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Immunocytochemistry : a practical guide for biomedical research / Richard W. Burry. — New York ; London : Springer, c2010. – (58.691/B972) |
Contents
Contents
1 Introduction
What Is Immunocytochemistry?
What Can Immunocytochemistry Tell Us?
An Outline of the Immunocytochemistry Procedure
What Is Included in This Book?
2 Antibodies
Introduction
Antibody Molecules
Making Antibodies
Talking About Antibodies
Finding and Getting Antibodies
Choice of Primary (1°) Antibodies
Antibodies Handling and Storing
3 Sample Preparation/Fixation
Introduction
Fixation Theory
Chemical Fixatives
Vehicle
Applying Fixatives
Protocol - Fixation
Procedure
4 Tissue Sectioning 29
Introduction 29
Embedding Tissue by Freezing 30
Theory of Freezing Tissue
Freezing Tissue
Cryostat Sectioning
Tissue Processing.
Vibratome， Freezing Microtome， and Microwave
Fresh Frozen Tissue
Embedding Tissue with Paraffin
Cryostat Protocol
5 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
6 Labels for Antibodies
Introduction
Fluorescence Theory
Four Generations of Fluorescent Labels
Immunocytochemistry Fluorophores and Flow Cytometry
Enzyme Theory
Enzyme Substrates
Particulate Label
Choice of Fluorescent or Enzymes for Immunocytochemistry
7 Application Methods
Introduction
Direct Immunocytochemistry
Indirect Immunocytochemistry
Avidin-Biotin Molecules
Direct Avidin-Biotin Immunocytochemistry
Indirect Avidin-Biotin Immunocytochemistry
Avidin-Biotin Complex (ABC) Immunocytochemistry
Tyramide Signal Amplification (TSA) Immunocytochemistry
ABC with TSA
8 Controls
Introduction
Three Immunocytochemistry Controls
9 Method and Label Decision
Introduction
Choose Application Label and Method
Experimental Design Chart
10 Single Antibody Procedure
Introduction
Experimental Design Chart
Incubation Conditions
Antibody Dilutions
Antibody Dilution Matrix
2° Antibody Controls
Rinses
Mounting Media
Final Procedure
11 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
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
Combine Two 1° Antibodies from the Same
Species with Zenon
Experimental Design Chart for the Same Species with Zenon 130
Design the Antibody Control for the Same Species with Zenon 133
Final Procedure for Two 1 ° Antibody from the Same Species with Zenon
13 Fluorescent Microscopy and Imaging
Introduction
Filter Sets in Fluorescence Microscopy
Fluorescent Bleed-Through
Fluorescence Quench and Photobleach
Image Parameters - Contrast and Pixel Saturation
Ethics of Image Manipulation
14 Troubleshooting
Introduction
Procedural Errors
Method of Troubleshooting
Troubleshooting Unique to Multiple Primary Antibodies
Bad Antibodies
15 Electron Microscopic Immunocytochemistry
Protocol - Pre-embedding Electron Microscopic Immunocytochemistry
Introduction
Need for Electron Microscopic Immunocytochemistry
Pre-embedding Electron Microscopic Immunocytochemistry
Postembedding Electron Microscopic Immunocytochemistry
Choice of a Method
Protocol - Pre-embedding Electron Microscopic Immunocytochemistry
Solutions
Appendix
References
Glossary
Index
