Galectins: Methods and Protocols (Methods in Molecular Biology, 2442)

Preface
Acknowledgments
Contents
Contributors
Chapter 1: Galectins: An Ancient Family of Carbohydrate Binding Proteins with Modern Functions
	Abbreviations
	1 Introduction
		1.1 The Evolution of Carbohydrate Recognition
		1.2 Galectin Evolution and Biochemistry
			1.2.1 Galectin Evolution
			1.2.2 The Galectin CRD
			1.2.3 Galectin Quaternary Structure and Function
			1.2.4 Synthesis of Galectin Carbohydrate Ligands
	2 Mammalian Galectin Expression and Localization
	3 Modern Galectin Functions
		3.1 Nuclear and Cytoplasmic Galectins
		3.2 Regulation of Pre-mRNA Splicing and mRNA Stability
		3.3 Intracellular Signaling: Proliferation and Apoptosis
		3.4 Galectin Secretion
		3.5 The Galectin Lattice
		3.6 Homeostasis
		3.7 Epithelial Barrier
		3.8 Vascular Endothelium
		3.9 Hemostasis
	4 Cancer
		4.1 Transformation
		4.2 Tumor Microenvironment
		4.3 Roles in the Mammalian Immune System
			4.3.1 Engagement and Crosslinking of Surface Receptors
			4.3.2 Regulation of Immune Cell Turnover
			4.3.3 Modulation of Inflammatory Intracellular Signaling Pathways
			4.3.4 Intracellular Pattern Recognition and Damage Sensing
			4.3.5 Galectin-Pathogen Interactions
			4.3.6 Bacteria
			4.3.7 Viruses
	5 Summary and Conclusions
	References
Chapter 2: Cloning, Expression, and Purification of Galectins for In Vitro Studies
	1 Introduction
	2 Materials
		2.1 Cloning of Galectins
		2.2 Expression and Purification of GST-Galectins
		2.3 Preparation of GST-Galectins for Pull-Down Assays
		2.4 Preparation of Immunopurified Proteins
		2.5 Special Equipment
	3 Methods
		3.1 Cloning
		3.2 Expression and Purification of GST-Galectins
		3.3 Preparation of GST-Galectins for Pull-Down Assays
		3.4 Pull-Down Assays with GST-Galectins
	4 Notes
	References
Chapter 3: Purification of Recombinant Galectins from Different Species Using Distinct Affinity Chromatography Methods
	1 Introduction
	2 Materials
		2.1 Expression and Purification of Fungal Galectin CGL2 Using Lactosyl-Sepharose Affinity Chromatography
		2.2 Preparation of Luria-Bertani Broth for Bacterial Cultivation
		2.3 Purification of Recombinant Fungal Galectin
			2.3.1 Day 1: Starter Culture
			2.3.2 Day 2: Secondary Culture
			2.3.3 Day 3: Pelleting of Bacterial Cultures
		2.4 Processing of Bacterial Pellets
		2.5 Coupling of Lactose to Sepharose 4B Using Divinyl Sulfone
		2.6 Packing of Lactosyl-Sepharose Chromatography (LSC) Column
		2.7 Galectin Purification
		2.8 Removal of Lactose from Purified Galectin Solution Using PD10-Desalting Column
		2.9 Expression and Purification of Human Galectin-7 Using Nickel-NTA Chromatography
			2.9.1 Reagents and Solutions
		2.10 Purification of Human Galectin-7 by Glutathione S-Transferase Affinity Chromatography
			2.10.1 Reagents and Solutions
	3 Methods
		3.1 Expression and Purification of Galectins Using Lactosyl-Sepharose Chromatography (LSC) Column
			3.1.1 Protocol for Bacterial Culture
				3.1.1.1 Day 1: Starter Culture
				3.1.1.2 Day 2: Secondary Culture
				3.1.1.3 Day 3: Pelleting of Bacterial Cultures
