Thông tin chi tiết tài liệu

Quốc hội

Thủ tướng Chính phủ

Liên bộ

Danh sách các Bộ, Ngành

Click vào tìm kiếm

Danh sách các tỉnh & thành phố trực thuộc Trung ương

Chi tiết tài liệu

Extracellular Matrix

Tác giả: Leach, Jennie B.; Powell, Elizabeth M.

Chủ đề:

Chủ đề: Medicine; Neuromethods; Extracellular Matrix

Nhà xuất bản: Humana Press

Năm xuất bản: 2015

Loại tài liệu: Book

Mô tả vật lý: 225 p.

Định danh: 978-1-4939-2083-9

Ngôn ngữ: en

Liên kết: Neuromethods;Book 93

Đọc trực tuyến

Tải tài liệu

Divided into four parts, each focusing on different aspects of the extracellular matrix and the nervous system, Extracellular Matrix covers methods to analyze native tissue, in vitro models for investigating cell-extracellular matrix interactions in a variety of contexts, protocols to investigate the role of the extracellular matrix in nervous system injury, degeneration, and regeneration, as well as therapeutics and engineered systems. Each chapter is written by leading experts and presents established protocols in a concise format, encompassing current technologies as well as methods developed over years of research. Beginning with an introduction to the method, chapters continue with a listing of the materials and equipment, step-by-step protocols, and a Notes section with troubleshooting tips, supplemental details and alternative approaches, as well as a list of references for further information.

