Immunocytochemistry

Due to the dramatic increase in productivity in biomedical research, immunocytochemistry has emerged as a major technique. This book is the first practical guide to planning, performing and evaluating immunocytochemistry experiments.

Description:

In biomedical research, because of a dramatic increase in productivity, immunocytochemistry has emerged as a major technique. The proposed book will provide the first practical guide to planning, performing, and evaluating immunocytochemical experiments.

In today's graduate education the emphasis is on doing research and not on formal class work. Graduate students therefore lack the background in many essential techniques necessary to perform research in fields in which they were not trained. As director of a university core microscopy facility which sees students and faculty from dozens of laboratories each year, Dr. Burry has surmised the vast majority of these novice microscope users need considerable help. In an attempt to educate users, Dr. Burry has initiated immunocytochemistry seminars and workshops which serve to train people in this powerful research tool. The proposed book is an outgrowth of these presentations and conversations with, by now, hundreds of people who have asked for help.

The philosophy which separates this book from other books in this field is that it is practical, rather than academic. In looking at other important immunocytochemistry titles, the predominant orientation is academic, with the author attempting to comprehensively discuss the topic. For example, one book with sample preparation lists ten fixatives which can be used; however, only two such fixatives are commonly used today. In this particular title, the detailed discussion of old methods might be seen as important in establishing the author as an expert. By contrast, the approach for Burry's book would be to discuss methods based on what works in animal research laboratories today, and focus only on the most productive methods.

An additional distinction with this proposed book is the focus on animal research and not human pathology. There is a certification program for pathology technicians which requires them tolearn a set body of material based on processing human tissue for examination by a pathologist. Many of the books on immunocytochemistry aim at this large pathology user base. Due to historical reasons, pathology laboratories process human tissues in a specific way and embed the tissue in paraffin, as has been done for over a century. In the last ten years, the power of immunocytochemistry in clinical diagnosis has become clear and has accordingly been adapted to pathology. However, the extensive processing needed for paraffin sections is not needed if the tissues are from research animals. Processing for animal-based tissues takes about a third of the time and results in higher quality images. The focus of this book is on processing these animal research tissues for immunocytochemistry. Today, there are no technique books which are aimed at this user base.

As a subject matter expert in the area of the proposed book, Dr. Burry will make recommendations and offer opinions. Because this field is new and is emerging, there are numerous advantages of specific methods over other, more generalized methods. The purpose of this book is to show a novice how to do immunocytochemistry without engaging in a discussion of possible advanced methods. For the advanced user, there are several good books which discuss the unusual methods, yet for the novice there are currently none.

Main Author :

Richard W. Burry, The Ohio State University (United States).

The Outline of the Book :

Each chapter supplies a set of important principals and steps necessary for good immunocytochemistry. The information is distilled down to include only the most important points and does not attempt to cover infrequently used procedures or reagents. At the end of most chapters is a section on trouble-shooting many of the common problems using the Sherlock Holmes method. Each chapter also includes specific protocols which can be used. The goal of each chapter is to present the reader with enough information to successfully design experiments and solve many of the problems one may encounter. Using immunocytochemical protocols without the understanding of their workings is not advised, as the user will need to evaluate his or her results to determine whether the results are reliable. Such evaluation is extremely important for users who need reliable images which will clearly answer important scientific questions.

1. Introduction

   Definitions (immunocytochemistry and immunohistochemistry)

   Scope: animal research and not human pathology, paraffin sections, epitope retrieval, or immunohistochemistry

   Focus: fluorescence and enzyme detection

   Why do immunocytochemistry?

   Immunocytochemistry "individual study" rather than "population study"

   Example of a two-label experiment

   What is included in these chapters?

   Overview of the theory

   Background with enough information to help solve common problems.

   Advantages and disadvantages of different options

   Opinions and suggestions

   2. Fixation and Sectioning

   Chemistry of fixation

   Denaturing vs cross-linking fixatives

   Application of fixative

   Perfusion, drop-in, cultures, fresh-frozen

   Selection of sample section type

   Sectioning tissue

   Rapid freezing, cryostat, freezing microtome, vibratome

   Storage of tissue

   Protocols

   3. Antibodies

   Introduction

   Isoforms, structure, reactivity

   Generation

   Polyclonal vs monoclonal

   Antibodies as reagents

   Antibody specificity and sources

   Storage and handling

   4. Labels for antibodies

   Fluorescence, enzymes and particulates

   Fluorescence theory

   Fluorescent labels - four generations

   Enzymes theory

   Selecting enzymes vs. fluorescence

   Selecting a label- advantages and disadvantages

   Protocols

   5. Methods of applying antibodies

   Direct method

   Indirect method

   Antibody amplification methods

   ABC

   TSA

   Protocols

   6. Blocking and Permeability

   Theory of blocking

   Theory of detergents

   Protocols

   7. Procedure- Single primary antibody

   Planning steps

   Sample, fixation, sectioning

   Vehicle

   Antibody dilutions

   Controls

   Protocols

   8. Multiple primary antibodies - primary antibodies of different species

   Procedure

   Controls

   Protocols

   9. Multiple primary antibodies-primary antibodies of same species

   Block-between

   Zenon

   HRP-chromogen development

   High-titer incubations

   Controls

   Protocols

   10. Microscopy

   Wide-field fluorescence microscope

   Confocal microscope

   Bright fieldenzyme chromogen

   Choice

   Problems

   11. Images

   Size, intensity, and pixels

   Manipulationwhat is ethical?

   Manuscript Figures

   11. Planning and Troubleshooting

   Scheme for discussion-making in planning experiments

   Case studies with Sherlock Holmes detective work

   12. So you want to do electron microscopic ICC?

   Criteria in decision-making

   Summary of the two techniques

   The first practical guide to planning, performing, and evaluating immunocytochemical experiments Emphasis on practical, rather than academic, focusing only on the most productive methods Strong focus on animal research over human pathology, as processing for animal-based tissues takes about a third of the time and results in higher quality images Because this field is new and is emerging, there are numerous advantages of specific methods over other, more generalized methods The purpose of this book is to show a novice how to do immunocytochemistry without engaging in a discussion of possible advanced methods Includes supplementary material: sn.pub/extras

   Klappentext

Immunocytochemistry is a microscopic method that uses antibodies to localize proteins in tissue sections and cell cultures. Dr. Burry's book is the first practical guide to planning, performing, and evaluating immunocytoc…