Molecular Morphology in Human Tissues: Techniques and Applications (Advances in Pathology, Microscopy, & Molecular Morphology)

Front cover......Page 1
DEDICATION......Page 6
FOREWORD......Page 8
PREFACE......Page 10
THE EDITORS......Page 14
ACKNOWLEDGMENTS......Page 16
CONTRIBUTORS......Page 18
CONTENTS......Page 22
1.2 SINGLE IMMUNOSTAINING ON CONSECUTIVE SECTIONS......Page 24
1.3 DOUBLE OR TRIPLE IMMUNOSTAINING ON THE SAME SECTION 1.3.1 Bright-Field Light Microscopy......Page 25
1.3.2 Fluorescence Microscopy......Page 32
1.4.2 Single–PAP Combined with Triple Immunofluorescence Staining......Page 41
1.6 PROTOCOLS I, II, III, IV, V, AND VI......Page 42
1.6.4 Protocol IV: Double Immunofluorescence Staining with Primary Antibodies Raised in Different Animal Species Using the Catalyzed Reporter Deposition (CARD) Method and an Indirect Method [70–73]......Page 43
1.6.6 Protocol VI: Double Immunofluorescence Staining with Primary Antibodies Raised in the Same Animal Species (e.g., Rabbit) Using a Double Catalyzed Reporter Deposition (CARD) Method [73, 85]......Page 44
2.1.1 Serial Sections......Page 50
2.1.2 Co-Localization: The Definition......Page 51
2.1.3 Aims for Double and Triple Staining......Page 52
2.1.4 The Demands for Successful Multiple Staining......Page 53
2.2.1 Sequential Techniques......Page 55
2.2.2 Simultaneous Technique:......Page 57
2.2.3 Simultaneous Technique:......Page 58
2.2.4 Multistep Techniques:......Page 60
2.3 MULTIPLE STAINING STRATEGY AND CONTROLS 2.3.1 General Strategy......Page 62
2.3.2 Double Staining Control Experiments......Page 63
2.4 SELECTING A CONCEPT, DETECTION SYSTEM, AND COLOR COMBINATION FOR DOUBLE STAINING 2.4.1 Selecting a Double Staining Concept......Page 64
2.4.2 Selecting Two Individual Detection Systems......Page 66
2.4.3 Selecting Antibody Tracers and Color Combinations......Page 67
2.4.4 Double Staining: Possibilities for Computerized Imaging of Two Individual Colors?......Page 69
2.5 ADDITIONAL PRACTICAL TIPS AND COMMON MISTAKES WITH DOUBLE STAINING 2.5.1 Which Antigen, Which Color?......Page 70
2.5.2 Be Aware Of......Page 71
2.5.4 Order of Enzymatic Development......Page 72
2.5.6 Restrictions for the Visualization of a Mixed Color at Sites of Co-Localization Using Immunoenzyme Double Staining Techniques......Page 73
2.6.2 Protocol B for General Immunohistochemistry: Cryostat Sections......Page 74
2.6.4 Protocol D for Sequential Immunoenzyme Double Staining......Page 75
2.6.6 Protocol F for Simultaneous Immunoenzyme Double Staining:......Page 76
2.6.8 Protocol H for Multistep Immunoenzyme Double Staining: Indirect/Direct Concept......Page 77
2.6.10 Suggested Immunoreagent List......Page 79
2.6.10 Suggested Immunoreagent List (continued)......Page 80
2.6.12 Commercial Visualization Systems HRP – AEC: DakoCytomation AEC+......Page 82
 3.1 INTRODUCTION......Page 88
3.2 PROTOCOLS Enzyme-Based Fluorescence Amplification 3.2.1 Protocol A: TSA IHC......Page 92
3.2.2 Protocol B: TSA ISH......Page 93
3.2.3 Protocol C: ELF-97 IHC......Page 94
3.2.4 Protocol D: Double IHC with ELF 97......Page 95
3.2.5 Protocol E: TSA-ELF-97 ISH......Page 96
3.2.6 Protocol F: Double ISH with TSA-ELF-97......Page 97
3.3 RESULTS......Page 98
3.4 TECHNICAL HINTS AND DISCUSSION 3.4.1 Choice of TSA Detection Methods......Page 99
