Download Alzheimer's Disease and Frontotemporal Dementia - Methods and Protocols - E. Roberson (Humana, 2011) WW PDF

TitleAlzheimer's Disease and Frontotemporal Dementia - Methods and Protocols - E. Roberson (Humana, 2011) WW
TagsMedical
LanguageEnglish
File Size36.0 MB
Total Pages290
Table of Contents
                            front-matter.pdf
	Alzheimer’s Diseaseand Frontotemporal Dementia
		Preface
		Contents
		Contributors
fulltext
	Chapter 8: Cell-Based Assays for Regulators of Tau Biology
		1. Introduction
			1.1. In-Cell Western
			1.2. Western Blotting with the Odyssey System
		2. Materials
			2.1. In-Cell Western
				2.1.1. Cell Culture and Treatment
				2.1.2. In-Cell Western Assay
			2.2. Western Validation of Hits
				2.2.1. Cell Lysis
				2.2.2. Protein Estimation
				2.2.3. Western Blotting with the Odyssey System
		3. Methods
			3.1. In-Cell Western
				3.1.1. Cell Culture and Treatments
				3.1.2. In-Cell Western Assay (Per Four Plates)
			3.2. Western Validation of Hits
				3.2.1. Cell Culture and Treatment
				3.2.2. Cell Lysis
				3.2.3. Protein Estimation
				3.2.4. Western Blotting with the Odyssey System
		4. Notes
		References
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	Chapter 1: Contemporary Approaches to Alzheimer’s Disease and Frontotemporal Dementia
		1. Alzheimer’s Disease
		2. Frontotemporal Dementia
		3. Molecules Involved in AD and FTD
		4. Experimental Systems for AD and FTD
		References
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	Chapter 2: Preparing Synthetic Ab in Different Aggregation States
		1. Introduction
			1.1. Overview of Experimental Methods to Prepare and Characterize Defined Ab Assemblies (Unaggregated, Oligomers, Fibrils,
			1.2. Preparation and Use of Fluorophore-Labeled Ab42 Assemblies
		2. Materials
			2.1. Preparation of HFIP-Treated Ab Peptide Stocks
			2.2. Unaggregated Ab42 Preparation
			2.3. Oligomeric Ab42 Preparation
			2.4. Fibrillar Ab42 Preparation
			2.5. “Plaques in a Dish” Preparation
			2.6. Fluorophore-Labeled Ab42 Oligomer Preparation
			2.7. Structural Characterization of Ab42 Preparations
				2.7.1. Western Blot Analysis by SDS-PAGE
				2.7.2. Atomic Force Microscopy Structural Analysis
			2.8. Functional Characterization of Ab42 Preparations
				2.8.1. Neurotoxicity Assay (For Example, Figs. 1c, d and 2b). (See Note 5)
				2.8.2. Neuronal Uptake Assay (Fig. 6b)
		3. Methods
			3.1. Preparation of HFIP-Treated Ab Peptide Stocks (Figs. 3 and 4)
			3.2. Unaggregated Ab Preparation (see Note 14)
			3.3. Oligomeric Ab Preparation
			3.4. Fibrillar Ab Preparation
			3.5. “Plaque in a Dish” Preparation
			3.6. Fluorophore-Labeled Ab42 Oligomer Preparation (Fig. 6)
			3.7. Structural Characterization of Ab42 Preparations
				3.7.1. Western Analysis by SDS-PAGE (see Note 16)
				3.7.2. Atomic Force Microscopy
			3.8. Functional Characterization of Ab42 Preparations
				3.8.1. Neurotoxicity/Viability Assay (Figs. 1c, d and 2b) (see Note 5)
				3.8.2. Neuronal Uptake Assay (Fig. 6b)
		4. Notes
		References
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	Chapter 3: Isolation of Low-n Amyloid b-Protein Oligomers from Cultured Cells, CSF, and Brain
		1. Introduction
		2. Materials
			2.1. Culture of 7PA2 Cells
			2.2. Serial Extraction of Human Brain Tissue
			2.3. Immuno­precipitation
			2.4. Size Exclusion Chromatography
