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During the last century, before the advent of microtomes capable of producing thin, even sections, whole-mounts and thick sections were used to study the peripheral nervous system. Tissues were either viewed directly, if sufficiently thin, or further separated into layers by acid maceration (1). Initially, histochemical staining, particularly methylene blue (2), was applied to wholemounts to characterize neuronal cell types, distributions, and innervation of various organs. In more recent times precise innervations were traced through the use of histochemical techniques specific for neuronal components. Histofluorescence techniques were introduced by Falk (3) for the localization of nonadrenaline-containing nerves, and the discovery of neuropeptides led to the application of immunohistochemical techniques in the late 1970s and early 1980s (4,5). Most recently, the application of monoclonal antibody technology has opened a new era in research and diagnosis such that in situ normal and pathological tissue constituents may be detected in whole-mounts ranging from tissues to entire organisms.

Part I. Antibody Preparation. Overview of Antibody Use in Immunocytochemistry, 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 Immunohistochemical 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. Fluorescence 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 Analyses. Fixation and Embedding, Constance Oliver and Maria Celia Jamur. 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 Immunohistochemistry Laboratory: Regulations and Troubleshooting Guidelines, Patricia A. Fetsch and Andrea Abati. Overview of Immunohistochemical Approaches to the Differential Diagnosis of Tumors,Dennis M. Frisman. Overview of Automated Immunostainers, Gilbert E. Herman, Edna A. Elfont, and Alton D. Floyd.