【资源】[共享]Adhesion Protein Protocols——（很有用的）
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Contents:
Table of Contents
Adhesion Protein Protocols
Preface
Contents
Contributors
1— Monoclonal Antibodies Specific for Leukocyte Adhesion Molecules Selective Protocols of Immunizati...
1— Introduction
2— Materials
3— Methods
4— Notes
References
2— Epitope Mapping
3— Sequencing of Antibodies
4— Purification through Affinity Chromatography and Microsequencing
5— Functional Analysis of Cell Adhesion Molecules
6— Application of Polymerase Chain Reaction for the Discovery of New Adhesion Molecule Family Member...
1— Introduction
2— Materials
3— Method
4— Notes
Acknowledgments
References
7— Construction and Purification of Adhesion Molecule Immunoglobulin Chimeric Proteins
1— Introduction
2— Materials
2.1— Construction of Adhesion Molecule—Ig Chimeras
2.2— Electroporation of Cells
2.3— Harvest and Culture of Transfectant Clones
2.4— Selection (ELISA) and Subcloning
2.5— Purification of Chimeric Proteins
3— Methods
3.1— Construction of Adhesion Molecule-Ig Chimeras
3.2— Electroporation of Cells
3.3— Harvest and Culture of Transfectant Clones
3.4— Selection (ELISA) and subcloning
3.5— Purification of Chimeric Proteins
4— Notes
References
8— Immunofluorescence of Cultured Cells
1— Introduction
2— Materials and Preparative Work
3— Methods
3.1— Fixation Procedure
3.2— Permeabilization Procedure
3.3— Fixation-Permeabilization Procedure
3.4— First Antibody Incubation
3.5— Secondary Antibody Incubation
3.6— Mounting
4— Notes
References
9— Detection of Adhesion Molecules by Immunohistochemistry on Human and Murine Tissue Sections
1— Introductrion
1.1— Principles
1.2— Paraffin vs Frozen Sections
1.3— Interpretation of the Results
1.4— Immunohistochemistry in Murine Tissues
2— Materials
2.1— Formalin Fixation for Paraffin Embedding
2.2— Tissue Freezing and Cryostat Sectioning
2.3— Immunostaining Reagents
2.4— Cromogen Substrates
2.5— Counterstaining and Mounting
3— Methods
3.1— Formalin Fixation for Paraffin Embedding
3.2— Freezing of Tissue Samples
3.3— Preparation of Tissue Sections
3.4— The Immunohistochemistry Reaction in Human Tissues
3.4.1— The Primary Antibody
3.4.2— The Development Kit
3.4.3— Cromogen Substrates
3.4.4— Counterstaining
3.4.5— Controls and Interpretation of the Results
3.4.6— Immunohistochemistry of Adhesion Proteins
3.5— Immunohistochemistry in Murine Tissues
4— Notes
References
10— In Situ Hybridization with RNA Probes
1— Introduction
2— Materials
2.1— Tissue Embedding
2.2— Sectioning and Postfixation
2.3— Preparation of RNA Probes
2.4— Section Pretreatment
2.5— Hybridization
2.6— Post-Hybridization Washes and RNase Digestion
2.7— Autoradiography
2.8— Developing and Staining of Slides
3— Methods
3.1— Tissue Embedding
3.2— Sectioning and Postfixation
3.3— Preparation of RNA Probes
3.3.1— Preparation of the Linearized DNA template
3.3.2— In Vitro Transcription
3.3.3— Removal of the DNA Template
3.3.4— Probe Purification
3.3.5— Hydrolysis of RNA Probe (Optional)
3.4— Section Pretreatment
3.5— Hybridization
3.6— Posthybridization Washes and RNase digestion
3.7— Autoradiography
3.8— Developing and Staining of Slides
4— Notes
References
11— Purification of Fibronectin from Human Plasma
1— Introduction
2— Materials
3— Methods
3.1— Purification of Fibronectin
3.2— Quantitation, Electrophoretic Analysis, and Dialysis of Purified Fibronectin
4— Notes
References
12— Adhesion to Matrix Proteins
1— Introduction
2— Materials
3— Methods
4— Notes
References
13— Leukocyte-Endothelial Monolayer Adhesion Assay (Static Conditions)
1— Introduction
2— Materials
3— Method
3.1— Isolation of Peripheral Blood Leukocytes
3.2— Loading Leukocytes with BCECF
3.3— Adhesion Assay
4— Notes
Acknowledgment
References
14— Cell—Cell Adhesive Interactions in an In Vitro Flow Chamber
1— Introduction
2— Materials
2.1— Flow Chamber
2.2— Video Equipment
2.3— Buffers
2.4— Cell Preparation
2.5— Disposable Supplies
3— Methods
3.1— Preparation for the Flow Chamber Assay
3.2— Using the Parallel Plate Flow Chamber