			2.5. SDS-Polyacrylamide Gel Electrophoresis and Western Blotting
		3. Methods
			3.1. Culture of 7PA2 Cells and Generation of Oligomer-Containing Conditioned Medium
			3.2. Preparation of Human Brain Extracts
			3.3. Immunopreci­-pitation
			3.4. Size Exclusion Chromatography
			3.5. Denaturing PAGE and Western Blotting
		4. Notes
		References
fulltext_004
	Chapter 4: Detecting Ab*56 Oligomers in Brain Tissues
		1. Introduction
		2. Materials
			2.1. Brain Tissues and Lysis
			2.2. Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis
			2.3. Transfer and Western Blotting
			2.4. Immunoprecipi-tation
		3. Methods
			3.1. Brain Tissue Lysis and Fractionation
			3.2. Tris–Tricine SDS-PAGE and Western Blotting
			3.3. Immunoprecipitation
		4. Notes
		References
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	Chapter 5: Assessing Ab Aggregation State by Atomic Force Microscopy
		1. Introduction
		2. Materials
		3. Methods
			3.1. Preparation of Ab  Samples
			3.2. Ex Situ Studies of Ab
			3.3. In Situ Studies of Ab
			3.4. Quantitative Analysis of AFM Images
			3.5. Incorporating Other Factors into AFM Studies of Ab
		4. Notes
		References
fulltext_006
	Chapter 6: Measuring APP Carboxy-Terminal Fragments
		1. Introduction
		2. Materials
			2.1. Brain Lysis and Fractionation for APP CTFs
			2.2. Tris/Tricine SDS-Polyacrylamide Gel Electrophoresis for APP C-Terminal Fragments
			2.3. Western Blot for APP C-Terminal Fragments
			2.4. Brain Lysis for Measurement of Ab Peptides
			2.5. Acetic Acid Urea PAGE for Measurement of Ab Peptides
		3. Methods
			3.1. Brain Lysis and Fractionation for APP CTFs
			3.2. Tris/Tricine SDS-Polyacrylamide Gel Electrophoresis for Measurement of APP C-Terminal Fragments
			3.3. Western Blot for APP C-Terminal Fragments
			3.4. Brain Lysis for Measurement of Ab Peptides
			3.5. Acetic Acid Urea PAGE for Measurement of Ab Peptides
		4.  Notes
		References
fulltext_007
	Chapter 7: Detection of APP Intracellular Domain in Brain Tissue
		1. Introduction
		2. Materials
			2.1. Brain Tissue and Homogenization
			2.2. SDS-Polyacrylamide Gel Electrophoresis
			2.3. Western Blotting
		3. Methods
			3.1. Brain Tissue Sample Preparation
			3.2. SDS-PAGE
			3.3. Antigen Retrieval
			3.4. Western Blotting
		4. Notes
		References
fulltext_008
	Chapter 8: Cell-Based Assays for Regulators of Tau Biology
		1. Introduction
			1.1. In-Cell Western
			1.2. Western Blotting with the Odyssey System
		2. Materials
			2.1. In-Cell Western
				2.1.1. Cell Culture and Treatment
				2.1.2. In-Cell Western Assay
			2.2. Western Validation of Hits
				2.2.1. Cell Lysis
				2.2.2. Protein Estimation
				2.2.3. Western Blotting with the Odyssey System
		3. Methods
			3.1. In-Cell Western
				3.1.1. Cell Culture and Treatments
				3.1.2. In-Cell Western Assay (Per Four Plates)
			3.2. Western Validation of Hits
				3.2.1. Cell Culture and Treatment
				3.2.2. Cell Lysis
				3.2.3. Protein Estimation
				3.2.4. Western Blotting with the Odyssey System
		4. Notes
		References
fulltext_009
	Chapter 9: Split GFP Complementation Assay for Quantitative Measurement of Tau Aggregation In Situ
		1. Introduction
		2. Materials
			2.1. Preparation of Tau–GFP11 Constructs
			2.2. Qualitative Imaging of In Situ GFP Complementation
			2.3. Identify Optimal Amounts of Tau-GFP11 to Transfect
			2.4. Confirm Sensitivity of the Assay to Changes in Tau Aggregation