		3.2 Lysis of Bacterial Pellets
			3.2.1 Preparation of LSC Lysis Buffer
			3.2.2 Method of Bacterial Lysis
		3.3 Coupling of Lactose to Sepharose 4B Using Divinyl Sulfone
			3.3.1 Packing and Preparation of Lactosyl-Sepharose Column
		3.4 Galectin Purification
		3.5 Re-equilibration and Storage of Lactose-Sepharose Column
		3.6 Removal of Lactose from Galectin Solution Using Desalting PD10 Column
		3.7 Purification of Human Galectin-7 Using Nickel-NTA Affinity Chromatography
			3.7.1 Day 1: Starter Culture
			3.7.2 Day 2: Main Culture
			3.7.3 Day 3: Pelleting of Bacterial Cultures and Lysis of Bacterial Pellets
			3.7.4 Nickel-NTA Column Purification of Human Galectin-7
		3.8 Purification of Human Galectin-7 Using Glutathione-Affinity Chromatography
	4 Notes
	References
Chapter 4: Alkylation of Galectin-1 with Iodoacetamide and Mass Spectrometric Mapping of the Sites of Incorporation
	1 Introduction
	2 Materials
		2.1 Iodoacetamide Alkylation of Free Sulfhydryls on Galectin-1
		2.2 Galectin Activity Assay
		2.3 Proteolytic Digestion of Alkylated Galectin-1
		2.4 HPLC Separation and Fraction Collection
		2.5 Mass Spectrometry Reagents and Software
			2.5.1 Mass Spectrometry Analysis Software
		2.6 Special Equipment
	3 Methods
		3.1 Iodoacetamide Alkylation of Free Sulfhydryls on Galectin-1
		3.2 Galectin Activity Assay
		3.3 Proteolytic Digestion of Alkylated Galectin-1
		3.4 HPLC Separation and Fraction Collection
		3.5 MALDI-TOF and TOF/TOF Analysis
		3.6 ESI-MS, Direct Infusion
		3.7 LC-MS Analysis (See Note 17)
		3.8 Data Processing and Analysis
	4 Notes
	References
Chapter 5: Rapid Detection and Purification of Galectin-3 by the Capture and Release (CaRe) Method
	1 Introduction
	2 Materials
		2.1 Extraction of Recombinant Gal-3 from E. coli Cells
		2.2 Preparation of Crude Extracts from Chicken Egg Whites (Source of Ovalbumin)
		2.3 Identification of Multivalent Inhibitors as Target Capturing Agents (TCAs) by Hemagglutination Inhibition
		2.4 Testing the Capturing Abilities of TCAs by Precipitation Assays
		2.5 Capturing of Targets (Lectins and GPs) in the Crude Extracts by TCAs and Separation of the Target-TCA Complex from Other C...
		2.6 Releasing the Captured Targets (Lectins and GPs) by Dissolving the Complex
		2.7 Separation of the Targets (Lectins and Glycoproteins) from Their Respective TCAs
		2.8 Verification of the Purity of the Isolated Targets (Lectins or GPs)
	3 Methods
		3.1 Extraction of Recombinant Gal-3 from E. coli Cells
		3.2 Preparation of Crude Extracts from Chicken Egg Whites (Source of Ovalbumin)
		3.3 Identification of Multivalent Inhibitors as Target Capturing Agents (TCAs) by Hemagglutination Inhibition
		3.4 Testing the Capturing Abilities of TCAs by Precipitation Assays
			3.4.1 Preparing the Spectrophotometer
			3.4.2 Precipitation of Target Lectin (Gal-3) from the Crude Solution
			3.4.3 Precipitation of Target Glycoprotein (Chicken Ovalbumin) from the Crude Solution
		3.5 Capturing of Targets (Lectins and GPs) in the Crude Extracts by TCAs and Separation of the Target-TCA Complex from Other C...