3.4.4 ELF Detection Protocols......Page 100
4.1 INTRODUCTION: WHY CLUSTER LABELS?......Page 104
4.2.2 Autometallography: Silver Enhancement......Page 105
4.2.4 Combined Fluorescent and Gold Probes......Page 107
4.2.5 Nanogold Autometallography......Page 108
4.2.7 Enzyme Metallography......Page 109
4.3 SPECIFIC GOLD CLUSTER PROBES AND METHODS 4.3.1 LI Silver Enhancement of Nanogold Conjugates in Light Microscopy......Page 110
4.3.2 Silver Acetate Autometallography of Nanogold Conjugates for LM Nanogold-Silver Immunostaining......Page 111
4.3.3 Gold Enhancement for Light Microscopy......Page 112
4.3.5 Gold Cluster Labels for Chromogenic Microarray and Biochip Detection......Page 114
4.3.6 Gold Cluster-Labeled Lipids......Page 115
4.3.7 Localization and Detection of DNA: In Situ Hybridization and In......Page 116
4.3.8 Combined Fluorescent and Gold Labeling Applications......Page 117
5.1 INTRODUCTION......Page 124
5.3 MATERIALS AND PROTOCOL PROCEDURE 5.3.1 Reagent Sources......Page 126
5.3.5 Scoring of Preparations and Interpretation......Page 127
6.1 INTRODUCTION AND BRIEF HISTORICAL OVERVIEW......Page 130
6.2 TYRAMIDE-AMPLIFIED NANOGOLD-SILVER/GOLD ISH......Page 131
6.2.2 The GOLDFISH Procedure (Gold-Facilitated Autometallographic......Page 136
6.2.3 Autometallography: General Guidelines......Page 138
6.3 DISCUSSION......Page 141
6.4 OUTLOOK......Page 142
7.1 INTRODUCTION......Page 146
7.2 MATERIALS AND PROTOCOLS 7.2.1 DNA Isolation......Page 148
7.2.3 DNA Labeling......Page 149
7.2.4 Hybridization......Page 151
7.3 TROUBLESHOOTING......Page 155
8.1 INTRODUCTION......Page 156
8.2 PROTOCOL A: FROZEN TISSUE SECTIONS 8.2.1 Materials and Reagents......Page 158
8.3 PROTOCOL B: PARAFFIN-EMBEDDED TISSUE SECTIONS 8.3.1 Materials and Reagents......Page 159
8.4 PROTOCOL C: CULTURED CELLS 8.4.1 Materials and Reagents......Page 160
8.5 RESULTS......Page 161
8.6.1 Points to Notice of Southwestern Histochemistry......Page 164
8.6.4 Concentration of Probe and Salt......Page 165
8.6.7 Prospects......Page 166
9 High Throughput Morphological Gene Expression Studies Using Automated mRNA In Situ Hybridization Applications and Tissue Microarrays for Post-Genomic and Clinical Research......Page 170
9.2 IMMUNOHISTOCHEMISTRY VS. ISOTOPIC MRNA ISH APPLICATIONS......Page 171
9.5 PROTOCOLS 9.5.1 Materials and Methods......Page 172
9.6 RESULTS 9.6.1 Tissue Fixation Conditions for AmpMap™ mRNA ISH......Page 174
9.7 TECHNICAL HINTS AND DISCUSSION......Page 175
9.8.6 Hybridization Temperature......Page 177
9.8.7 Signal Amplification......Page 178
10.1 INTRODUCTION......Page 180
10.2.2 Isolation and Fixation of Specimens......Page 181
10.2.3 Preparation of Digoxigenin- Labeled Anti-Sense cRNA......Page 184
10.2.5 Manual ISH......Page 185
10.2.6 Automated ISH......Page 186
10.3.1 Manual vs. Automated In Situ Hybridization......Page 188
11.1 INTRODUCTION......Page 190
11.2 TECHNICAL ASPECTS OF FISH......Page 192
11.3 LIQUID-BASED THIN-LAYER PREPARATION AND ITS SUITABILITY FOR FISH......Page 193
11.4.2 Reagent Preparation......Page 194
11.4.3 Other Laboratory Supplies......Page 196
11.4.7 Slide Pretreatment......Page 197
11.4.9 Rapid Wash Procedure......Page 198
11.5 PITFALLS AND TROUBLESHOOTING......Page 199
11.6 CONCLUSIONS......Page 200
12.1 INTRODUCTION......Page 202