			2.5. Using Split GFP Complementation to Examine Changes in Tau Aggregation
			2.6. Western Blotting for Tau Expression
		3. Methods
			3.1. Preparation of Tau–GFP11 Constructs
			3.2. Qualitative Imaging of In Situ GFP Complementation
			3.3. Identify Optimal Amounts of Tau-GFP11 to Transfect
			3.4. Confirm Sensitivity of the Assay to Changes in Tau Aggregation
			3.5. Using Split GFP Complementation to Examine Changes in Tau Aggregation
			3.6. Western Blotting for Tau Expression
		4. Notes
		References
fulltext_010
	Chapter 10: Apolipoprotein E Expression and Purification
		1. Introduction
		2. Materials
			2.1. Transform Bacteria with ApoE Construct
			2.2. Protein Production, Induction, Harvest and Cell Lysis
			2.3. Lipidation and Thrombin Cleavage
			2.4. Lyophilization and Delipidation
			2.5. Chromatographic Purification
			2.6. SDS-PAGE and Coomassie Staining
		3. Methods
			3.1. Transform Bacteria with the ApoE Construct
			3.2. Protein Production, Induction, Harvest, and Cell Lysis
			3.3. Lipidation and Thrombin Cleavage
			3.4. Lyophilization and Delipidation
			3.5. Chromatographic Purification
			3.6. SDS-PAGE and Coomassie Staining
		4. Notes
		References
fulltext_011
	Chapter 11: Ab Toxicity in Primary Cultured Neurons
		1. Introduction
		2. Materials
			2.1. Coverslip and Culture Dish Preparations
			2.2. Cell Culture and Media
			2.3. Ab Aggregation
		3. Methods
			3.1. Preparation of Coverslips
			3.2. Coating of Culture Dishes and Coverslips with Poly-l-Lysine
			3.3. Cortical Astroglia Cell Culture
				3.3.1. Dissection
				3.3.2. Dissociation and Plating
				3.3.3. Maintenance
			3.4. Neuronal Culture
				3.4.1. Dissection
				3.4.2. Dissociation and Plating
				3.4.3. Maintenance
			3.5. Ab Aggregation
			3.6. Separation of Oligomeric and Fibrillar Ab
			3.7. Ab Treatment
		4. Notes
		References
fulltext_012
	Chapter 12: Manipulation of Gene Expression in the Central Nervous System with Lentiviral Vectors
		1. Introduction
		2. Materials
			2.1. Preparation of HEK293T Cells
			2.2. Transfection of Lentivirus-Encoding Plasmids
			2.3. Virus Concentration/Purification
			2.4. Virus Titration
			2.5. Primary Neuronal Cultures
			2.6. Stereotaxic Injection
		3. Methods
			3.1. Preparation of HEK293T Cells
			3.2. Transfection of Lentivirus-Encoding Plasmids
			3.3. Virus Concentration/Purification
			3.4. Virus Titration
				3.4.1. Biological Transduction Units (TU)
				3.4.2. Quantification of p24
			3.5. Virus-Mediated Gene Transfer in Primary Neural Cultures
				3.5.1. Neuronal Culture of P0 Rat Cortex
				3.5.2. Viral Infection of Primary Neural Cells
			3.6. Virus-Mediated Gene Transfer in Special Brain Regions
				3.6.1. Stereotaxic Injection
				3.6.2. Analysis of Gene Expression in the Brain
		4. Notes
		References
fulltext_013
	Chapter 13: Selecting a Mouse Model of Alzheimer’s Disease
		1. Introduction
		2. Major Mouse Models of AD
			2.1. Evaluating APP and APP/PS1 Transgenic Mice
			2.2. Evaluating Tau and APP/Tau Transgenic Mice
		3. Considerations in Selecting a Mouse Model of AD
		4. For More Information
		References
fulltext_014
	Chapter 14: Monitoring Spatial Learning and Memory in Alzheimer’s Disease Mouse Models Using the Morris Water Maze
		1. Introduction
		2. Materials
			2.1. Essential Components
			2.2. Useful Accessories
		3. Methods
			3.1. Planning
			3.2. Preparing the Tank
