Beschreibung
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 Biologya''¢ 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.
Inhaltsverzeichnis
Part I: Molecular Toolbox 1. Protein Antigen Expression in E. coli for Antibody Production David M. Rancour, Steven K. Backues, and Sebastian Y. Bednarek 2. Expression of Epitope-Tagged Proteins in Plants Takuya Furuichi 3. Expression of Epitope-Tagged Proteins in Arabidopsis Leaf Mesophyll Protoplasts Young-Hee Cho and Sang-Dong Yoo 4. Transient Expression of Epitope-Tagged Proteins in Mammalian Cells Melanie L. Styers, Jason Lowery, and Elizabeth Sztul 5. Production and Purification of Polyclonal Antibodies Masami Nakazawa, Mari Mukumoto, and Kazutaka Miyatake 6. Production and Purification of Monoclonal Antibodies Masami Nakazawa, Mari Mukumoto, and Kazutaka Miyatake 7. Production of Antipetide Antibodies Bao-Shiang Lee, Jin-Sheng Huang, G.D. Lasanthi P. Jayathilaka, Syed S. Lateef, and Shalini Gupta 8. Preparation of Colloidal Gold Particles and Conjugation to Protein A, IgG, F(ab¿)2 and Strepavidin Sadaki Yokota Part II: Microscopy Toolbox 9. Immunoelectron Microscopy of Chemically Fixed Developing Plant Embryos Tetsuaki Osafune and Steven D. Schwartzbach 10. Pre-Embedding Immunogold Localization of Antigens in Mammalian Brain Slices Thomas Schikorski 11. Pre-Embedding Immunoelectron Microscopy of Chemically Fixed Mammalian Tissue Culture Cells Haruo Hagiwara, Takeo Aoki, Takeshi Suzuki, and Kuniaki Takata 12. Immunoelectron Microscopy of Cryofixed and Freeze-Substituted Plant Tissues Miyuki Takeuchi, Keiji Takabe, and Yoshinobu Mineyuki 13. In vivo Cryotechniques for Preparation of Animal Tissues for Immunoelectron Microscopy Shinichi Ohno, Nobuhiko Ohno, Nobuo Terada, Sei Saitoh, Yurika Saitoh, and Yasuhisa Fujii 14. Immunoelectron Microscopy of Cryofixed Freeze Substituted Mammalian Tissue Culture Cells Akira Sawaguchi 15. Immunoelectron Microscopy of Cryofixed Freeze Substituted Saccharomyces cerevisiae Jindriska Fiserova and Martin W. Goldberg 16. High Resolution Molecular Localization by Freeze-Fracture Replica Labeling Akikazu Fujita and Toyoshi Fujimoto 17. Pre-Embedding Electron Microscopy Methods for Glycan Localization in Chemically Fixed Mammalian Tissue Using Horseradish Peroxidase-Conjugated Lectin Yoshihiro Akimoto and Hayato Kawakami 18. Pre-Embedding Nanogold Silver and Gold Intensification Akitsugu Yamamoto and Ryuichi Masaki 19. The Post-Embedding Method for Immunoelectron Microscopy of Mammalian Tissues: A Standardized Procedure Based on Heat-Induced Antigen Retrieval Shuji Yamashita 20. Double-Label Immunoelectron Microscopy for Studying the Colocalization of Proteins in Cultured Cells Haruo Hagiwara, Takeo Aoki, Takeshi Suzuki, and Kuniaki Takata 21. Serial Section Immunoelectron Microscopy of Algal Cells Tetsuaki Osafune and Steven D. Schwartzbach 22. Freeze-Etch Electron Tomography for the Plasma Membrane Interface Nobuhiro Morone 23. Localization of rDNA at Nucleolar Structural Components by Immunoelectron Microscopy Seiichi Sato and Yasushi Sato 24. Immunogold Labeling for Scanning Electron Microscopy Martin W. Goldberg and Jindriska Fiserova 25. Horseradish Peroxidase as a Reporter Gene and as a Cell-Organelle-Specific Marker in Correlative Light-Electron Microscopy Thomas Schikorski 26. Monitoring Rapid Endocytosis in the Electron Microscope via Photoconversion of Vesicles Fluorescently Labeled with FM1-43 Thomas Schikorski