12.2.1 Kary Mullis’ Invention: The Polymerase Chain Reaction......Page 204
12.2.3 Oligonucleotide Primers......Page 205
12.2.4 Denaturation and Annealing......Page 206
12.2.6 Second Thermal Cycle......Page 207
12.2.8 Reverse Transcription: Making cDNA from RNA......Page 208
12.2.9 How to Design Primers......Page 210
12.2.12 Sources for Sequence Data and Computerized Design of Primers......Page 212
12.3.1 Preparation of Tissues......Page 213
12.3.2 In Situ PCR......Page 214
13.1 INTRODUCTION......Page 222
13.2 MATERIAL AND REAGENTS 13.2.1 Fixation......Page 223
13.2.3 Embedding in Epon......Page 224
13.4 STAINING PROCEDURE 13.4.1 Removing the Resin......Page 225
13.4.3 Immunohistochemistry for BrdU......Page 226
13.4. 4 Immunohistochemistry with Transglutaminase......Page 227
13.5 RESULTS......Page 228
13.6 DISCUSSION......Page 230
14.1 INTRODUCTION......Page 232
14.2 METHODOLOGY 14.2.1 General Features......Page 233
14.2.2 The DOM System......Page 234
14.2.3 Commercial Sources......Page 235
14.3.1 Applications in Immunohistochemistry......Page 240
14.3.2 Applications in Molecular Morphology......Page 241
14.5 THE FUTURE......Page 242
15.1 INTRODUCTION......Page 248
15.3.1 Protocol A: Preparation of IgG-Opsonized Glass Beads......Page 249
15.3.2 Protocol B: Preparation of IgG-Opsonized Sheep Red Blood Cells (SRBC)......Page 251
15.3.3 Protocol C: Transient Transfection of RAW Cells......Page 252
15.4.1 Rate of Ingestion......Page 254
15.5 PROTOCOLS FOR THE VIRAL TRANSDUCTION OF HUMAN AND MOUSE MACROPHAGES......Page 256
15.5.1 Protocol D: Viral Plasmid Construction......Page 258
15.5.2 Protocol E: Transduction of Human and Mouse Cells......Page 260
15.5.3 Protocol F: Characterization of Transduced Cells by Dual Parameter Flow Cytometry......Page 262
15.5.4 Protocol for TNF-α Production......Page 263
15.6 DISCUSSION......Page 265
Visualization of Signal Transduction Pathways in Real Time......Page 266
16.1 INTRODUCTION......Page 268
16.2.2 Protocol B: Immunogold-Silver Staining......Page 269
16.2.3 Protocol C: Staining FIZ Slide with Ponceau 3R......Page 270
16.4 TECHNICAL HINTS AND DISCUSSION 16.4.1 Incubation Time of Film In Situ Zymography......Page 271
16.4.3 Estimation of Inhibition Effects in Film In Situ Zymography......Page 272
16.4.4 Immunohistochemistry......Page 273
17.1 INTRODUCTION......Page 276
17.2.2 The Challenge: Ethical Considerations on Immunization Protocols......Page 277
17.3.2 Ethical Considerations Related to Monoclonal Antibodies......Page 281
17.4 RECOMBINANT ANTIBODIES......Page 283
17.4.2 Advantages of Phage Display- Based Recombinant Antibody Technology......Page 284
17.4.3 Drawbacks of Phage Display- Based Recombinant Antibody Technology......Page 285
17.4.4 Antibodies Produced in Plants......Page 286
17.6 LEGAL ASPECTS 17.6.1 General Legal Aspects......Page 287
17.6.2 Legal Aspects in the United States......Page 288
17.6.3 Legal Aspects in the European Union......Page 289
17.6.6 Legal Aspects in Switzerland......Page 291
17.7 CONCLUDING REMARKS......Page 293
18.1 INTRODUCTION......Page 298
18.2 PRESERVATION OF TISSUES......Page 300
18.4 THE ASSAY......Page 301
18.5 EVALUATION OF RESULTS......Page 302
18.7.1 Quality Assurance......Page 303
18.8 THE FUTURE......Page 312
Index......Page 318
Back cover......Page 344