Immunocytochemistry and in situ hybridization are widely used biomedical sciences. They are essential in medical diagnosis and in cell biology research. Affinity labeling is the central goal of the experimental strategy involving a series of techniques in a logical order; from the effects of specimen fixation, through specimen preparation to expose the antigen, to optimizing immunolabeling, to assessing the result and finally to safety considerations. Numerous examples of these techniques in biomedical sciences are included, as well as experimental assays and practical tips. This survey of methods will serve as an invaluable reference source in any laboratory setting (academic, industrial or clinical) involved in research in almost every branch of biology or medicine, as well as in pharmaceutical, biotechnological and clinical applications.
In Situ Molecular Pathology and Co-Expression Analyses explains, in easy-to-understand language, simplified ways of understanding and performing in situ hybridization and immunohistochemistry tests. The book also focuses on straightforward protocols used to simultaneously detect two or more proteins/nucleic acids within intact tissue by doing co-expression analyses. The fields of in situ hybridization and immunohistochemistry have expanded rapidly due to the use of computer-based analysis. To get the most out of these automated platforms, researchers and diagnostic biomedical investigators must have a solid understanding of the basics of in situ-based tests, protocols, and regimens for troubleshooting. Practicing molecular pathologists, clinical chemists, and toxicologists, as well as clinicians and researchers in training, will benefit from this book's clear presentation of protocols and theoretical framework. - Includes over 200 easy-to-follow experimental protocols - Features chapter-ending summaries of "Key Points to Remember" to bring beginners up to speed with any seasoned veteran in the field - Offers two chapters written by industry leaders in the fields of in situ hybridization, immunohistochemistry, and computer software for co-expression analyses
The past several decades have witnessed an impressive array of conceptual and techno logical advances in the biomedical sciences. Much of the progress in this area has developed directly as a result of new morphology-based methods that have permitted the assessment of chemical, enzymatic, immunological, and molecular parameters at the cellular and tissue levels. Additional novel approaches including laser capture microdissection have also emerged for the acquisition of homogeneous cell popula tions for molecular analyses. These methodologies have literally reshaped the approaches to fundamental biological questions and have also had a major impact in the area of diagnostic pathology. Much of the groundwork for the development of morphological methods was estab th lished in the early part of the 19 century by Francois-Vincent Raspail, generally acknowledged as the founder of the science of histochemistry. The earliest work in the field was primarily in the hands of botanists and many of the approaches to the under standing of the chemical composition of cells and tissues involved techniques such as microincineration, which destroyed structural integrity. The development of aniline th dyes in the early 20 century served as a major impetus to studies of the structural rather than chemical composition of tissue. Later in the century, however, the focus returned to the identification of chemical constituents in the context of intact cell and tissue structure.
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 to learn 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
The most complete, up-to-date reference on antigen retrieval and immunohistochemistry An antigen is a substance that prompts the generation of antibodies and can cause an immune response. The antigen retrieval (AR) technique is in wide use across the globe, and is a critical technique used in medical diagnosis of disease, particularly clinical targeted cancer treatment. Antigen Retrieval Immunohistochemistry Based Research and Diagnostics discusses several scientific approaches to the standardization of quantifiable immunohistochemistry (IHC). Based on the development and application of AR by the editors, this volume summarizes recent achievements in AR-IHC and analyzes numerous cutting-edge issues for future research projects. Featuring contributions from a worldwide group of leading experts and research scientists in the field, this important work: Summarizes the key problems in the four fields of antigen retrieval Discusses the advances of AR techniques and their applications Provides practical methods and protocols in AR-IHC, such as extraction of nucleic acids and proteins for molecular analysis, cell/tissue sample preparation, and standardization and development of various techniques to meet the future needs of¿clinical and research molecular analysis Encourages further research in AR and IHC, particularly how AR methods might be employed for improved test performance and the development of greater reliability and reproducibility of IHC Includes an appendix of related laboratory protocols Antigen Retrieval Immunohistochemistry Based Research and Diagnostics is intended for clinical pathologists, molecular cell biologists, basic research scientists, technicians, and graduate students who undertake tissue/cell morphologic and molecular analysis and wish to use and extend the power of immunohistochemistry. It is also pertinent for most biotechnology companies majoring in development of IHC products. Wiley Series in Biomedical Engineering and Multi-Disciplinary Integrated Systems / Kai Chang, Series Editor