Contents
Contributors
Preface
Series Preface
Cover
Neuromethods
Title pages
Part I: Native Tissue
- Chapter 1: Immunohistochemical Characterization of Brain Neural and Vascular Basement Membranes
  - 1 Introduction
  - 2 Materials
    - 2.1 Brain Sectioning Components
    - 2.2 Immunostaining Components
  - 3 Methods
    - 3.1 Brain Fixation and Sectioning
    - 3.2 Immunofluoresưcent Staining
  - 4 Notes
  - References
- Chapter 2: The Use of Synaptic Basal Lamina and Its Components to Identify Sites of Recent Morphological Alterations at Mammalian Neuromuscular Junctions
  - 1 Introduction
  - 2 Materials
    - 2.1 Reagents for Conjugation of Alexa Fluor 647 to Fasciculin 2
    - 2.2 Reagents for Purification of Fasciculin 2-Alexa Fluor 647 Dye Conjugate
    - 2.3 Antibodies and Solutions for Immunohistocheưmistry
    - 2.4 Electron Microscopy (EM) Reagents
  - 3 Methods
    - 3.1 Preparation of Bio-Gel P2 Column (3 Columns): The Purification Resin (Component E) Is Replaced with Bio-Rad Bio-Gel P2 Gel (See Note 3)
    - 3.2 Toxin-Dye Reaction
    - 3.3 Purifying the Toxin-Dye Conjugate
    - 3.4 Fluorescent Labeling of Whole-Mount Skeletal Muscle
    - 3.5 Processing Muscle Tissues for EM
    - 3.6 EM Imaging of NMJs
  - 4 Notes
  - References
- Chapter 3: Perineuronal Nets: A Special Structure in the Central Nervous System Extracellular Matrix
  - 1 Introduction
  - 2 Materials
    - 2.1 GAGs Extraction
    - 2.2 Dialysis
    - 2.3 Cetylpyridinium Chloride (CPC) Turbidity Assay
    - 2.4 Measurement
  - 3 Methods
    - 3.1 Day 1: Homogenization and Dialysis
    - 3.2 Day 2: Pronase Treatment
    - 3.3 Day 3: Protein Precipitation
    - 3.4 Day 4: Glycan Precipitation
    - 3.5 Day 5: Glycan Collection and Quantification by CPC Turbidimetry
  - 4 Notes
  - References
Part II: In Vitro Models
- Chapter 4: Isolation of Perinatal Mouse Cerebral Cortical Astrocytes
  - 1 Introduction
  - 2 Materials
    - 2.1 Dissection of Cerebral Cortex
    - 2.2 Tissue Culture
    - 2.3 For Culturing on Glass Coverslips
    - 2.4 Immunocytocheưmistry
    - 2.5 General Supplies
    - 2.6 Equipment
  - 3 Methods
    - 3.1 Dissection of Mouse Cerebral Cortex
    - 3.2 Growth and Maintenance of Astrocyte Cultures
    - 3.3 Replating Purified Astrocytes
    - 3.4 Poly-l-lysine-ưCoated Coverslips
    - 3.5 Immunocytocheưmistry to Assess Astrocyte Purity
  - 4 Notes
  - References
- Chapter 5: Techniques for Examining the Effect of Substratum-Bound Proteoglycans on Neurite Outgrowth In Vitro
  - 1 Introduction
  - 2 The Stripe Assay for Examining Neurite Outgrowth
    - 2.1 Materials
      - 2.1.1 Petri-Dish Glass Well Preparation
      - 2.1.2 Petri-Dish Substratum Preparation
      - 2.1.3 Application of Chondroitin Sulfate Proteoglycan
      - 2.1.4 Preparation of Embryonic Chicken DRG Explants
    - 2.2 Methods
      - 2.2.1 Petri-Dish Glass Well Preparation
      - 2.2.2 Petri-Dish Substratum Preparation
      - 2.2.3 Application of Chondroitin Sulfate Proteoglycan (CSPG) for Stripe Assay
      - 2.2.4 Dorsal Root Ganglion (Sensory) Neuron Dissection
  - 3 The Step Gradient Assay
    - 3.1 Materials
      - 3.1.1 Application of Chondroitin Sulfate Proteoglycan Gradient
    - 3.2 Methods
      - 3.2.1 Application of Chondroitin Sulfate Proteoglycan Gradient
  - 4 Notes
  - 5 Commentary
  - References
- Chapter 6: High-Throughput Quantitative Assay for Analyzing Neurite Outgrowth on a Uniform Substratum: The Cell-Substratum Assay
  - 1 Introduction
  - 2 Materials
    - 2.1 Materials for Chick DRG Dissociation
    - 2.2 Materials for the Cell-ưSubstratum Assay
    - 2.3 Materials for the Cell-ưSubstratum Assay Immunocytochemistry
    - 2.4 Materials for the Cell-ưSubstratum Assay Imaging Protocol
  - 3 Methods
    - 3.1 Chick DRG Dissociation
    - 3.2 The Cell-ưSubstratum Assay