			3.5.1 Capturing the Targets (Lectins or GPs) (Fig. 2)
			3.5.2 Washing the Insoluble Precipitate
		3.6 Releasing the Captured Targets (Lectins and GPs) by Dissolving the Complex
		3.7 Separation of the Targets (Lectins and Glycoproteins) from Their Respective TCAs
			3.7.1 Separating the Galectin-3-TCA Complexes
			3.7.2 Separating the Ovalbumin-ConA Complexes
		3.8 Verification of the Purity of the Isolated Targets (Lectins or GPs)
	4 Notes
	References
Chapter 6: Introducing 77Se NMR Spectroscopy to Analyzing Galectin-Ligand Interaction
	1 Introduction
	2 Materials
		2.1 Chemical Synthesis
		2.2 Equipment (Hardware, Software) and Materials Used for NMR Measurements
	3 Methods
		3.1 Synthesis of  SeDG
		3.2 Synthesis of Diselenodigalactoside (DSeDG, DSeDGal, 6b)
		3.3 77Se NMR-Spectroscopical Monitoring and Competition Assay
	4 Notes
	References
Chapter 7: Evaluation of Galectin Binding by Surface Plasmon Resonance
	1 Introduction
	2 Materials (See Notes 1 and 2)
		2.1 Starting the BIAcore 3000
		2.2 Preparing the Sensor Surface
		2.3 Galectin Binding
		2.4 Data Analysis
	3 Methods
		3.1 Starting Up BIAcore 3000 (See Notes 7 and 8)
		3.2 Preparing the Sensor Surface
		3.3 Galectin Binding
		3.4 Data Analysis
	4 Notes
	References
Chapter 8: Revealing the Identity of Human Galectin-3 as a Glycosaminoglycan-Binding Protein
	1 Introduction
	2 Materials
		2.1 Preparation of E. coli Cell Lysates Containing Human Galectin-3 (Gal-3)
		2.2 Preparation of 2% v/v Red Blood Cell (RBC) Suspension
		2.3 Detection of Lectin Activity of Gal-3 in the Crude Cell Extracts
		2.4 Purification of Gal-3 by Affinity Chromatography
		2.5 Dialysis of Gal-3 for Removal of Lactose
		2.6 Determination of the Concentration of Purified Gal-3
		2.7 Hemagglutination Inhibition Assays
		2.8 Isothermal Microtitration Calorimetry (ITC) of Gal-3 with Glycosaminoglycans (GAGs) and Proteoglycan
		2.9 UV-Vis Spectroscopic Analysis of Gal-3 and GAGs Precipitation (Complex Formation Through Noncovalent Cross-linking) and Te...
	3 Methods
		3.1 Preparation of E. coli Cell Lysates Containing Human Galectin-3 (Gal-3)
		3.2 Methods
			3.2.1 Washing the Red Blood Cells
			3.2.2 Making a 2% v/v RBC Suspension
		3.3 Methods
			3.3.1 Activity Assay
		3.4 Methods
			3.4.1 Affinity purification of Gal-3
		3.5 Methods
			3.5.1 Dialysis of Purified Gal-3
		3.6 Methods
			3.6.1 Determination of Gal-3 Concentration
		3.7 Methods
			3.7.1 Inhibition Assay
		3.8 Methods
			3.8.1 ITC Studies
		3.9 Methods
			3.9.1 Cross-linking Studies
	References
Chapter 9: Examining Galectin Binding Specificity Using Glycan Microarrays
	1 Introduction
	2 Materials
		2.1 Galectin Preparation
			2.1.1 Special Equipment
			2.1.2 Buffers
		2.2 Galectin Derivitiztion
		2.3 Glycan Array Screening
			2.3.1 Special Equipment
			2.3.2 Buffers
	3 Methods
		3.1 Galectin Preparation
			3.1.1 Purification
		3.2 Galectin Biotinylation
		3.3 Microarray Probing with Biotinylated Galectin
	4 Notes
	References
Chapter 10: Mechanism of Mucin Recognition by Lectins: A Thermodynamic Study
	1 Introduction
		1.1 General Overview
		1.2 Affinities of SBA and Vatairea Macrocarpa Lectin (VML) for Mucins
		1.3 Thermodynamics of SBA Binding Tn-PSM
		1.4 Thermodynamics of SBA Binding the 81-mer Tn-PSM
		1.5 Thermodynamics of SBA Binding 38/40-mer Tn-PSM
		1.6 Thermodynamics of SBA Binding Fd-PSM
		1.7 Thermodynamics of VML Binding Tn-PSM
		1.8 Thermodynamics of VML Binding 81-mer Tn-PSM and 38/40-mer Tn-PSM
		1.9 Thermodynamics of VML Binding Fd-PSM
		1.10 Analysis of the Stoichiometry of Binding of SBA to the Mucins
