Note: Front Cover -- Methods in Cell Biology, Volume 37 -- Copyright Page -- Contents -- Contributors -- Preface -- Chapter 1. The Immune Response: An Overview -- I. Clonal Selection -- II. Size of the Immune Repertoire -- III. Specificity and Affinity Maturation -- IV. Idea of Cross-Reactivity -- V. Monoclonality and Multispecificity -- VI. A Cautionary Note -- Suggested Readings -- Chapter 2. Production of Polyclonal Antibodies against Proteins and Peptides -- I. Introduction -- II. Properties of Immunoglobulins -- III. Polyclonal versus Monoclonal Antibodies -- IV. Processing of Antigens for Immunization -- V. Adjuvants -- VI. Routes of Immunization -- VII. General Schedule for Immunization -- VIII. Collection of Blood -- IX. Preparation, Storage, and Shipping of Serum -- X. Determination of Antibody Titer (Test of Reactivity) -- XI. Test of Specificity -- XII. Production and Selection of Isofom-Specific Immunoglobulins -- XIII. Purification of Immunoglobulins -- References -- Chapter 3. Making Monoclonal Antibodies -- I. lntroduction -- II. First Principles -- III. Overview of Protocol -- IV. Hybridoma Protocols -- V. Perspective -- References -- Chapter 4. Purification of Mouse Antibodies and Fab Fragments -- I. Introduction -- II. Purification of Murine IgG Monoclonal Antibodies -- III. Generation of Fab and Fab' Fragments -- IV. Fab Purification -- V. Further Purification of Fab Fragments -- VI. Crystallization of Fab Fragments -- VII. Summary -- References -- Chapter 5. Blot-Affinity Purification of Antibodies -- I. Introduction -- II. SDS-Polyacrylamide Gel Electrophoresis and Blotting -- Ill. Antibody Adsorption, Elution, and Storage -- IV. Quality Evaluation of the Eluted Antibodies -- V. Discussion -- References -- Chapter 6. Immunoblotting -- I. Introduction -- II. Semidry and Wet Blotting of Gels. , III. Staining Nitrocellulose with Antibodies -- IV. Miscellaneous Information -- V. Perspectives -- References -- Chapter 7. Immunoprecipitation Methods -- I. Introduction -- II. Reagents and Solutions -- III. Protocol for Immunoprecipitation -- IV. Troubleshooting -- References -- Chapter 8. A Quantitative Solid-Phase Binding Assay for Tubulin -- I . Introduction -- II. Materials -- III. Methods -- IV. Typical Results and Discussion -- V. Development of a Competitive ELISA -- References -- Chapter 9. Confocal Immunofluorescence Microscopy of Microtubules in Oocytes, Eggs, and Embryos of Algae and Amphibians -- I . Introduction -- II . Immunofluorescence Microscopy of Pelvetia and Xenopus -- III. Confocal Microscopy: Advantages and Considerations -- IV. Conclusion -- Appendix I: Recipes and Reagents -- Appendix II: Antibodies -- Appendix III: Supplier Information -- References -- Chapter 10. Immunolabeling of Antigens in Plant Cells -- I. Introduction -- II. Reagents -- III. Immunofluorescence Microscopy of Cytoskeletal Elements in Isolated Root Tip Cells -- IV. Preparation of Leaf Epidemial Sheets for Immunolabeling -- V. Cryosectioning for Light Microscopy Immunolabeling -- VI. Troubleshooting/Variations on Protocols -- VII. Perspectives -- References -- Chapter 11. Antigen Localization in Fission Yeast -- I . Introduction -- II. Immunofluorescence Microscopy -- III. Solutions and Notes for Immunofluorescence Microscopy -- IV. Fluorescence Microscopy -- V. Solutions and Notes for Fluorescence Microscopy -- VI. Immunogold Labeling of Yeast Ultrathin Sections -- References -- Chapter 12. Nonfluorescent Immunolocalization of Antigens in Mitotic Sea Urchin Blastomeres -- I. Introduction -- II. Immunolocalization and Sea Urchin Embryos -- III. Reagents and Buffers -- IV. Antibody Preparation -- V. Preparation of Sea Urchin Embryos -- VI. Immunostaining. , VII. Alternative Techniques -- VIII. Kesults and Perspectives -- References -- Chapter 13. Ultrasmall Gold Probes: Characteristics and Use in Immuno(cyto)chemical Studies -- I. Introduction -- II. Improvement of Detection System -- III. Characteristics of Ultrasmall Gold Probes -- IV. Application of Ultrasmall Gold Probes -- V. Discussion -- References -- Chapter 14. Immunogold Electron Microscopy: Mapping Tubulin Isotypes on Neurite Microtubules -- I. Introduction -- II. Scope and Application -- III. Labeling Protocols (Preembedding) -- IV. Results and Perspectives -- V. Controls and Critical Considerations -- VI. Troubleshooting