This is a brand new edition of the leading reference work on histological techniques. It is an essential and invaluable resource suited to all those involved with histological preparations and applications, from the student to the highly experienced laboratory professional. This is a one stop reference book that the trainee histotechnologist can purchase at the beginning of his career and which will remain valuable to him as he increasingly gains experience in daily practice. Thoroughly revised and up-dated edition of the standard reference work in histotechnology that successfully integrates both theory and practice.Provides a single comprehensive resource on the tried and tested investigative techniques as well as coverage of the latest technical developments. Over 30 international expert contributors all of whom are involved in teaching, research and practice.Provides authoritative guidance on principles and practice of fixation and staining. Extensive use of summary tables, charts and boxes.Information is well set out and easy to retrieve. Six useful appendices included (SI units, solution preparation, specimen mounting, solubility). Provides practical information on measurements, preparation solutions that are used in daily laboratory practice. Color photomicrographs used extensively throughout. Better replicates the actual appearance of the specimen under the microscope. Brand new co-editors. New material on immunohistochemical and molecular diagnostic techniques.Enables user to keep abreast of latest advances in the field.
Electron microscopy is frequently portrayed as a discipline that stands alone, separated from molecular biology, light microscopy, physiology, and biochemistry, among other disciplines. It is also presented as a technically demanding discipline operating largely in the sphere of "black boxes" and governed by many absolute laws of procedure. At the introductory level, this portrayal does the discipline and the student a disservice. The instrumentation we use is complex, but ultimately understandable and, more importantly, repairable. The procedures we employ for preparing tissues and cells are not totally understood, but enough information is available to allow investigators to make reasonable choices concerning the best techniques to apply to their parti cular problems. There are countless specialized techniques in the field of electron and light microscopy that require the acquisition of specialized knowledge, particularly for interpretation of results (electron tomography and energy dispersive spectroscopy immediately come to mind), but most laboratories possessing the equipment to effect these approaches have specialists to help the casual user. The advent of computer operated electron microscopes has also broadened access to these instruments, allowing users with little technical knowledge about electron microscope design to quickly become operators. This has been a welcome advance, because earlier instru ments required a level of knowledge about electron optics and vacuum systems to produce optimal photographs and to avoid "crashing" the instruments that typically made it difficult for beginners.
This volume contains a comprehensive compilation of chromogenic and fluorescent RNA in situ hybridization (ISH) technology in many of its various shades, forms, and applications. The book is organized into a number of parts and chapters focusing on the application of ISH methodologies to different animal species as used in Evolutionary Development (EvoDevo) and Biomedical research, and covering new developments in RNA visualization by fluorescent ISH (FISH). The described (F)ISH protocols employ effective strategies for signal enhancement and target amplification allowing for high signal intensities and drastically improved signal-to-noise ratios. Chromogenic and fluorescent ISH, as specified in the various chapters, are most essential for RNA expression profiling, applied to many fields of research including cellular, developmental, and evolutionary biology, neurobiology and neuropathology. Written for the popular Neuromethods series, chapters include the kind of detail and key implementation advice that ensures successful results in the laboratory. Essential and authoritative, In Situ Hybridization Methods provides detailed protocols for newcomers to ISH, and inspires researchers familiar with the technique to seek and find up-to-date methodology for new and specialized applications.
This leading reference work on histological techniques is an essential and invaluable resource no matter what part you play in histological preparations and applications, whether you're a student or a highly experienced laboratory professional.