		1.11 Mechanisms of Binding of SBA and VML to PSM: The ``Bind and Jump Model´´
	2 Materials
	3 Method
		3.1 Hemagglutination Inhibition Assay to Determine the Binding Affinity of Mucins
		3.2 ITC Studies of Lectin-Mucin Interactions
	4 Notes
	References
Chapter 11: Examination of Whole-Cell Galectin Binding by Solid Phase and Flow Cytometric Analysis
	1 Introduction
	2 Materials
		2.1 Biotinylation and Fixation of HL-60 Cells
		2.2 Glycosidase Treatments of Biotinylated and Fixed HL-60 Cells
		2.3 Fluorescence Labeling of Gal-1 Through Primary Amines
		2.4 Fluorescence Labeling of Gal-1 Through Cysteines
		2.5 Fluorescence Labeling of Tomato Lectin (LEA) Through Carbohydrates
		2.6 Gal-1, LEA, and GS-II Binding to Immobilized HL-60 Cells
		2.7 Determination of Gal-1 Binding Using Flow Cytometric Analysis
		2.8 Determination of Apparent Binding Affinity of Gal-1 for Immobilized HL-60 Cells
	3 Methods
		3.1 Biotinylation and Fixation of HL-60 Cells
		3.2 Fixation Alone of HL-60 Cells (for Enzymatic Treatment Followed by Flow Cytometric Analysis)
		3.3 Glycosidase Treatments of Biotinylated and Fixed (or Fixed Alone) HL-60 Cells
		3.4 Fluorescence Labeling of Gal-1 Through Primary Amines
		3.5 Fluorescence Labeling of Gal-1 Through Cysteines
		3.6 Fluorescence Labeling of Tomato Lectin (LEA) Through Carbohydrates
		3.7 Gal-1, LEA, and GS-II Binding to Immobilized HL-60 Cells
		3.8 Determination of Gal-1 Binding to HL-60 Cells Using Flow Cytometric Analysis
		3.9 Determination of Apparent Binding Affinity of Gal-1 for Immobilized HL-60 Cells
	4 Notes
	References
Chapter 12: Dissecting Context-Specific Galectin Binding Using Glycoengineered Cell Libraries
	1 Introduction
	2 Materials
		2.1 Galectin Preparation
		2.2 Culturing CHO Wild-Type and KO Cells
		2.3 Cell Surface Binding: Flow Cytometry
		2.4 Single-Molecule Binding: Fluorescence Anisotropy
	3 Methods
		3.1 Galectin Preparation
		3.2 Culturing CHO KO Cells
			3.2.1 Cell Surface Binding: Flow Cytometry
			3.2.2 Data Analysis: Flow Cytometry
			3.2.3 Single-Molecule Binding: Fluorescence Anisotropy
			3.2.4 Data Analysis: Fluorescence Anisotropy
	4 Notes
	References
Chapter 13: Method for Identifying Galectin Ligands on Lymphocyte Membrane Glycoproteins
	1 Introduction
	2 Materials
		2.1 NK Cell Isolation and Validation (See Note 1)
		2.2 NK Cell Lysis
		2.3 CD16a Immunoprecipitation
		2.4 CD16a Proteolysis and Glycopeptide Enrichment
		2.5 Liquid Chromatography and Mass Spectrometry (See Note 4)
		2.6 Data Analysis
	3 Method
		3.1 NK Cell Isolation
		3.2 NK Cell Lysis
		3.3 CD16a Immunoprecipitation
		3.4 CD16a Proteolysis and Glycopeptide Enrichment
		3.5 Liquid Chromatography and Mass Spectrometry
		3.6 Data Analysis
	4 Notes
	References
Chapter 14: Transformation of Agrocybe cylindracea Galectin into αGalNAc-Specific Lectin
	1 Introduction
	2 Materials
		2.1 Primers
		2.2 PCR
		2.3 Transformation and Bacterial Expression
		2.4 Glycoconjugate Microarray
		2.5 Purification of N46A Mutant by Affinity Chromatography
		2.6 Frontal Affinity Chromatography (FAC)
	3 Methods
		3.1 Site-Directed Mutagenesis
			3.1.1 Amplification of the Whole Vector Using Mutagenic Primers
			3.1.2 Transformation of Competent Cells
			3.1.3 Plasmid Extraction and Sequencing
		3.2 Analysis of Sugar-Binding Functions of ACG Mutants by Glycoconjugate Microarray Using Crude Samples
		3.3 Purification of N46A Mutant by Affinity Chromatography
		3.4 Quantitative Analysis of Sugar-Binding Specificity of N46A by Frontal Affinity Chromatography (FAC) Using Purified Samples
	4 Notes
	References
Chapter 15: What Happens If a Human Galectin Enters the Endoplasmic Reticulum?