3.3— Analyses of Leukocyte-Endothelial Adhesion Events
4— Notes
Acknowledgments
References
15— Adhesion of Tumor Cells to Endothelium Under Static Conditions
1— Introduction
2— Materials
3— Methods
4— Notes
Acknowledgment
References
16— Adhesion of Tumor Cells Under Flow
1— Introduction
2— Materials
3— Methods
3.1— Adhesion Assay
3.2— Image Acquisition and Analysis
3.3— Functional Blockade of Rolling and Adhesion
References
17— Platelet Adhesion to the Subendothelium Under Flow
1— Introduction
2— Materials
2.1— Perfusion Chambers
2.2— Flow Device
2.3— Blood
2.4— Surfaces
3— Methods
3.1— Preparation of a Perfusion Experiment
3.2— The Actual Perfusion Experiment
3.3— The Termination of a Perfusion Experiment
3.4— Between Two Perfusion Experiments
3.5— Evaluation of Platelet Adhesion
4— Notes
References
18— Leukocyte Transmigration Through Vascular Endothelium: An In Vitro Method
1— Introduction
2— Materials
3— Methods
3.1— Culture of Endothelial Cells on Transwell Membrane Inserts
3.2— Culture of Endothelial Cells on PVP-Free Polycarbonate Filters
3.3— Leukocyte Preparation and Radiolabeling
3.4— Leukocyte Transmigration with Transwell Inserts
3.5— Leukocyte Transmigration in Boyden Chambers
3.6— Identification of Adhesion Molecules
4— Notes
References
19— Cell Migration into a Wounded Area In Vitro
1— Introduction
2— Materials
3— Methods
4— Notes
References
20— Determination of the Endothelial Barrier Function In Vitro
1— Introduction
2— Materials
2.1— Cell-Culture Media
2.2— Materials for Culture of Human Endothelial Cells on Porous Filters
3— Methods
3.1— Culture of Human Endothelial Cells on Porous Filters According to the Method of Langeler and Va...
3.2— Determination of the Permeability of Human Endothelial Cell Monolayers on Porous Filters
4— Notes
Acknowledgment
References
21— Induction of Intracellular Signals Through Binding of Adhesive Molecules: Activation of p125FAK ...
1— Introduction
2— Materials
2.1— Adhesion-Induced Tyrosine Phosphorylation
2.2— Tyrosine Phosphorylation Following Integrins Clustering in Nonadherent Cells
Table of Contents
Adhesion Protein Protocols
Preface
Contents
Contributors
1— Monoclonal Antibodies Specific for Leukocyte Adhesion Molecules Selective Protocols of Immunizati...
1— Introduction
2— Materials
3— Methods
4— Notes
References
2— Epitope Mapping
3— Sequencing of Antibodies
4— Purification through Affinity Chromatography and Microsequencing
5— Functional Analysis of Cell Adhesion Molecules
6— Application of Polymerase Chain Reaction for the Discovery of New Adhesion Molecule Family Member...
1— Introduction
2— Materials
3— Method
4— Notes
Acknowledgments
References
7— Construction and Purification of Adhesion Molecule Immunoglobulin Chimeric Proteins
1— Introduction
2— Materials
2.1— Construction of Adhesion Molecule—Ig Chimeras
2.2— Electroporation of Cells
2.3— Harvest and Culture of Transfectant Clones
2.4— Selection (ELISA) and Subcloning
2.5— Purification of Chimeric Proteins
3— Methods
3.1— Construction of Adhesion Molecule-Ig Chimeras
3.2— Electroporation of Cells
3.3— Harvest and Culture of Transfectant Clones
3.4— Selection (ELISA) and subcloning
3.5— Purification of Chimeric Proteins
4— Notes
References
8— Immunofluorescence of Cultured Cells
1— Introduction
2— Materials and Preparative Work
3— Methods
3.1— Fixation Procedure
3.2— Permeabilization Procedure
3.3— Fixation-Permeabilization Procedure
3.4— First Antibody Incubation
3.5— Secondary Antibody Incubation
3.6— Mounting
4— Notes
References
9— Detection of Adhesion Molecules by Immunohistochemistry on Human and Murine Tissue Sections
1— Introductrion
1.1— Principles
1.2— Paraffin vs Frozen Sections
1.3— Interpretation of the Results
1.4— Immunohistochemistry in Murine Tissues
2— Materials
2.1— Formalin Fixation for Paraffin Embedding
2.2— Tissue Freezing and Cryostat Sectioning
2.3— Immunostaining Reagents
2.4— Cromogen Substrates
2.5— Counterstaining and Mounting
3— Methods
3.1— Formalin Fixation for Paraffin Embedding
3.2— Freezing of Tissue Samples
