| Foreword | 5 |
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| Preface | 7 |
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| Contents | 9 |
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| Contributors | 11 |
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| Research Governance | 15 |
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| INTRODUCTION | 15 |
| WHY WE NEED RESEARCH GOVERNANCE | 16 |
| HISTORY OF DEVELOPMENT OF RESEARCH GOVERNANCE1,2 | 16 |
| SALIENT FEATURES OF THE RESEARCH GOVERNANCE FRAMEWORK | 19 |
| CONCLUSION | 20 |
| References | 20 |
| Designing Health Studies | 22 |
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| ESSENTIALS OF A STUDY DESIGN Specifying the Research Question | 22 |
| Selection of Subjects | 22 |
| Specifying the Primary Outcome | 22 |
| Inclusion of a Control Group | 23 |
| Confounding | 23 |
| Sample Size | 24 |
| Bias | 24 |
| Writing a Protocol | 25 |
| TWO TYPES OF STUDIES | 25 |
| Randomized Controlled Trials | 25 |
| Types of Trials | 26 |
| Observational Studies | 27 |
| References | 30 |
| Immunohistochemistry | 32 |
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| INTRODUCTION | 32 |
| BASIC IMMUNOHISTOCHEMISTRY | 32 |
| METHODOLOGY | 34 |
| Fixation | 35 |
| Antigen Retrieval | 35 |
| Antibodies | 36 |
| Staining Methods/Detection Systems | 37 |
| Enzyme Labels and Chromogens | 40 |
| Animal Tissue | 41 |
| TROUBLESHOOTING/OPTIMIZATION Optimizing Primary Antibodies | 41 |
| Endogenous Enzyme Activity | 42 |
| Endogenous Biotin | 43 |
| Washing | 43 |
| References | 43 |
| Cell Culturing: A Beginner’s Guide to Understanding the Basics of Cell Culturing | 45 |
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| INTRODUCTION | 45 |
| Primary Cells | 45 |
| Cell Lines | 45 |
| FUNDAMENTALS OF CELL CULTURING | 46 |
| What Happens When an Infection Occurs? | 46 |
| Media: Their Function in Cell Culturing | 47 |
| Support Cells | 48 |
| Incubation | 49 |
| CELL EXTRACTION | 49 |
| Handling Tissue Biopsies | 49 |
| Fibroblast Extraction | 49 |
| CONCLUSION | 51 |
| References | 51 |
| Flow Cytometry | 52 |
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| WHAT IS FLOW CYTOMETRY? | 52 |
| HISTORICAL ASPECTS OF FLOW CYTOMETRY AND THE FLOW CYTOMETER | 53 |
| FLUORESCENCE | 53 |
| LIGHT SCATTER | 55 |
| COMPONENTS OF THE MODERN FLOW CYTOMETER Fluidic System | 55 |
| Optical System and Analysis | 55 |
| Color Assignment | 56 |
| DATA ANALYSIS Histograms and Dot Plots | 57 |
| Gating and Negative Controls | 58 |
| PRINCIPLES OF FLOW CYTOMETRY SAMPLE PREPARATION | 60 |
| References | 60 |
| Western, Northern, and Southern Blotting | 62 |
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| INTRODUCTION: THE HISTORY OF SOUTHERN, NORTHERN, AND WESTERN BLOTTING | 62 |
| BASIC PRINCIPLES AND METHODS: ELECTROPHORESIS, GEL BLOTTING, AND DETECTION | 63 |
| Gel Electrophoresis: Size Separation | 63 |
| Sample Preparation: Southern and Northern | 63 |
| Sample Preparation: Western | 63 |
| Agarose Electrophoresis: Southern and Northern | 64 |
| Sodium Sodecylsulfate–Polyacrylamide Gel Electrophoresis: Western | 65 |
| Blotting: Transfer to Solid Support | 66 |
| Detection and Localization: Complementarity and Hybridization | 66 |
| Blocking | 68 |
| Hybridization | 68 |
| Washing | 68 |
| Detection | 68 |
| Stripping | 70 |
| KEY POINTS | 70 |
| References | 70 |
| Fluorescent In Situ Hybridization | 72 |
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| INTRODUCTION | 72 |
| BASIC PRINCIPLES | 72 |
| METHODOLOGICAL ASPECTS OF FISH Probe and Sample Preparation | 73 |
| DNA UNMASKING: PRETREATMENT AND DIGESTION De- waxing and Rehydration | 74 |
| Acid Permeabilization | 74 |
| Reducing Agents | 74 |
| Protease Digestion | 74 |
| Probe and Target DNA Denaturation | 75 |
| Probe and Target DNA Hybridization | 75 |
| POSTHYBRIDIZATION WASH AND VISUALIZATION Posthybridization Wash | 76 |
| Visualization and Scoring/Interpretation of Results | 76 |
| QUALITY ASSURANCE | 76 |
| Slide Pretreatment | 76 |
| Acid Permeabilization | 77 |
| Pretreatment (Reducing Agent) | 77 |
| Protease Digestion | 77 |
| Denaturation | 78 |
| Hybridization | 78 |
| Posthybridization Wash | 78 |
| Visualization and Scoring Slides | 79 |
| References | 79 |
| Quantitative Reverse Transcriptase Polymerase Chain Reaction | 80 |
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| INTRODUCTION | 80 |
| UNDERSTANDING THE FUNDAMENTALS | 81 |
| DISCUSSION OF METHODS TO QUANTIFY REAL-TIME POLYMERASE CHAIN REACTION RESULTS | 84 |
| CONTROLS IN QUANTITATIVE REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION: USE OF THE HO
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