	1 Introduction
	2 Materials
		2.1 Ligation
		2.2 Transformation of Chemically Competent E. coli One Shot TOP10 Cells
		2.3 Cloning of Galectin-Coding cDNA into a Pichia Expression Vector with Signal Sequence (pPICZα A)
		2.4 Modified QuikChange Site-Directed Mutagenesis Protocol
		2.5 Electroporation of Pichia Strain X-33
			2.5.1 Preparation of Plasmid DNA for Electroporation
			2.5.2 Preparation of Electrocompetent Pichia X-33 Cells
			2.5.3 Electroporation Procedure of Pichia X-33 Cells
		2.6 Analysis of Colonies
		2.7 Expression of Galectin in P. pastoris
		2.8 Purification of Galectin from P. pastoris Supernatant by Affinity Chromatography on Lactosylated Sepharose 4B Resin
		2.9 Electrophoresis and Transfer
		2.10 Western Blotting
		2.11 Lectin Blotting
		2.12 Sample Preparation of Galectins for Electrospray Mass Spectrometry (ESMS) Analysis
			2.12.1 Sample Preparation for Analysis of Protein
			2.12.2 Sample Preparation with Prior Protein Deglycosylation
		2.13 ESMS Analysis of Galectin
		2.14 Sample Preparation for LC/MSMS Analysis of Galectin
			2.14.1 Deglycosylation
			2.14.2 Tryptic Digestion
		2.15 LC/MSMS Analysis of Galectin
		2.16 Radioiodination of Galectin
		2.17 Cell Culture and Cell Binding Studies
		2.18 Proliferation Assay
		2.19 Cloning of Galectin-Coding cDNA into Mammalian Expression Vectors (pSecTag2 B, pcDNA3.1+/C-HA) with Signal Peptide
		2.20 Culture of Human Embryonic Kidney 293T Cells (HEK293T)
			2.20.1 Preparation of Plastic and Glass Substrata for Cell Culture
			2.20.2 Cell Cultures for Biochemical Analysis or for Immunocytochemical Staining
		2.21 Density Gradient Fractionation of Subcellular Membrane Vesicles
			2.21.1 Preparation of Total Membrane Extracts from HEK293T Cells for Subcellular Fractionation
			2.21.2 Preparation of Density Gradients, Membrane Fractionation, and Fraction Protein Precipitation
			2.21.3 Precipitation of Proteins from Fractions from the Density Gradient
			2.21.4 Analysis of Fractions by Western Blotting
		2.22 Immunocytochemistry (ICC)
	3 Methods
		3.1 Ligation
		3.2 Transformation of Chemically Competent E. coli One Shot TOP10 Cells
		3.3 Cloning of Galectin-Coding cDNA into a Pichia Expression Vector with Signal Sequence (pPICZα A)
		3.4 Modified QuikChange Site-Directed Mutagenesis Protocol
		3.5 Electroporation of Pichia Strain X-33
			3.5.1 Preparation of Plasmid DNA for Electroporation
			3.5.2 Preparation of Electrocompetent Pichia X-33 Cells
			3.5.3 Electroporation Procedure of Pichia X-33 Cells
		3.6 Analysis of Colonies
		3.7 Expression of Galectin in P. pastoris
		3.8 Purification of Galectin from P. pastoris Supernatant by Affinity Chromatography on Lactosylated Sepharose 4B Resin
		3.9 Electrophoresis and Transfer
		3.10 Western Blotting
		3.11 Lectin Blotting
		3.12 Sample Preparation for ESMS Analysis for Galectins
			3.12.1 Sample Preparation for Analysis of Protein
			3.12.2 Sample Preparation with Deglycosylation
		3.13 ESMS Analysis
		3.14 Sample Preparation for LC/MSMS Analysis of Galectin
			3.14.1 Deglycosylation
			3.14.2 Tryptic Digestion
		3.15 LC/MSMS Analysis of Galectin
		3.16 Radioiodination of Galectin
		3.17 Neuroblastoma Cell Culture and Binding Studies
		3.18 Proliferation Assay
