Immunocytochemical Methods and Protocols (3^rd Ed., 3rd ed. 2010) Methods in Molecular Biology Series, Vol. 588

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.

Antibody Preparation.- Overview of Antibodies for Immunochemistry.- to the Purification of Antibodies.- Antibody Purification: Ammonium Sulfate Fractionation or Gel Filtration.- Antibody Purification: Ion-Exchange Chromatography.- Antibody Purification: Affinity Chromatography – Protein A and Protein G Sepharose.- Conjugation of Fluorochromes to Antibodies.- Biotinylation of Antibodies.- Tissue Preparation for Light Microscopic Analysis.- Cell Fixatives for Immunostaining.- Permeabilization of Cell Membranes.- Preparation of Frozen Sections for Analysis.- Processing of Cytological Specimens.- Processing of Tissue Culture Cells.- Processing of Tissue Specimens.- Heat-Induced Antigen Retrieval for Immunohistochemical Reactions in Routinely Processed Paraffin Sections.- Bright Field Detection Systems.- Fluorochromes: Properties and Characteristics.- Direct Immunofluorescent Labeling of Cells.- Fluorescence Labeling of Surface Antigens of Attached or Suspended Tissue-Culture Cells.- Fluorescence Labeling of Intracellular Antigens of Attached or Suspended Tissue-Culture Cells.- Fluorescent Visualization of Macromolecules in Drosophila Whole Mounts.- Overview of Conventional Fluorescence Photomicrography.- Overview of Confocal Microscopy.- Overview of Laser Microbeam Applications as Related to Antibody Targeting.- Immuno-Laser Capture Microdissection of Rat Brain Neurons for Real Time Quantitative PCR.- Overview of Antigen Detection Through Enzymatic Activity.- The Peroxidase–Antiperoxidase (PAP) Method and Other All-Immunologic Detection Methods.- The Avidin–Biotin Complex (ABC) Method and Other Avidin–Biotin Binding Methods.- Avidin-Biotin Labeling of Cellular Antigens in Cryostat-Sectioned Tissue.- Multiple Antigen Immunostaining Procedures.- ImmunoenzymaticQuantitative Analysis of Antigens Expressed on the Cell Surface (Cell-ELISA).- Use of Immunogold with Silver Enhancement.- Fluorescence-Activated Cell Sorter (FACS) Analyses.- Overview of Flow Cytometry and Fluorescent Probes for Flow Cytometry.- Tissue Disaggregation.- Indirect Immunofluorescent Labeling of Viable Cells.- Indirect Immunofluorescent Labeling of Fixed Cells.- Fluorescent Labeling of DNA.- Deparaffinization and Processing of Pathologic Material.- Colloidal Gold Detection Systems for Electron Microscopic Analysis.- Fixation and Embedding.- Preparation of Colloidal Gold.- Conjugation of Colloidal Gold to Proteins.- Colloidal Gold/Streptavidin Methods.- Pre-embedding Labeling Methods.- Postembedding Labeling Methods.- The Clinical Laboratory.- The Clinical Immunohistochemistry Laboratory: Regulations and Troubleshooting Guidelines.

Fully updated from the first edition to offer cutting-edge methods in immunocytochemistry Includes methods applicable for the basic research laboratory as well as the clinical laboratory Provides the theoretical basis behind the methods Contains methods that have multidisciplinary applications Suitable for beginning and experienced investigators