Immunoelectron Microscopy, 2010 Methods and Protocols Methods in Molecular Biology Series, Vol. 657

Immunoelectron microscopy is a key technique that bridges the information gap between biochemistry, molecular biology, and ultrastructural studies placing macromolecular functions within a cellular context. In Immunoelectron Microscopy: Methods and Protocols, expert researchers combine the tools of the molecular biologist with those of the microscopist. From the molecular biology toolbox, this volume presents methods for antigen production by protein expression in bacterial cells, methods for epitope tagged protein expression in plant and animal cells allowing protein localization in the absence of protein specific antibodies as well as methods for the production of anti-peptide, monoclonal, and polyclonal antibodies. From the microscopy toolbox, sample preparation methods for cells, plant, and animal tissue are presented. Both cryo-methods, which have the advantage of retaining protein antigenicity at the expense of ultrastructural integrity, as well as chemical fixation methods that maintain structural integrity while sacrificing protein antigenicity have been included, with chapters examining various aspects of immunogold labeling. Written in the highly successful Methods in Molecular Biology? series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and essential, Immunoelectron Microscopy: Methods and Protocols seeks to facilitate an increased understanding of structure function relationships.

Molecular Toolbox.- Protein Antigen Expression in Escherichia coli for Antibody Production.- Expression of Epitope-Tagged Proteins in Plants.- Expression of Epitope-Tagged Proteins in Arabidopsis Leaf Mesophyll Protoplasts.- Transient Expression of Epitope-Tagged Proteins in Mammalian Cells.- Production and Purification of Polyclonal Antibodies.- Production and Purification of Monoclonal Antibodies.- Production of Antipeptide Antibodies.- Preparation of Colloidal Gold Particles and Conjugation to Protein A, IgG, F(ab’)2, and Streptavidin.- Microscopy Toolbox.- Immunoelectron Microscopy of Chemically Fixed Developing Plant Embryos.- Pre-embedding Immunogold Localization of Antigens in Mammalian Brain Slices.- Pre-embedding Immunoelectron Microscopy of Chemically Fixed Mammalian Tissue Culture Cells.- Immunoelectron Microscopy of Cryofixed and Freeze-Substituted Plant Tissues.- In Vivo Cryotechniques for Preparation of Animal Tissues for Immunoelectron Microscopy.- Immunoelectron Microscopy of Cryofixed Freeze-Substituted Mammalian Tissue Culture Cells.- Immunoelectron Microscopy of Cryofixed Freeze-Substituted Saccharomyces cerevisiae.- High-Resolution Molecular Localization by Freeze-Fracture Replica Labeling.- Pre-embedding Electron Microscopy Methods for Glycan Localization in Chemically Fixed Mammalian Tissue Using Horseradish Peroxidase-Conjugated Lectin.- Pre-embedding Nanogold Silver and Gold Intensification.- The Post-embedding Method for Immunoelectron Microscopy of Mammalian Tissues: A Standardized Procedure Based on Heat-Induced Antigen Retrieval.- Double-Label Immunoelectron Microscopy for Studying the Colocalization of Proteins in Cultured Cells.- Serial Section Immunoelectron Microscopy of Algal Cells.- Freeze-Etch Electron Tomography for the PlasmaMembrane Interface.- Localization of rDNA at Nucleolar Structural Components by Immunoelectron Microscopy.- Immunogold Labelling for Scanning Electron Microscopy.- Horseradish Peroxidase as a Reporter Gene and as a Cell-Organelle-Specific Marker in Correlative Light-Electron Microscopy.- Monitoring Rapid Endocytosis in the Electron Microscope via Photoconversion of Vesicles Fluorescently Labeled with FM1-43.

Presents a wide range of Cryo and chemical fixation methods for single cells, plant, and animal tissue Contains Immunogold labeling methods for transmission and scanning electron microscopy Includes comprehensive pre and post embedding immunolabeling methods Includes supplementary material: sn.pub/extras