		3.19 Cloning of Galectin-Coding cDNA into Mammalian Expression Vectors (pSecTag2 B, pcDNA3.1+/C-HA) with Signal Peptide
		3.20 Culture of Human Embryonic Kidney 293T Cells (HEK293T)
			3.20.1 Preparation of Plastic and Glass Substrata for Cell Culture
			3.20.2 Cell Cultures for Biochemical Analysis or for Immunocytochemical Staining
		3.21 Density Gradient Fractionation of Subcellular Membrane Vesicles
			3.21.1 Preparation of Total Membrane Extracts from HEK293T Cells for Subcellular Fractionation
			3.21.2 Preparation of Density Gradients, Membrane Fractionation, and Fraction Protein Precipitation
			3.21.3 Precipitation of Proteins from Fractions from the Density Gradient
			3.21.4 Analysis of Fractions by Western Blotting
		3.22 Immunocytochemistry
	4 Notes
	References
Chapter 16: Investigation of Galectins in Frozen Tissue and Mammalian Cell Culture Using Confocal Miccroscopy
	1 Introduction
	2 Materials
		2.1 Tissue Technique
			2.1.1 Preparation of Glass Slides
			2.1.2 Processing of Tissue
			2.1.3 Cryogenic Technique
			2.1.4 Preparation of Sample Sectioning and Mounting
			2.1.5 Tissue Immunofluorescence
		2.2 Cell Culture Immunofluorescence Technique
	3 Methods
		3.1 Tissue Technique
			3.1.1 Preparation of Glass Slides
			3.1.2 Processing of Tissue
			3.1.3 Cryogenic Technique
			3.1.4 Preparation of Sample Sectioning and Mounting
			3.1.5 Tissue Immunofluorescence
		3.2 Cell Culture Immunofluorescence Technique
	4 Notes
	References
Chapter 17: Exploring the Galectin Network by Light and Fluorescence Microscopy
	1 Introduction
	2 Materials
		2.1 Preparation of Non-cross-Reactive Antibodies
		2.2 Measurement of Galectin Activity
			2.2.1 Preparation of Solutions of Galectins
			2.2.2 Preparation of Solutions of Carbohydrate Ligands
			2.2.3 Instrumentation and Software
		2.3 Preparation of Biotinylated Galectins
		2.4 Preparation of Fluorescent Galectins
		2.5 Fixation, Embedding, and Tissue Sectioning
		2.6 Light Microscopy
		2.7 Fluorescence Microscopy (See Note 6)
		2.8 Synthesis of a Glycocluster
			2.8.1 Reagents for Click Chemistry
			2.8.2 Reagents for Zemplén Deacetylation and Purification of Glycocluster
		2.9 Evaluation of the Inhibitory Capacity of Glycocluster in Galectin Histochemistry
	3 Methods
		3.1 Preparation of Non-cross-Reactive Antibodies
		3.2 Measurement of Galectin Activity (See Note 13)
			3.2.1 Preparation of Solutions of Galectins
			3.2.2 Preparation of Carbohydrate Ligand
			3.2.3 Isothermal Titration Calorimetry (ITC)
			3.2.4 Data Analysis
		3.3 Preparation of Biotinylated Galectins
		3.4 Preparation of Fluorescent Galectins
		3.5 Fixation, Embedding, and Tissue Sectioning
		3.6 Light Microscopy
			3.6.1 Immunohistochemistry: Detection of Endogenous Galectins
			3.6.2 Galectin Histochemistry: Detection of Accessible Binding Sites
		3.7 Fluorescence Microscopy (See Note 6)
			3.7.1 Immunohistochemistry: Detection of Endogenous Galectins
			3.7.2 Galectin Histochemistry: Detection of Accessible Binding Sites
			3.7.3 Detection of Endogenous Galectins and Accessible Binding Sites by Fluorescence Microscopy
		3.8 Synthesis of Glycocompounds
			3.8.1 Preparation of Intermediate 3 by Click Chemistry (Scheme 1)
