Cell biology is the science of correlating cell structure and function. The electron microscopist obtains high-resolution pictures of the intricate structures found in cells. Electron tomography and serial sections can be used to reconstruct the three-dimensional structure of the cell and its organelles. Pictures are worth a thousand words, but they are unable to provide information regarding the function of the intricate structures found in cells and their macromolecular composition.
The biochemist and molecular biologist determine the functions of the molecules, macromolecular complexes, and organelles found within cells. They isolate individual cellular constituents and reconstruct vital cellular processes. These in vitro experiments provide a detailed understanding of cellular function. Organelle isolation provides a method to place macromolecular functions within a structural context. Understanding structure–function relationships through organelle isolation has restricted utility because organelles cannot be isolated from every organism, not every organelle can be isolated free of contamination by other organelles, suborganellular compartments often cannot be purified for biochemical characterization, and, when a protein is recovered in multiple organelles, it is often difficult to distinguish true localization from contamination artifacts.
Immunoelectron microscopy is the technique that bridges the information gap between biochemistry, molecular biology, and ultrastructural studies placing macromolecular functions within a cellular context. Immunoelectron microscopy can be used on virtually every unicellular and multicellular organism. The only requirements are suitable fixation protocols and the availability of an antibody to the molecule whose structural location is to be determined. Structure–function relationships can be determined even when it is impossible to purify the organelle or suborganellular compartment containing the macromolecules being studied. Most importantly, immunoelectron microscopy is a totally objective procedure that is not dependent on conjectures as to where the protein is localized and thus which organelles to isolate for biochemical studies.
The successful application of immunoelectron microscopy requires combining the tools of the molecular biologist with those of the microscopist. From the molecular biology toolbox, this volume will present methods for antigen production by protein expression in bacterial cells and by expression of epitope tagged proteins in plant and animal cells. Methods for production of anti-peptide, monoclonal, and polyclonal antibodies will be presented. From the microscopy toolbox, this volume will present methods for cryoultramicrotomy and rapid freeze-replacement fixation 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. Plants and algae contain cell walls, vacuoles, and other structures which present barriers to antibody penetration and complicate fixation. Due to these problems, separate chapters will discuss fixation and immunolabeling protocols for animals, plants, and algae. Preand post-embedding immunogold labeling protocols will be presented. Pre-embedding methods perform immunogold labeling before ultrathin sections are prepared from resinembedded samples resulting in greater sensitivity and better microstructure preservation. Post-embedding methods perform immunolabeling after ultrathin sections are prepared from resin-embedded samples resulting in decreased antigenicity. The detailed methods and notes will facilitate choosing the best method for the antibody and biological material to be studied. Finally, methods will be presented for immunogold labeling of two antigens for protein colocalization studies, for three-dimensional reconstruction of intracellular antigen distribution, for immunogold labeling of DNA, and for immunogold scanning electron microscopy. The toolbox created by this volume will facilitate an increased understanding of structure–function relationships.
Contents
PART I MOLECULAR TOOLBOX
- 1. Protein Antigen Expression in Escherichia coli for Antibody Production
- 2. Expression of Epitope-Tagged Proteins in Plants
- 3. Expression of Epitope-Tagged Proteins in Arabidopsis Leaf Mesophyll Protoplasts
- 4. Transient Expression of Epitope-Tagged Proteins in Mammalian Cells
- 5. Production and Purification of Polyclonal Antibodies
- 6. Production and Purification of Monoclonal Antibodies
- 7. Production of Antipeptide Antibodies
- 8. Preparation of Colloidal Gold Particles and Conjugation to Protein A, IgG, F(ab’)2, and Streptavidin
PART II MICROSCOPY TOOLBOX
- 9. Immunoelectron Microscopy of Chemically Fixed Developing Plant Embryos
- 10. Pre-embedding Immunogold Localization of Antigens in Mammalian Brain Slices
- 11. Pre-embedding Immunoelectron Microscopy of Chemically Fixed Mammalian Tissue Culture Cells
- 12. Immunoelectron Microscopy of Cryofixed and Freeze-Substituted Plant Tissues
- 13. In Vivo Cryotechniques for Preparation of Animal Tissues for Immunoelectron Microscopy
- 14. Immunoelectron Microscopy of Cryofixed Freeze-Substituted Mammalian Tissue Culture Cells
- 15. Immunoelectron Microscopy of Cryofixed Freeze-Substituted Saccharomyces cerevisiae
- 16. High-Resolution Molecular Localization by Freeze-Fracture Replica Labeling
- 17. Pre-embedding Electron Microscopy Methods for Glycan Localization in Chemically Fixed Mammalian Tissue Using HorseradishPeroxidase-Conjugated Lectin
- 18. Pre-embedding Nanogold Silver and Gold Intensification
- 19. The Post-embedding Method for Immunoelectron Microscopy of Mammalian Tissues: A Standardized Procedure Based on Heat-Induced Antigen Retrieval
- 20. Double-Label Immunoelectron Microscopy for Studying the Colocalization of Proteins in Cultured Cells
- 21. Serial Section Immunoelectron Microscopy of Algal Cells
- 22. Freeze-Etch Electron Tomography for the Plasma Membrane Interface
- 23. Localization of rDNA at Nucleolar Structural Components by Immunoelectron Microscopy
- 24. Immunogold Labelling for Scanning Electron Microscopy
- 25. Horseradish Peroxidase as a Reporter Gene and as a Cell-Organelle-Specific Marker in Correlative Light-Electron Microscopy
- 26. Monitoring Rapid Endocytosis in the Electron Microscope via Photoconversion of Vesicles Fluorescently Labeled with FM1-43
Subject Index
From the Back Cover
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.
Product Details
- Hardcover: 352 pages
- Publisher: Humana Press; 1st edition (July 20, 2010)
- Language: English
- ISBN-10: 160761782X
- ISBN-13: 978-1607617822
- Product Dimensions: 10.1 x 6.9 x 1 inches
List Price: $139.00