Contents Introduction Antibodies Sample Preparation/Fixation Tissue Sectioning

Transcription

1 Contents 1 Introduction... 1 What Is Immunocytochemistry? What Can Immunocytochemistry Tell Us? An Outline of the Immunocytochemistry Procedure What Is Included in This Book? Antibodies... 7 Introduction Antibody Molecules Making Antibodies Talking About Antibodies Finding and Getting Antibodies Choice of Primary (1 ) Antibodies Antibodies Handling and Storing Recommended Storage Freezer, 20 C Recommended Storage Refrigerator, 4 C Sample Preparation/Fixation Introduction Fixation Theory ChemicalFixatives Vehicle ApplyingFixatives DissectingtheAreaofInterest Protocol Fixation Components for Paraformaldehyde Fixative Procedure Perfusion Procedure PerfusionEquipment Drop-in-Fixation Tissue Sectioning Introduction Embedding Tissue by Freezing ix

2 x Contents Theory of Freezing Tissue Freezing Tissue CryostatSectioning TissueProcessing Vibratome, Freezing Microtome, and Microwave Fresh Frozen Tissue Embedding Tissue with Paraffin Cryostat Protocol Blocking and Permeability Introduction Nonspecific Antibody Binding to Tissue and Cells Blocking for Nonspecific Antibody Binding Permeabilize Tissue and Cells to Allow Antibody Penetration Effects of Blocking Agents on Antibody Penetration Combined Incubation Step Labels for Antibodies Introduction Fluorescence Theory Four Generations of Fluorescent Labels Immunocytochemistry Fluorophores and Flow Cytometry Choosing Fluorochromes Enzyme Theory Enzyme Substrates Particulate Label Choice of Fluorescent or Enzymes for Immunocytochemistry Application Methods Introduction Direct Immunocytochemistry Direct Immunocytochemistry Advantages Direct Immunocytochemistry Disadvantages Indirect Immunocytochemistry Indirect Immunocytochemistry Advantages Indirect Immunocytochemistry Disadvantages Avidin Biotin Molecules Direct Avidin Biotin Immunocytochemistry Direct Avidin Biotin Method Advantages Direct Avidin Biotin Method Disadvantages Indirect Avidin Biotin Immunocytochemistry Indirect Avidin Biotin Advantages Indirect Avidin Biotin Disadvantages Avidin Biotin Complex (ABC) Immunocytochemistry Avidin Biotin Complex (ABC) Advantages Avidin Biotin Complex (ABC) Disadvantages

3 Contents xi Tyramide Signal Amplification (TSA) Immunocytochemistry Tyramide Signal Amplification Advantages Tyramide Signal Amplification Disadvantages ABCwithTSA ABC with TSA Advantages ABC with TSA Disadvantages Controls Introduction Three Immunocytochemistry Controls Antibody Controls Antibody Controls LabelingControls Method and Label Decision Introduction Choose Application Label and Method Experimental Design Chart Single Antibody Procedure Introduction Experimental Design Chart Incubation Conditions Antibody Dilutions Antibody Dilution Matrix Antibody Controls Rinses MountingMedia Final Procedure Steps in a Single 1 Antibody Indirect Immunocytochemistry Experiment Steps in a Single 1 Antibody Immunocytochemistry Experiment for Ag A Multiple Antibodies Different Species Introduction Combining Two 1 Antibody Incubations Experimental Design Chart Designing 2 Antibody Controls Rules for Multiple Label Experiments Complete Final Procedure (D) Block and Permeabilize (E) Rinse after Block and Permeabilize (F) 1 Antibodies (G) Rinse After 1 Antibody (H) 2 Antibody (I) Rinse After 2 Antibody

4 xii Contents 12 Multiple Antibodies from the Same Species Introduction Combine Two 1 Antibodies from the Same Species with Block-Between Method Experimental Design Chart for Block-Between Method Design the 2 Antibody Control for the Same Species with Block-Between Final Procedure for Two 1 Antibody Same Species with Block-Between (A) Prepare Cell Culture (B)FixCulture (C) Block and Permeabilize (D) Rinse After Block and Permeabilize (E) Incubate First 1 Antibody (F) Rinse After First 1 Antibody (G) Incubate First 2 Antibody (H) Rinse After First 2 Antibody (I) Block Antibodies in First Set (J) Incubate Second 1 Antibody (K) Rinse After Second 1 Antibody (L) Incubate Second 2 Antibody (M) Rinse After Second 2 Antibody (N) Mount Coverslip (O) Examine in Microscope (P)EvaluateResults Combine Two 1 Antibodies from the Same Species with Zenon Experimental Design Chart for the Same Species with Zenon Design the Antibody Control for the Same Species with Zenon Final Procedure for Two 1 Antibody from the Same Species with Zenon (A) Prepare Cell Culture (B)FixCulture (C) Block and Permeabilize (D) Rinse after Block and Permeabilize (E) Prepare the Zenon Reagents (F) Incubate with Labeled Antibody(ies) (G) Rinse After Antibody Incubation (H)Fixwith4%Paraformaldehyde (I) Rinse after Antibody Incubation (J) Mount Coverslip (K) Examine in Microscope (L)EvaluateResults

5 Contents xiii 13 Fluorescent Microscopy and Imaging Introduction Filter Sets in Fluorescence Microscopy Fluorescent Bleed-Through Fluorescence Quench and Photobleach ImageParameters ContrastandPixelSaturation EthicsofImageManipulation Do DoNot Troubleshooting Introduction Procedural Errors Method of Troubleshooting CaseNo CaseNo CaseNo CaseNo CaseNo Troubleshooting Unique to Multiple Primary Antibodies Bad Antibodies Bad 1 Antibodies Bad 2º Antibodies Electron Microscopic Immunocytochemistry Protocol Pre-embedding Electron Microscopic Immunocytochemistry 175 Introduction Need for Electron Microscopic Immunocytochemistry Pre-embedding Electron Microscopic Immunocytochemistry Postembedding Electron Microscopic Immunocytochemistry ChoiceofaMethod Advantages and Disadvantages Protocol Pre-embedding Electron Microscopic Immunocytochemistry 185 Solutions Stock Solutions to Make Ahead and Store Solutions Made on the First Day of the Experiment NPG Silver Enhancement Solution and Silver Lactate TestStrip Appendix References Glossary Index

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