			3.8.2 Preparation of the NaOMe Solution in MeOH for Deacetylation
			3.8.3 Preparation of Glycocluster 4 Via Zemplén Deacetylation (Scheme 2)
		3.9 Evaluation of the Inhibitory Capacity of Glycocompounds in Galectin Histochemistry (See Note 13)
	4 Notes
	References
Chapter 18: Molecular Imaging for In Vivo Tracking and Detection of Galectin Binding Partners
	1 Introduction
	2 Materials
		2.1 Bioluminescent Galectins
			2.1.1 Cloning Strategy: Expression Vector for Recombinant Bioluminescent Galectin
			2.1.2 Expression and Purification of Bioluminescent Galectin
			2.1.3 Mouse Injection of Bioluminescent Galectin for In Vivo Tracking
		2.2 Radiolabeling Galectins
			2.2.1 Galectin Conjugation to NHS-HYNIC
			2.2.2 Galectin-HYNIC Purification
			2.2.3 Radiolabeling Galectin-HYNIC Using 99mTc
			2.2.4 Galectin-HYNIC-99mTc Purification
			2.2.5 Mouse Injection of Galectin-HYNIC-99mTc for In Vivo Tracking
	3 Methods
		3.1 Bioluminescent Galectins
			3.1.1 Cloning Strategy: Expression Vector for Recombinant Bioluminescent Galectin
			3.1.2 Expression and Purification of Recombinant Bioluminescent Galectin
			3.1.3 Mouse Injection of Rluc-mGal3 for In Vivo Tracking
		3.2 Radiolabeling Galectins
			3.2.1 Galectin Conjugation to NHS-HYNIC
			3.2.2 Galectin-HYNIC Purification
			3.2.3 Radiolabeling Galectin-HYNIC Using 99mTc
			3.2.4 Galectin-HYNIC-99mTc Purification
			3.2.5 Mouse Injection of Galectin-HYNIC-99mTc for In Vivo Tracking
	4 Notes
	References
Chapter 19: Visualization of Cytosolic Galectin Accumulation Around Damaged Vesicles and Organelles
	1 Introduction
	2 Materials
		2.1 Examination of Cytosolic Galectin Accumulation Around Endosomes Damaged by a Light-Illuminated Photosensitizer Using Fluor...
			2.1.1 Cell Culture
			2.1.2 Plasmids
			2.1.3 Reagents
			2.1.4 Equipment
		2.2 Examination of Cytosolic Galectin Accumulation Around Endosomes Damaged by a Light-Illuminated Photosensitizer Using APEX2...
			2.2.1 Plasmids
			2.2.2 Reagents
		2.3 Examination of Cytosolic Galectin Accumulation Around L. monocytogenes-Containing Phagosomes in Mouse Bone Marrow-Derived ...
			2.3.1 Cell Culture
			2.3.2 Bacterial Culture
			2.3.3 Reagents
			2.3.4 Equipment
		2.4 Examination of Cytosolic Galectin Accumulation Around L. monocytogenes-Containing Phagosomes in Mouse Bone Marrow-Derived ...
			2.4.1 Reagents
		2.5 Examination of Cytosolic Galectin Accumulation Around Damaged Lysosomes in H. pylori-Infected Gastric Epithelial Cells Usi...
			2.5.1 Cell Culture
			2.5.2 Bacteria Culture
			2.5.3 Reagents
			2.5.4 Equipment
	3 Methods
		3.1 Examination of Cytosolic Galectin Accumulation Around Endosomes Damaged by a Light-Illuminated Photosensitizer Using Fluor...
		3.2 Examination of Cytosolic Galectin Accumulation Around Endosomes Damaged by a Light-Illuminated Photosensitizer Using APEX2...
		3.3 Examination of Cytosolic Galectin Accumulation Around L. monocytogenes-Containing Phagosomes in Mouse Bone Marrow-Derived ...
		3.4 Examination of Cytosolic Galectin Accumulation Around L. monocytogenes-Containing Phagosomes in Mouse Bone Marrow-Derived ...
		3.5 Examination of Cytosolic Galectin Accumulation Around  Damaged Lysosomes in H. pylori-Infected Human Gastric Epithelial Ce...