    - 3.3 The Cell-ưSubstratum Assay Immunocytochemistry
    - 3.4 Neurite Outgrowth Imaging Protocol (Specific for the Zeiss Axiovert 200M Microscope)
    - 3.5 Neurite Outgrowth Data Analysis
  - 4 Notes
  - 5 Commentary
  - References
- Chapter 7: Extracellular Matrix Components as a Substrate for Outgrowing Motoneurons
  - 1 Introduction
  - 2 Materials
    - 2.1 Motoneuron Isolation and Culture
    - 2.2 Cell Culture and Matrix Preparation
  - 3 Methods
    - 3.1 Motoneuron Isolation
    - 3.2 Maintenance of Cell Lines
    - 3.3 Matrix Preparation
    - 3.4 Co-culture of Motoneurons and Cell-Derived Matrices
  - 4 Notes
  - References
- Chapter 8: Replicating the In Vivo Environment: Organotypic and Submerged Three-Dimensional Culture Methods
  - 1 Introduction
  - 2 Materials
    - 2.1 Collagen Gel
    - 2.2 Cell or Tissue Culture
    - 2.3 For Culturing the 3D Gels on the Surface of Media
    - 2.4 For Culturing on Glass Coverslips
    - 2.5 General Supplies
    - 2.6 Equipment
  - 3 Methods
    - 3.1 Nuclepore Membrane Preparation for Floating Culture Option
    - 3.2 Glass Coverslip Preparation for Submerged Culture Option
    - 3.3 Collagen Gel Preparation
    - 3.4 Collagen Gel Preparation for Floating Culture Option
    - 3.5 Collagen Gel Preparation for Submerged Culture Option
    - 3.6 Collagen Gel Preparation for Tissue Explants
  - 4 Notes
  - References
- Chapter 9: Culturing Neurons, Glia, and Progenitor Cells in Three-ưDimensional Hydrogels
  - 1 Introduction
  - 2 Materials (see Notes 1 and 2)
    - 2.1 Buffers, Media, and Biological Solutions
    - 2.2 Other Chemicals
    - 2.3 Supplies and Equipment
  - 3 Methods
    - 3.1 Culture in 3D Hydrogels
    - 3.2 Viability Staining
    - 3.3 Fixation and DAPI Staining
    - 3.4 Isolation of Live Cells Following Encapsulation and Culture
  - 4 Notes
  - References
- Chapter 10: Investigating Cell-ECM Interactions and ECM Synthesis in Three-Dimensional Hydrogels
  - 1 Introduction
  - 2 Materials (see Notes 1 and 2)
    - 2.1 Buffers and Biological Solutions
    - 2.2 Other Chemicals
    - 2.3 Supplies and Equipment
  - 3 Methods
    - 3.1 Immunofluoresưcence Labeling and Imaging in 3D Hydrogels
      - 3.1.1 Immunofluoresưcence Labeling
      - 3.1.2 Confocal Microscopy
    - 3.2 Extraction of Proteins from 3D Hydrogels
  - 4 Notes
  - References
Part III: Injury, Degeneration, and Regeneration
- Chapter 11: Characterization and Analysis of Extracellular Matrix in Malignant Brain Tumors and Their Cellular Derivatives
  - 1 Introduction
  - 2 Materials
    - 2.1 General Stock Solutions and Materials for Subcellular Fractionation
    - 2.2 Deglycosylation Components
    - 2.3 SDS-ưPolyacrylamide Gel Electrophoresis (SDS-PAGE) Components
    - 2.4 Western Blotting Components
    - 2.5 General Stock Solutions and Materials for Immunochemistry
  - 3 Methods
    - 3.1 Subcellular Fractionation for Biochemical Studies
    - 3.2 Extraction of Aggregated or Poorly Soluble ECM Molecules
    - 3.3 Deglycosylation of Protein Samples Before SDS-PAGE
    - 3.4 Deglycosylation of Blotted Proteins After SDS-PAGE
    - 3.5 SDS-PAGE and Western Blotting
    - 3.6 Brain Tissue Preparation for ECM Immunochemistry
    - 3.7 Tumorsphere Preparation for ECM Immunochemistry
    - 3.8 Immunochemical Staining of ECM
  - 4 Notes
  - References
- Chapter 12: Chondroitinase Gene Therapy for Spinal Cord Injury
  - 1 Introduction
    - 1.1 Ch’ase Application as a Therapeutic Strategy for SCI
  - 2 Materials
    - 2.1 Reagents
    - 2.2 Buffers and Reagents
  - 3 Methods
    - 3.1 Preparation of Samples
    - 3.2 Preparation of ELISA Plates
    - 3.3 Sample Addition: Since This Is an Enzymatic Reaction, Have All Samples Diluted and Ready to Be Added
  - References
