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Methods in Neurosciences, Volume 1: Gene Probes is a compendium of papers that deals with the developments in molecular biology, cell biology, and electrophysiology. Section I… Read more
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Contributors to Volume 1
Preface
Section I Gene Expression
Chapter 1: Using the Xenopus Oocyte System for Expression and Cloning of Neuroreceptors and Channels
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Introduction
Methods
Chapter 2: Expression of Neurotransmitter Receptors and Voltage-Activated Channels from Brain mRNA in Xenopus Oocytes
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Extraction and Purification of mRNA
Preparation and Injection of Oocytes
Electrophysiological Recording from mRNA-Injected Oocytes
Other Applications
Chapter 3: Expression of Mammalian Plasma Membrane Receptors in Xenopus Oocytes: Studies of Thyrotropin-Releasing Hormone Action
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Introduction
Methods
Results
Chapter 4: Expression of Exogenous Voltage-Gated Calcium Channels in Xenopus Oocytes
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RNA Isolation Procedures
Preparation and Injection of Oocytes
Electrophysiology of Ca2+ Channels Expressed in Oocytes
Section II In Situ and Solution Hybridization
Chapter 5: Quantification of in Situ Hybridization Histochemistry for Analysis of Brain Function
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Introduction
Oligonucleotide Probes for ISHH
Standards for ISHH
Image Analysis of ISHH
Quantify Procedures
Chapter 6: In Situ Hybridization: A Methodological Guide
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Introduction: Basic Principles
Laboratory Environment
Tissue Preparation
Prehybridization and Hybridization
Posthybridization Treatments
Preparation of Autoradiograms
Controls
Analysis and Troubleshooting
Combination with Other Techniques, and Use of Nonisotopically Labeled Probes
Appendix
Chapter 7: Combination of in Situ Hybridization with Immunohistochemistry and Retrograde Tract-Tracing
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Introduction
Methodology
Results
Summary
Chapter 8: Localization of Neuronal mRNAs by Hybridization Histochemistry
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Introduction
Solutions and Materials
Experimental Procedures
Quantitation of Results
Controls for Specificity
Conclusions
Chapter 9: In Situ Detection of Peptide Messenger RNA Using Complementary RNA Probes
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Introduction
Methodology
Localization of Peptide mRNAs in the Neuroendocrine System
Conclusion
Chapter 10: Quantification of mRNA in Discrete Cell Groups of Brain by in Situ Hybridization Histochemistry
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Introduction
Chapter 11: In Situ Hybridization Approaches to Human Neurological Disease
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Maintenance of RNA Integrity in Human Postmortem Brain Tissue
Tissue Preparation
In Situ Hybridization Procedure
Application of Quantitative In Situ Hybridization Methods for Study of Gene Expression in Human Neurological Disease
Chapter 12: Localization of Peptide Gene Expression by in Situ Hybridization at Electron Microscopic Level
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Introduction
Preparations of cDNA Probes
Fixation and Tissue Preparation
Hybridization Procedure and Embedding
Combination of Immunocytochemistry and in Situ Hybridization
Localization of PRL mRNA
Localization of POMC and Preprosomatostatin mRNAs
Discussion
Chapter 13: In Situ Hybridization for Detecting Gonadotropin-Releasing Hormone Messenger RNA and Measuring Physiologically Stimulated Changes
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Introduction
Experimental Procedures
Conclusion
Future Directions
Chapter 14: Location of Gene Expression in Tissue Sections by Hybridization Histochemistry Using Oligodeoxyribonucleotide Probes
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Methodology
Discussion of Method
Comparison of Methods
Conclusion
Chapter 15: In Situ mRNA Hybridization: Standard Procedures and Novel Approaches
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Introduction
General Methods
Emerging Techniques
Chapter 16: Nonradioactive in Situ Hybridization Histochemistry with Digoxigenin–Deoxyuridine 5′-Triphosphate-Labeled Oligonucleotides
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Introduction
Solutions
Resolution
Concluding Remarks
Chapter 17: Quantitation of Nuclear Low-Level Gene Expression in Central Nervous System Using Solution Hybridization and in Situ Hybridization
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Introduction
Solution Hybridization/Nuclease Protection Assay
Isolation of Cell Nuclei and Cytoplasm
Quantitation of Nuclear hnRNA and mRNA
Intervening Sequence in Situ Hybridization
Conclusion
Section III Screening, Sequencing, and Cloning
Chapter 18: Immunoscreening λgt11 cDNA Library
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Theory
Choice of Antiserum
Preparation of Plates
Preparation of Top Agar
Selection of Bacterial Host
Preparation and Plating of Phage
Amplification by Plate Lysis
Preparation of Membrane Blots of Plaques
Immunoscreening of the Blotted Membrane
What Is a Positive Result?
Plaque Purification
Chapter 19: Rapid Identification of DNA Clones: Utilization of Same Degenerate Oligonucleotides for Both Screening and Sequencing
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Introduction
Materials and Methods
Results
Discussion
Chapter 20: Molecular Cloning of Nicotinic Acetylcholine Receptor Genes
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Introduction
Cloning of cDNAs for Muscle and Neuronal Nicotinic Acetylcholine Receptors
Genomic Cloning of Nicotinic Acetylcholine Receptors
Section IV Lineage Analysis
Chapter 21: Lineage Analysis in the Vertebrate Nervous System by Retrovirus-Mediated Gene Transfer
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Introduction
Overall Strategy
Vector Designs
Infection of Target Cells
Detection of Infected Cells via X-Gal Histochemistry
Evaluation of Lineage Data
Chapter 22: Use of Transgenic Models to Access Neural Lineages in Mammals
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Designing Transgenes Which Exhibit Neuronal Expression
Purification of Fragments for Microinjection
Preparation of Pseudopregnant Host Mice
Production of Fertilized Mouse Eggs
Preparation of Holding and Injection Pipettes
Pronuclear Microinjections
Surgical Reimplantations of Injected Eggs
Analysis of Neural Lineages Using Transgenic Mice
Section V Molecular Pathology
Chapter 23: Characterization of Molecular Pathology of Alzheimer’s Disease
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Amyloid Fibril and Its Precursor
Paired Helical Filament
Procedures: cDNA Library Construction from Human Brain
Section VI Appendixes
Appendix I: Restriction Endonuclease Sites of Cleavage
Appendix II: Amino Acids and Genetic Code
Index
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