Technical considerations 21610.4 Advantages and disadvantages of LCM 21710.5 Applications of LCM 22210.6 Future perspectives 22910.7 Acknowledgements 22910.8 References 22911 The In-situ Polymerase Chain Reaction 233John J. O’Leary, Cara Martin and Orla Sheils11.1 Introduction 23311.2 Overview of the methodology 23411.3 In-cell PCR technologies 23511.4 In-cell amplification of DNA 23811.5 Detection of amplicons 24211.6 Reaction, tissue and detection controls for use with in-cell DNA PCR assays 24311.7 In-cell RNA amplification 24411.8 Problems encountered with in-cell PCR amplification 24611.9 Amplicon diffusion and back diffusion 24711.10 Future work with in-cell PCR-based assays 24711.11 References 24912 TaqMan® Technology and Real-Time Polymerase Chain Reaction 251John J. O’Leary, Orla Sheils, Cara Martin, and Aoife Crowley12.1 Introduction 25112.2 Probe technologies 25212.3 TaqMan® probe and chemistry (first generation) 25412.4 Second generation TaqMan® probes 25612.5 Hybridisation 25812.6 TaqMan® PCR conditions 25912.7 Standards for quantitative PCR 26012.8 Interpretation of results 26112.9 End-point detection 26212.10 Real-time detection 26312.11 Relative quantitation 26312.12 Reference genes 26412.13 Specific TaqMan® PCR applications 26512.14 References 26813 Gene Expression Analysis Using Microarrays 269Sophie E. Wildsmith and Fiona J. Spence13.1 Introduction 26913.2 Microarray experiments 26913.3 Data analysis 27313.4 Recent examples of microarray applications 28413.5 Conclusions 28413.6 Acknowledgements 28413.7 References 28413.8 Further Reading 28613.9 Useful websites 28614 Comparative Genomic Hybridisation in Pathology 287Marjan M. Weiss, Mario A.J.A. Hermsen, Antoine Snijders, Horst Buerger, Werner Boecker, Ernst J. Kuipers, Paul J. van Diest andGerritA.Meijer14.1 Introduction 28714.2 Technique 28914.3 Data analysis 29214.4 Applications 29314.5 Clinical applications 29914.6 Screening for chromosomal abnormalities in fetal and neonatal genomes 29914.7 Future perspectives 30014.8 Acknowledgements 30114.9 References 30115 DNA Sequencing and the Human Genome Project 307Philip Bennett15.1 Introduction 30715.2 DNA sequencing: the basics 30815.3 Applications of DNA sequencing 31815.4 The Human Genome Project 32015.5 References 32715.6 Further Reading 32715.7 Useful websites 32816 Monoclonal Antibodies: The Generation and Application of ‘Tools of the Trade’ Within Biomedical Science 329Paul N. Nelson, S. Jane Astley and Philip Warren16.1 Introduction 32916.2 Antibodies and antigens 33116.3 Polyclonal antibodies 33216.4 Monoclonal antibody development 33316.5 Monoclonal antibody variants 33816.6 Monoclonal antibody applications 34116.7 Therapy 34516.8 Specific applications 34616.9 Conclusions 34716.10 Acknowledgements 34716.11 References 34717 Proteomics 351Kathryn Lilley, Azam Razzaq and Michael J. Deery17.1 Introduction 35117.2 Definitions and applications 35217.3 Stages in proteome analysis 35217.4 Future directions 36817.5 References 368Index 371