Main description:

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 (TM) 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.

Contents:

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 ReplicaLabeling
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