3.3— Preparation of Tissue Sections
3.4— The Immunohistochemistry Reaction in Human Tissues
3.4.1— The Primary Antibody
3.4.2— The Development Kit
3.4.3— Cromogen Substrates
3.4.4— Counterstaining
3.4.5— Controls and Interpretation of the Results
3.4.6— Immunohistochemistry of Adhesion Proteins
3.5— Immunohistochemistry in Murine Tissues
4— Notes
References
10— In Situ Hybridization with RNA Probes
1— Introduction
2— Materials
2.1— Tissue Embedding
2.2— Sectioning and Postfixation
2.3— Preparation of RNA Probes
2.4— Section Pretreatment
2.5— Hybridization
2.6— Post-Hybridization Washes and RNase Digestion
2.7— Autoradiography
2.8— Developing and Staining of Slides
3— Methods
3.1— Tissue Embedding
3.2— Sectioning and Postfixation
3.3— Preparation of RNA Probes
3.3.1— Preparation of the Linearized DNA template
3.3.2— In Vitro Transcription
3.3.3— Removal of the DNA Template
3.3.4— Probe Purification
3.3.5— Hydrolysis of RNA Probe (Optional)
3.4— Section Pretreatment
3.5— Hybridization
3.6— Posthybridization Washes and RNase digestion
3.7— Autoradiography
3.8— Developing and Staining of Slides
4— Notes
References
11— Purification of Fibronectin from Human Plasma
1— Introduction
2— Materials
3— Methods
3.1— Purification of Fibronectin
3.2— Quantitation, Electrophoretic Analysis, and Dialysis of Purified Fibronectin
4— Notes
References
12— Adhesion to Matrix Proteins
1— Introduction
2— Materials
3— Methods
4— Notes
References
13— Leukocyte-Endothelial Monolayer Adhesion Assay (Static Conditions)
1— Introduction
2— Materials
3— Method
3.1— Isolation of Peripheral Blood Leukocytes
3.2— Loading Leukocytes with BCECF
3.3— Adhesion Assay
4— Notes
Acknowledgment
References
14— Cell—Cell Adhesive Interactions in an In Vitro Flow Chamber
1— Introduction
2— Materials
2.1— Flow Chamber
2.2— Video Equipment
2.3— Buffers
2.4— Cell Preparation
2.5— Disposable Supplies
3— Methods
3.1— Preparation for the Flow Chamber Assay
3.2— Using the Parallel Plate Flow Chamber
3.3— Analyses of Leukocyte-Endothelial Adhesion Events
4— Notes
Acknowledgments
References
15— Adhesion of Tumor Cells to Endothelium Under Static Conditions
1— Introduction
2— Materials
3— Methods
4— Notes
Acknowledgment
References
16— Adhesion of Tumor Cells Under Flow
1— Introduction
2— Materials
3— Methods
3.1— Adhesion Assay
3.2— Image Acquisition and Analysis
3.3— Functional Blockade of Rolling and Adhesion
References
17— Platelet Adhesion to the Subendothelium Under Flow
1— Introduction
2— Materials
2.1— Perfusion Chambers
2.2— Flow Device
2.3— Blood
2.4— Surfaces
3— Methods
3.1— Preparation of a Perfusion Experiment
3.2— The Actual Perfusion Experiment
3.3— The Termination of a Perfusion Experiment
3.4— Between Two Perfusion Experiments
3.5— Evaluation of Platelet Adhesion
4— Notes
References
18— Leukocyte Transmigration Through Vascular Endothelium: An In Vitro Method
1— Introduction
2— Materials
3— Methods
3.1— Culture of Endothelial Cells on Transwell Membrane Inserts
3.2— Culture of Endothelial Cells on PVP-Free Polycarbonate Filters
3.3— Leukocyte Preparation and Radiolabeling
3.4— Leukocyte Transmigration with Transwell Inserts
3.5— Leukocyte Transmigration in Boyden Chambers
3.6— Identification of Adhesion Molecules
4— Notes
References
19— Cell Migration into a Wounded Area In Vitro
1— Introduction
2— Materials
3— Methods
4— Notes
References
20— Determination of the Endothelial Barrier Function In Vitro
1— Introduction
2— Materials
2.1— Cell-Culture Media
2.2— Materials for Culture of Human Endothelial Cells on Porous Filters
3— Methods
3.1— Culture of Human Endothelial Cells on Porous Filters According to the Method of Langeler and Va...
3.2— Determination of the Permeability of Human Endothelial Cell Monolayers on Porous Filters
4— Notes
Acknowledgment
References
21— Induction of Intracellular Signals Through Binding of Adhesive Molecules: Activation of p125FAK ...
1— Introduction
2— Materials
2.1— Adhesion-Induced Tyrosine Phosphorylation
2.2— Tyrosine Phosphorylation Following Integrins Clustering in Nonadherent Cells