			3.3. Pretraining
			3.4. Cued Platform Training
			3.5. Hidden Platform Training
			3.6. Probe Trials
			3.7. Reversal Training
			3.8. Data Analysis
		4. Notes
		References
fulltext_015
	Chapter 15: Step-by-Step In Situ Hybridization Method for Localizing Gene Expression Changes in the Brain
		1. Introduction
		2. Materials
			2.1. Avoiding RNase Contamination
			2.2. Perfusing, Fixing, and Sectioning Brain Tissue
			2.3. Synthesis of Digoxigenin-Labeled Riboprobes
			2.4. RNA In Situ Hybridization
		3. Methods
			3.1. Avoiding RNase Contamination
			3.2. Perfusing, Fixing, and Sectioning Brain Tissue
				3.2.1. Perfusing and Fixing Brain Tissue
				3.2.2. Microtome Sectioning
				3.2.3. Cryostat Sectioning
			3.3. Generating Digoxigenin-Labeled Riboprobes
				3.3.1. Selecting cDNA/EST Clones
				3.3.2. Synthesizing Digoxigenin-Labeled Riboprobes
			3.4. RNA In Situ Hybridization
				3.4.1. RNA In Situ Hybridization for Floating Microtome Sections
				3.4.2. RNA In Situ Hybridization for Cryostat Sections
				3.4.3. Combined Fluorescence In Situ Hybridization and Immunohistochemistry
		4. Notes
		References
fulltext_016
	Chapter 16: Real-Time Visualization of Axonal Transport in Neurons
		1. Introduction
		2. Materials
			2.1. Preparation of Microfluidic Chamber
			2.2. Cell Culture
			2.3. Real-time Imaging of Axonal Transport
		3. Methods
			3.1. Preparation of Microfluidic Neuronal Culture Chamber
				3.1.1. Fabrication of Master Pattern on Si Wafer
				3.1.2. Preparation of PDMS Chamber by Replica Molding
				3.1.3. PLL Coating of Coverglass Substrate
			3.2. Culture DRG Neurons Inside Microfluidic Chambers
				3.2.1. Assembly of MNC Chamber
				3.2.2. Harvest DRG Neurons from Embryonic Rats
				3.2.3. Plating DRG or Cortical Neurons in MNC Chamber
				3.2.4. Selection and Maintenance of DRG Neurons in MNC Chamber
			3.3. Real-time Imaging of Axonal Transport of Qdot-NGF
				3.3.1. Preparation of Qdot-NGF
				3.3.2. Loading Qdot-NGF in the Distal Axon Compartment
				3.3.3. Fluorescence Imaging of Qdot-NGF Transport in Axons
		4. Notes
		References
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	Chapter 17: Quantifying Biomarkers of Cognitive Dysfunction and Neuronal Network Hyperexcitability in Mouse Models of Alzheime
		1. Introduction
		2. Materials
			2.1. Perfusion and Fixation of Brain Tissue
			2.2. Sectioning of Brain Tissue
			2.3. Immunohisto-chemistry
			2.4. Quantification of Results
		3. Methods
			3.1. Perfusion and Fixation of Brain Tissue
			3.2. Sectioning of Brain Tissue
			3.3. Immunohisto-chemical Staining
			3.4. Quantification of Results
				3.4.1. Quantification of Calbindin Immunoreactivity
				3.4.2. Quantification of NPY Immunoreactivity
				3.4.3. Quantification of Fos and Arc Immunoreactivity
		4. Notes
		References
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	Chapter 18: Epigenetic Changes in the Brain: Measuring GlobalHistone Modifications
		1. Introduction
		2. Materials
			2.1. Extracting Histone Proteins
			2.2. Western Blotting for Posttranslational Histone Modifications
			2.3. Analysis of Western Blots
		3. Methods
			3.1. Extracting Histone Proteins
			3.2. Western Blotting for Posttranslational Histone Modifications
			3.3. Analysis of Western Blots
		4. Notes
		References
back-matter
	Index
                        

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