HIGH-RESOLUTION IMAGING OF CELLULAR PROTEINS. METHODS AND PROTOCOLS

1. Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture
2. Antibody Production with Synthetic Peptides
3. Production and Purification of Polyclonal Antibodies
4. Preparation of Colloidal Gold Particles and Conjugation to Protein A/G/L, IgG, F(ab')
5. Helper-Dependent Adenoviral Vectors and Their Use for Neuroscience Applications
6. Localizing Proteins in Fixed
7. Using Fluorescent Protein Fusions to Study Protein Subcellular Localization and Dynamics in Plant Cells
8. Using FRAP or FRAPA to Visualize the Movement of Fluorescently Labeled Proteins or Cellular Organelles in Live Cultured Neurons Transformed with Adeno-Associated Viruses
9. Bimolecular Fluorescence Complementation (BiFC) Analysis of Protein–Protein Interactions and Assessment of Subcellular Localization in Live Cells
10. Viral Injection and Cranial Window Implantation for In Vivo Two-Photon Imaging
11. Imaging Synaptic Vesicle Exocytosis-Endocytosis with pH-Sensitive Fluorescent Proteins
12. Immunogold Protein Localization on Grid-Glued Freeze-Fracture Replicas
13. Pre-embedding Double-Label Immunoelectron Microscopy of Chemically Fixed Tissue Culture Cells
14. Immunoelectron Microscopy of Cryofixed and Freeze-Substituted Plant Tissues
15. Immunoelectron Microscopy of Cryofixed Freeze-Substituted Yeast
16. Pre-embedding Method of Electron Microscopy for Glycan Localization in Mammalian Tissues and Cells Using Lectin Probes
17. Pre-embedding Nanogold Silver and Gold Intensification
18. Post-embedding Mammalian Tissue for Immunoelectron Microscopy: A Standardized Procedure Based on Heat-Induced Antigen Retrieval
19. Pre- and Post-embedding Immunogold Labeling of Tissue Sections
20. Immunogold Labeling for Scanning Electron Microscopy
21. Monitoring Synaptic Vesicle Protein Sorting with Enhanced Horseradish Peroxidase in the Electron Microscope
22. Functional Nanoscale Imaging of Synaptic Vesicle Cycling with Superfast Fixation

This volume presents authoritative and cutting-edge methods and protocols focusing on three tool boxes covering the increasingly diverse methodologies used to image selected proteins and to investigate their function by light and electron microscopy. The first tool box includes the development of a wide range of molecular and immunological probes to target specific proteins. The second details the use of these probes for high resolution fluorescence microscopy and the third focuses on applications for transmission and scanning electron microscopy. 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 tips on troubleshooting and avoiding known pitfalls thus ensuring successful results in the further study of this vital field.

Features
• Includes cutting-edge methods and protocols
• Provides step-by-step detail essential for reproducible results
• Contains key notes and implementation advice from the experts