Full Description
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.
Contents
Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture.- Antibody Production with Synthetic Peptides.- Production and Purification of Polyclonal Antibodies.- Preparation of Colloidal Gold Particles and Conjugation to Protein A/G/L, IgG, F(ab')2 and Streptavidin.- Helper Dependent Adenoviral Vectors and their use for Neuroscience Applications.- Localizing Proteins in Fixed Giardia Lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy.- Using Fluorescent Protein Fusions to Study Protein Subcellular Localization and Dynamics in Plant Cells.- Using FRAP Or FRAPA to Visualize the Movement of Fluorescently-Labeled Proteins or Cellular Organelles in Live Cultured Neurons Transformed with Adeno-Associated Viruses.- Bimolecular Fluorescence Complementation (BiFc) Analysis of Protein-Protein Interactions and Assessment of Subcellular Localization in Live Cells.- Viral Injection and Cranial Window Implantation for In Vivo Two-Photon Imaging.- Imaging SynapticVesicle Exocytosis-Endocytosis with pH Sensitive Fluorescent Proteins.- Immunogold Protein Localization on Grid-Glued Freeze-Fracture Replicas.- Pre-Embedding Double-Label Immunoelectron Microscopy of Chemically Fixed Tissue Culture Cells.- Immunoelectron Microscopy of Cryofixed and Freeze- Substituted Plant Tissues.- Immunoelectron Microscopy of Cryofixed Freeze Substituted Yeast Saccharomyces cerevisiae.- Pre-embedding Method of Electron Microscopy for Glycan Localization in Mammalian Tissues and Cells Using Lectin Probes.- Pre-Embedding Nanogold Silver and Gold Intensification.- Post-Embedding Mammalian Tissue for Immunoelectron Microscopy: A Standardized Procedure Based On Heat-Induced Antigen Retrieval.- Pre- and Post-Embedding Immunogold Labeling of Tissue Sections.- Immuno-Gold Labelling for Scanning Electron Microscopy.- Monitoring Synaptic Vesicle Protein Sorting with Enhanced Horseradish Peroxidase in the Electron Microscope.- Functional Nanoscale Imaging Of Synaptic VesicleCycling With Superfast Fixation.
