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Lorette Javois' timely new 2nd edition revises and updates her widely acclaimed collection of step-by-step immunocyhemical methods, one that is now used in many biological and biomedical research programs. The methods are designed for researchers and clinicians who wish to visualize molecules in plant or animal embryos, tissue sections, cells, or organelles. In addition to cutting-edge prools for purifying and preparing antibodies, light microscopic analysis, confocal microscopy, FACS, and electron microscopy, this revised edition contains many new methods for applying immunocyhemical techniques in the clinical laboratory and in combination with in situ hybridization.
The book contains black-and-white illustrations.
Immunocytochemical Methods and Protocols offers a powerful collection of step-by-step immunocytochemical methods that can be used in many biological and biomedical disciplines in both research and clinical settings. Each protocol includes troubleshooting tips and alternative procedures, as well as informative explanations of why certain steps are necessary, and comprehensive lists of reagents and suppliers aids not usually found in standard journal recipes. Among the especially valuable protocols presented are those geared to preparing antibodies for staining procedures, light microscopic analysis, FACS, electron microscopy, and confocal microscopy. There are also protocols for nucleic acid immunocytochemistry, electron microscopic evaluation of antibody labeling using colloidal gold, and the use of antibodies for the differential diagnosis of tumors. Special applications arising from recent improvements in equipment design and technology among them laser microbeam workstations and automated immunostainers are also thoroughly presented.
Part I. Antibody Preparation. Overview of Antibody Use in Immunocyhemistry, Su-Yau Mao, Lorette C. Javois, and Ute M. Kent. Purification of Antibodies Using Ammonium Sulfate Fractionation or Gel Filtration, Ute M. Kent. Purification of Antibodies Using Ion-Exchange Chromatography, Ute M. Kent. Purification of Antibodies Using Affinity Chromatography, Ute M. Kent. Purification of Antibodies Using Protein A-Sepharose and FPLC, Ute M. Kent. Conjugation of Fluorochromes to Antibodies, Su-Yau Mao. Biotinylation of Antibodies, Su-Yau Mao. Part II. Tissue Preparation for light Microscopic Analyses. Overview of Cell Fixatives and Cell Membrane Permeants, Melissa A. Melan. Preparation of Frozen Sections for Analysis, Gary L. Bratthauer.
Processing of Cytological Specimens, Gary L. Bratthauer. Processing of Tissue Culture Cells, Gary L. Bratthauer. Processing of Tissue Specimens, Gary L. Bratthauer. Antigen Retrieval for Immunohishemical Reactions in Routinely Processed Paraffin Sections, Laszlo Krenacs, Tibor Krenacs, and Mark Raffeld. Part III. Light Microscopic Detection Systems. Overview of Fluorochromes, J. Michael Mullins. Direct Immunofluorescent Labeling of Cells, Lorette C. Javois. Fluorescence Labeling of Surface Antigens on Attached or Suspended Tissue-Culture Cells, Mark C. Willingham. Fluorescence Labeling of Intracellular Antigens in Attached or Suspended Tissue-Culture Cells, Mark C. Willingham. Fluorescent Labeling of Surface or Intracellular Antigens in Whole-Mounts, Lorette C. Javois. TUNEL Assay for Apoptotic Cells, Virginia M. Heatwole. Overview of Fluorescence Analysis with the Confocal Microscope, Liana Harvath. Overview of Laser Microbeam Applications as Related to Antibody Targeting, P. Scott Pine. Overview of Fluorescence Photomicography, Lorette C. Javois and J. Michael Mullins. Overview of Antigen Detection Through Enzymatic Activity, Gary L. Bratthauer. The Peroxidase Antiperoxidase (PAP) Method and Other All-Immunologic Detection Methods, Gary L. Bratthauer. The Avidin Biotin Complex (ABC) Method and Other Avidin Biotin Binding Methods,
Gary L. Bratthauer. Avidin Biotin Labeling of Cellular Antigens in Cryostat Sectioned Tissue, Mark Raffeld and Elaine S. Jaffe.
Multiple Antigen Immunostaining Procedures, Laszlo Krenacs, Tibor Krenacs, and Mark Raffeld. ELISA on Attachment Dependent or Suspension Grown Cells, Julie Ann Brent.. Use of Immunogold with Silver Enhancement ,Constance Oliver. Part IV. Flourescence Activated Cell Sorter (FACS) Analyses. Overview of Flow Cytometry and Fluorescent Probes for Cytometry,
Robert E. Cunningham. Tissue Disaggregation, Robert E. Cunningham. Indirect Immunofluorescent Labeling of Viable Cells,
Robert E. Cunningham. Indirect Immunofluorescent Labeling of Fixed Cells, Robert E. Cunningham. Fluorescent Labeling of DNA,
Robert E. Cunningham. Deparaffinization and Processing of Pathologic Material, Robert E. Cunningham. Assay for Phagocytosis,
Liana Harvath and Douglas A. Terle. Assay for Filamentous Actin, Liana Harvath. Assay for Chemoattractant Binding, Liana Harvath, Robert R. Aksamit, and Robert E. Cunningham. Assay for Oxidative Metabolism, Liana Harvath and Douglas A. Terle.
Part V. Colloidal Gold Detection Systems For Electron Microscopic Analysis. Fixation and Embedding, Constance Oliver. Preparation of Colloidal Gold, Constance Oliver. Conjugation of Colloidal Gold to Proteins, Constance Oliver. Colloidal Gold/Streptavidin Methods, Constance Oliver. Pre-Embedding Labeling Methods, Constance Oliver. Postembedding Labeling Methods, Constance Oliver. Part VI.Applications For Nucleic Acid Localization By In Situ Hybridization. Overview of In Situ Hybridization, Roland M. Nardone. In Situ Hybridization to Human Chromosomes of an Alkaline Phosphatase Labeled Centromeric Probe, Roland M. Nardone. Fluorescence In Situ Hybridization Using Whole Chromosome Library Probes,
Roland M. Nardone. In Situ RT-PCR and Hybridization Techniques, Vishakha Thaker. Part VII. Special Applications For the Clinical Laboratory. Overview of the Clinical Immunohishemistry Laboratory: Regulations and Troubleshooting Guidelines,
Patricia A. Fetsch and Andrea Abati. Overview of Immunohishemical Approaches to the Differential Diagnosis of Tumors,
Dennis M. Frisman. Overview of Automated Immunostainers, Gilbert E. Herman, Edna A. Elfont, and Alton D. Floyd.