Immunocytochemistry

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.

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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 field—enzyme chromogen


Choice


Problems





11. Images


Size, intensity, and pixels


Manipulation—what 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