Immunocytochemistry of plant cells is the first book exclusively dedicated to this topic. The first and largest portion of the book is concerned with a group of proven protocols and variations on these protocols that might prove useful, many developed or modified in the author's laboratory. The second portion of the book covers the studies that have been published previously on each of the plant organelles. Numerous state of the art micrographs from researchers around the world are included to demonstrate typical results.
Immunocytochemistry is classically defined as a procedure to detect antigens in cellular contexts using antibodies. However, over the years many aspects of this procedure have evolved within a plethora of experimental setups. There are different ways to prepare a given specimen, different kinds of antibodies to apply, different techniques for imaging, and different methods of analyzing the data. In this book, various ways of performing each individual step of immunocytochemistry in different cellular contexts are exemplified and discussed. Applications of Immunocytochemistry offers technical and background information on different steps of immunocytochemistry and presents the application of this technique and its adaptations in cell lines, neural tissue, pancreatic tissue, sputum cells, sperm cells, preimplantation embryo, arabidopsis, fish gonads, and Leishmania.
Various methodologies designed to study cell walls are compiled in this book. Methods in Cell Wall Cytochemistry covers the use of modern dyes, fluorescent chemicals, lectins, and antibody technology (immunocytochemisty.) Cell wall morphology and chemical composition is covered as well as light and fluorescent cytochemistry; transmission electron microscopic cytochemistry; lectin cytochemistry; and, special emphasis on immunocytochemistry. Addressing an emerging area of research and technology, this book will appeal to plant pathologists, cell biologists, as well as workers interested in stress response and those employing cell walls for biotechnological research.
It is a pleasure to contribute the foreword to Introduction to Cell and Tissue Culture: The ory and Techniques by Mather and Roberts. Despite the occasional appearance of thought ful works devoted to elementary or advanced cell culture methodology, a place remains for a comprehensive and definitive volume that can be used to advantage by both the novice and the expert in the field. In this book, Mather and Roberts present the relevant method ology within a conceptual framework of cell biology, genetics, nutrition, endocrinology, and physiology that renders technical cell culture information in a comprehensive, logical for mat. This allows topics to be presented with an emphasis on troubleshooting problems from a basis of understanding the underlying theory. The material is presented in a way that is adaptable to student use in formal courses; it also should be functional when used on a daily basis by professional cell culturists in a- demia and industry. The volume includes references to relevant Internet sites and other use ful sources of information. In addition to the fundamentals, attention is also given to mod ern applications and approaches to cell culture derivation, medium formulation, culture scale-up, and biotechnology, presented by scientists who are pioneers in these areas. With this volume, it should be possible to establish and maintain a cell culture laboratory devot ed to any of the many disciplines to which cell culture methodology is applicable.
A proper understanding of the structural organization of the plant body is essential to any study in plant biology. Experimental studies in vivo and in situ will lead to structural, physiological, and cellular changes of the experimental material. To study macroscopic and microscopic changes, different histological methods and microtechniques can be used as they provide valuable information of the experimental system. In addition, the observed structural changes allow investigators to set hypothesis for further studies based on one’s own observation. Thus, proper selection and utilization of microtechniques are a must for the success of a research program. At present, an up-to-date collection of protocols are not readily available in the literature. The latest work in plant microtechniques was published in 1999 by Ruzin but many others are no longer in print [e.g., Jensen (1964); O’Brien and McCully (1981)]. Furthermore, a majority of published works focus on techniques related to general processing and staining procedures. A comprehensive treatment that encompasses broader applications of microtechniques to other disciplines is lacking [e.g., archeology, wood science, etc.]. There is a need to create a comprehensive volume of botanical methods and protocols which includes traditional and novel techniques that can be used by researchers in plant science and investigators in other disciplines that require plant microtechniques in their research and teaching. This book covers a wide variety of applications and brings them up-to-date to make them understandable and relevant, especially to students using the methods for the first time. It is our intention to create a useful reference for plant histology and related methods that will serve as a foundation for plant scholars, researchers, and teachers in the plant sciences.
The first compilation of a wealth of knowledge on tobacco BY-2 cells, often cited as the HeLa cell line of higher plants. Basic issues of cell cycle progression, cytokinesis, cell organization and factors that are involved in these processes are covered in detail. Since the tobacco cell line is used as a tool for research in molecular and cellular biology, several chapters on such studies are also included. Further, changes of primary and secondary metabolites during culture and factors that affect these processes are treated. Last but not least, the so far unpublished historical background of the BY-2 cell line is described. This volume is a must for any scientist working in the field of plant biology.
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
Hands-on experimentalists describe the cutting-edge microscopical methods needed for the effective study of plant cell biology today. These powerful techniques, all described in great detail to ensure successful experimental results, range from light microscope cytochemistry, autoradiography, and immunocytochemistry, to recent developments in fluorescence, confocal, and dark-field microscopies. Important advances in both conventional and scanning electron microscopies are also fully developed, together with such state-of-the-art ancillary techniques as high-resolution autoradiography, immunoelectron microscopy, X-ray microanalysis, and electron systems imaging. Easy-to-use and up-to-date, Methods in Plant Electron Microscopy and Cytochemistry offers today's plant scientists a first class collection of readily reproducible light and electron microscopical methods that will prove the new standard for all working in the field.
Antibodies tagged with fuorescent markers have been used in histochemistry for over 50 years. Although early applications were focused on the detection of microbial antigens in tissues, the use of immunocytochemical methods now has spread to include the det- tion of a wide array of antigens including proteins, carbohydrates, and lipids from virtually any organism. Today, immunohistochemistry is widely used to identify, in situ, various components of cells and tissues in both normal and pathological conditions. The method gains its strength from the extremely sensitive interaction of a specifc antibody with its antigen. For some scientifc areas, books have been published on applications of immu- cytochemical techniques specifc to that area. What distinguished Immunocytochemical Methods and Protocols from earlier books when it was frst published was its broad appeal to investigators across all disciplines, including those in both research and clinical settings. The methods and protocols p- sented in the frst edition were designed to be general in their application; the accompa- ing “Notes” provided the reader with invaluable assistance in adapting or troubleshooting the protocols. These strengths continued to hold true for the second edition and again for the third edition. Since the publication of the frst edition, the application of immuno- tochemical techniques in the clinical laboratory has continued to rise and this third edition provides methods that are applicable to basic research as well as to the clinical laboratory.