- Chapter 13: Modulation of Neural Stem Cell Expressed Extracellular Matrix (ECM) by Targeting Glycosyltransferases
  - 1 Introduction
  - 2 Materials
    - 2.1 Cell Culture Materials
    - 2.2 Cell Culture Reagents
    - 2.3 Other Reagents and Equipment
    - 2.4 Preparation of Cell Culture Stock Solutions
  - 3 Methods
    - 3.1 Derivation of Neurospheres from Primary Mouse Tissue
    - 3.2 Nucleofection of Cortical Neurospheres
    - 3.3 Assays
      - 3.3.1 Differentiation
      - 3.3.2 Proliferation
      - 3.3.3 Notes
  - References
Part IV: Therapeutics and Engineered Systems
- Chapter 14: Evaluating the Spatial and Temporal Protein Production in Neural Tissue Engineering Constructs In Vitro
  - 1 Introduction
  - 2 Materials
    - 2.1 In Vitro Cell Culture System
    - 2.2 Western Blot
      - 2.2.1 Gel Casting Components
      - 2.2.2 Cell Lysis and Protein Quantification Components
      - 2.2.3 Electrophoresis Components
      - 2.2.4 Transfer Components
      - 2.2.5 Antibody Visualization
    - 2.3 Immunocytocheưmistry
      - 2.3.1 Fixation
      - 2.3.2 Cryoembedding
      - 2.3.3 Cryosectioning
      - 2.3.4 Immunostaining
  - 3 Methods
    - 3.1 In Vitro Cell Culture System
    - 3.2 Western Blot
      - 3.2.1 Cell Lysis and Protein Quantification
      - 3.2.2 Preparation of 6 % SDS-Polyacrylamide Gel
      - 3.2.3 SDS-PAGE Sample Preparation, Electrophoresis, and Membrane Transfer
      - 3.2.4 Visualization of Bands of Interest
    - 3.3 Immunocytocheưmistry
      - 3.3.1 Fixation
      - 3.3.2 Cryoembedding
      - 3.3.3 Cryosectioning
      - 3.3.4 Immunostaining
  - 4 Notes
  - References
- Chapter 15: Cultivation and Imaging of Astrocytes on Protein-Coated Fluorescent Topographies Constructed from Aligned PLLA Electrospun Fibers
  - 1 Introduction
  - 2 Materials
    - 2.1 Materials and Equipment for Electrospinning PLLA Fibers
    - 2.2 Materials for Isolation of Postnatal Day 1 Rat Astrocytes
    - 2.3 Components for Sterilizing and Coating PLLA Fibers with ECM Proteins
    - 2.4 Cell Culture Media and Components
    - 2.5 Staining and Imaging of Astrocytes on Fluorescent Fibers
  - 3 Methods
    - 3.1 Electrospinning PLLA Fibers
      - 3.1.1 Casting PLLA Films
      - 3.1.2 Electrospinning PLLA Fibers
    - 3.2 Sterilizing and Coating PLLA Fibers with ECM Proteins
    - 3.3 Isolation and Cultivation of Primary Rat Astrocytes
      - 3.3.1 Setup Procedures
      - 3.3.2 Brain Tissue Dissection
      - 3.3.3 Isolation of Brain Cells and Preparation of Primary Astrocyte Cultures
    - 3.4 Plating Primary Rat Astrocytes on PLLA Fibers for Physiology Studies
    - 3.5 Staining and Imaging of Astrocytes on Fluorescent Fibers
      - 3.5.1 Immunocyto-chemistry on Fiber-Coated Substrates
      - 3.5.2 Imaging of Immunostained Astrocytes
  - 4 Technical Notes
  - References
- Chapter 16: Engineered Microdevices to Study and Manipulate Neural Stem Cell Chemotaxis
  - 1 Introduction
  - 2 Materials
    - 2.1 Cell Culture
    - 2.2 Microfluidic Device Fabrication and Assembly
  - 3 Methods
    - 3.1 Microfluidic Device Fabrication
    - 3.2 Neural Progenitor Cell Culture and Cell Seeding Within Devices
    - 3.3 Device Assembly and Imaging
    - 3.4 Cell Tracking and Data Analysis
  - 4 Notes
  - References
- Chapter 17: Hydrolytically Degradable Polyethylene Glycol (PEG) Hydrogel: Synthesis, Gel Formation, and Characterization
  - 1 Introduction
  - 2 Materials
    - 2.1 Reagents
    - 2.2 Equipment
  - 3 Methods
    - 3.1 Azeotropic Distillation of 4-Arm PEG
    - 3.2 4-Arm PEG-Vinyl Sulfone (4-Arm PEG-VS) Synthesis
    - 3.3 PEG-Diester-ưDithiol Synthesis
    - 3.4 PEG Hydrogel Preparation for Cell Encapsulation
    - 3.5 PEG Hydrogel Preparation for Mass Loss and Swelling Measurements
    - 3.6 Mass Loss and Swelling Measurements
  - 4 Notes
  - References
Index