Guide -- VII. Conclusion -- VIII. Appendix -- References -- Chapter 15. Postembedding Immunocytochemical Techniques for Light and Electron Microscopy -- I. Introduction -- II. Protocols -- III. Recipes -- References -- Chapter 16. Electron Microscopy Immunocytochemistry Following Cryofixation and Freeze Substitution -- I. Introduction -- II. Systems and Goals -- III. Cryofixation, Freeze Substitution, and Embedding -- IV. Antibodies and Gold Markers -- V. Protocols -- VI. Conclusions and Perspectives -- References -- Chapter 17. Microinjection of Antibodies in the Analysis of Cellular Architecture and Motility -- I. Introduction: The Scope of the Play -- II. Actors and Requisites in the Play -- III. The Set -- IV. The Performance -- V. The Lessons to Be Learned -- VI. Perspectives -- References -- Chapter 18. Immunoselection and Characterization of cDNA Clones -- I . Introduction -- II. Overview of a Typical Immunoselection Project -- III. Preparation of Antibodies for Library Screening -- IV. Choice and Preparation of Expression Libraries -- V. Immunoselection of Clones from cDNA Librariesrs -- VI. Characterization of Cloned cDNAs -- VII. Cloned cDNAs as Hybridization Probes -- VIII. Fusion Protein Analysis. , IX. Confirmation of cDNA Clones -- X. Sequence Analysis of Cloned cDNAs -- XI. Materials and Reagents -- References -- Chapter 19. Antiidiotypic Antibodies: Methods, Applications, and Critique -- I . Introduction -- II. Structural Basis and Main Principles of Antiidiotypic Approach -- III. Production and Characterization of Antiidiotypic Antibodies -- IV. Receptor Identification by Antiidiotypic Antibodies -- V. Applications: Use of Antiidiotypic Antibodies to Study Molecular Interactions of Intermediate Filament Proteins -- VI. Conclusions -- References -- Appendix -- Index -- Volumes in Series.

Note: Cover -- Copyright Page -- Contents -- Contributors -- Preface -- Part I: Overview -- Chapter 1. Laboratory and Evolutionary History of Tetrahymena thermophila -- Summary -- I. Characters in Search of a Script -- II. Paramecium Precursors -- III. Tetrahymena Breeding Studies -- IV. Perils of Passage -- V. Doing Genetics by Evolution -- References -- Chapter 2. Cell Biology of Tetrahymena thermophila -- I. Introduction -- II. Membranes and Membrane-Bounded Compartments -- III. The Cytoskeleton and Membrane Skeleton -- IV. Dynamics of the Cell Cycle -- V. Spacial Patterning -- VI. Starvation-Mediated Development -- VII. Concluding Remarks -- References -- Chapter 3. Tetrahymena Genetics: Two Nuclei Are Better Than One -- I. Asexual and Sexual Reproduction -- II. Nuclear Dualism -- III. Special Features of Tetrahymena Genetics -- IV. DNA-Mediated Transformation -- V. Summary and Prospects -- References -- Part II: Methods -- Chapter 4. Tetrahymena as a Laboratory Organism: Useful Strains, Cell Culture, and Cell Line Maintenance -- I. Introduction -- II. Useful Tetrahymena Strains -- III. Culture Media -- IV. Culturing Tetrahymena Cells -- V. Cell Line Storage -- References -- Chapter 5. Long-Term Storage -- I. Introduction -- II. Supplies -- III. Preparation of Cells before Freezing -- IV. Freezing -- V. Thawing and Recovery -- VI. Conclusion -- References -- Chapter 6. Genetic Crosses: Setting Up Crosses, Testing Progeny, and Isolating Phenotypic Assortants -- I. Introduction -- II. Methods for Mating Cells -- III. How to Isolate and/or Select Progeny from a Cross -- IV. Mating Type Tests and Sexual Immaturity Tests -- V. Isolating Assortants -- References -- Chapter 7. Methods for Genetic Analysis -- I. Introduction -- II. Reagents -- III. Equipment -- IV. Strains -- V. Strategies -- References. , Chapter 8. Isolation of Micronuclear and Macronuclear DNA -- I. Introduction -- II. Cell Culture -- III. Percoll Method for Isolation of Nuclei -- IV. Isolation of High Molecular Weight DNA from Nuclei -- V. Solutions -- References -- Chapter 9. Genetically Sorting a Collection of Tetrahymena Mutants -- I. Introduction -- II. Mapping the Mutations to Chromosome Arms -- III. Genetic Complementation Tests -- References -- Chapter 10. Genetically Mapping New Mutants and Cloned Genes -- I. Introduction -- II. Genetically Mapping a Mutation -- III. Genetically Mapping a Cloned Gene or Sequence -- References -- Chapter 11. Selection of Motility Mutants -- I. Introduction -- II. Materials -- III. Special Procedures and Equipment -- IV. Strains -- V. Inducing and Screening for Temperature-Sensitive Mutations Affecting Cilia Regeneration -- VI. Mutagenesis by Targeted Gene Knockout -- References -- Chapter 12. Nuclear and Cytoskeletal Fluorescence Microscopy Techniques -- I. Introduction -- II. DAPI: A Fluorescent Nuclear Stain -- III. Immunofluorescence Labeling for Conventional Fluorescence Microscopy -- IV. Immunofluorescence Labeling for Confocal Fluorescence Microscopy -- References -- Chapter 13. Nuclear and Cortical Histology for Brightfield Microscopy -- I. Introduction -- II. Silver Staining -- III. Protargol Staining -- IV. Giemsa Staining -- References -- Chapter 14. Fixation of Tetrahymena Cells for Electron Microscopy -- I. Introduction -- II. Cell Culture -- III. Fixation -- IV. Critical Point Drying for SEM -- V. Embedding for TEM -- VI. Staining Sections (TEM) -- References -- Chapter 15. Immunoelectron Microscopy of Tetrahymena -- I. Introduction -- II. General Considerations -- III. Postembedding Labeling -- IV. Double Labeling with the Postembedding Technique -- V. Pre-embedding Labeling -- VI. Controls for Immunogold Labeling. , VII. Quantitative Analysis of Colloidal Gold Labeling -- VIII. Troubleshooting: Problems and Recommended Remedies -- References -- Part III: Cell Biology -- Chapter 16. Regulated Protein Secretion in Tetrahymena thermophila -- I. Introduction -- II. Isolation of the Released Contents of Dense-Core Secretory Vesicles (Mucocysts) -- III. Stimulation of Cells to Test Exocytic Capability or to Study Postexocytic Events -- IV. Isolation of Intact Secretory Vesicles -- References -- Chapter 17. Electrophysiology of Tetrahymena -- I. Introduction -- II. Materials and Methods -- III. Commentary and Summary -- References -- Chapter 18. Isolation and Characterization of in Vivo Modified Histones and an Activity Gel Assay for Identification of Histone Acetyltransferases -- I. Introduction -- II. Methods -- References -- Chapter 19. Tetrahymena Telomerase Activity, Purification, and Reconstitution -- I. Introduction -- II. Materials and Methods -- III. Commentary and Summary -- References -- Chapter 20. Studying the Telomerase RNA in Tetrahymena -- I. Introduction -- II. General Strategy and Overview -- III. Basic Methods -- References -- Chapter 21. Isolation and Characterization of 22S Outer Arm Dynein from Tetrahymena Cilia -- I. Introduction -- II. Methods -- III. Summary -- References -- Chapter 22. Preparation of Cytoskeletal Fractions from Tetrahymena thermophila -- I. Introduction -- II. Methods -- References -- Chapter 23. Immunoprecipitation Procedures -- I. Introduction -- II. Method -- References -- Chapter 24. Tetrahymena Calcium-Binding Proteins, TCBP-25 and TCBP-23 -- I. Introduction -- II. Methods -- References -- Part IV: Manipulating Genes -- Chapter 25. Microinjection of Tetrahymena thermophila -- I. Introduction -- II. The Technique of Microinjection -- III. Concluding Remarks -- References. , Chapter 26. Transient and Stable DNA Transformation of Tetrahymena thermophila by Electroporation -- I. Introduction -- II. Considerations -- III. Preparing Plasmid DNA for Electrotransformation -- IV. General Protocol for Conjugant Electrotransformation -- V. Transformation with Integrating Vectors: Gene Replacements and Gene Knockouts -- VI. Transformation with Autonomously Replicating Vectors -- References -- Chapter 27. Biolistic Transformation of Macro- and Micronuclei -- I. Introduction -- II. Cells -- III. Transformation -- References -- Chapter 28. Knockout Heterokaryons Enable Facile Mutagenic Analysis of Essential Genes in Tetrahymena -- I. Introduction -- II. Strategy -- III. Methods for Creating Knockout Heterokaryons -- References -- Chapter 29. Creation and Use of Antisense Ribosomes in Tetrahymena thermophila -- I. Introduction -- II. Using Antisense Ribosomes in Tetrahymena thermophila -- III. Considerations Particular to Antisense Ribosomes -- IV. Evaluation and Applications of the Antisense Ribosome System -- References -- Chapter 30. Protein Tagging in Tetrahymena -- I. Introduction -- II. Strategies for Gene Tagging in Tetrahymena -- III. Methods -- References -- Appendix I. Genetic Nomenclature Rules for Tetrahymena thermophila -- Appendix II. Codon Usage in Tetrahymena thermophila -- Index -- Volumes in Series.