	4 Notes
	References
Chapter 20: Transcytosis of Galectin-3 in Mouse Intestine
	1 Introduction
		1.1 General Introduction on Galectins
		1.2 Endogenous Galectin Expression in the Digestive Tract
			1.2.1 Galectin-2 (Glandular Stomach and Small Intestine)
			1.2.2 Galectin-3 (All Along the Gut)
			1.2.3 Galectin-4/6 (Glandular Stomach, Small Intestine, and Lower Digestive Tract)
			1.2.4 Galectin-7 (Stratified Squamous Epithelium)
		1.3 Transcytosis
		1.4 Gal3 Dynamics in the Small Intestine
	2 Materials
		2.1 Mucus Fixation with Carnoy´s Solution and Whole-Mount Immunofluorescence
		2.2 Preparation of Fluorescent or HRP Coupled Gal3 and LTF Conjugates
		2.3 DTT Treatment of Intestine
		2.4 Binding and Internalization Assay
		2.5 Tissue Fixation, Generation of Frozen Blocks, and Sections for Imaging
		2.6 Immunofluorescence
		2.7 Electron Microscopy
	3 Methods
		3.1 Mucus Fixation with Carnoy´s Solution and Whole-Mount Immunofluorescence
		3.2 Preparation of Fluorescent or HRP-Labeled Gal3 and LTF Conjugates
		3.3 DTT Treatment of Intestine
		3.4 Binding and Internalization Assay
			3.4.1 Binding (See Note 16)
			3.4.2 Internalization (See Note 18)
		3.5 Fixation, Generation of Frozen Tissue Blocks, and Sections for Imaging
		3.6 Immunofluorescence
		3.7 Electron Microscopy
	4 Notes
	References
Chapter 21: Evaluating the Role of Galectins in Clathrin-Independent Endocytosis
	1 Introduction
	2 Materials
		2.1 Cell Culture, Reagents, and Antibodies
		2.2 Lectin-Binding Assay
		2.3 Antibody Internalization Assay
		2.4 Transferrin Internalization Assay
		2.5 Galectin-3 ELISA Secretion Assay
		2.6 siRNA Transfection
		2.7 Plasmids and Transient Transfection
		2.8 FRAP Assays
		2.9 Cell-Spreading Assay
		2.10 GFP-Tagged Galectin-3 Secretion Assay
		2.11 Macropinosome Analysis
	3 Methods
		3.1 Cell Culture, Reagents, and Antibodies
		3.2 Lectin-Binding Assay
		3.3 Antibody Internalization Assay
		3.4 Transferrin Internalization Assay
		3.5 Galectin-3 ELISA Secretion Assay
		3.6 siRNA Transfection
		3.7 Plasmids and Transient Transfection
		3.8 FRAP Assays
		3.9 Cell-Spreading Assay
		3.10 GFP-Tagged Galectin-3 Secretion Assay
		3.11 Macropinosome Analysis
	4 Notes
	References
Chapter 22: Examination of Galectin-3 Recruitment into Multivesicular Bodies for Exosomal Secretion
	1 Introduction
	2 Materials
		2.1 Cell Lines
		2.2 Plasmids
		2.3 Antibodies
		2.4 Buffers
		2.5 Cell Culture Media
	3 Methods
		3.1 Cell Culture
		3.2 Generation of Stable MDCK Cell Lines
		3.3 Inhibition of Exosomal Secretion with Dimethylamiloride
		3.4 Purification of Exosomes from MDCK Cells
		3.5 Gold Labeling of Exosomal Galectin-3 for Electron Microscopy
		3.6 Material Preparation for Electron Microscopy
		3.7 Proteinase K Treatment of Purified Exosomes
	4 Notes
	References
Chapter 23: Manipulating Galectin Expression in Zebrafish (Danio rerio)
	1 Introduction
	2 Materials
		2.1 Preparation and Validation of Morpholino (MO) Oligos
		2.2 Preparation of sgRNA
		2.3 Embryo Preparation
		2.4 Embryo Microinjections
		2.5 Whole Mount Antibody Staining
		2.6 Rescuing the Phenotypes by Co-injection of Embryos with Drgal1-L2 mRNA and Drgal1-L2-MO (Ectopic Expression of Native Drga...
		2.7 Verification of Induced Indels
		2.8 Special Instruments
		2.9 General Instruments
	3 Methods
		3.1 Preparation and Validation of Morpholino (MO) Oligos
		3.2 Transformation and Preparation of sgRNA
		3.3 Embryo Preparation
		3.4 Embryo Microinjections
		3.5 Whole Mount Antibody Staining
		3.6 Rescuing the Phenotypes by Co-injection of Embryos with Drgal1-L2 mRNA and Drgal1-L2-MO (Ectopic Expression of Native Drga...
		3.7 Verifying the Induction of Genomic Indels
	4 Notes
	References
Chapter 24: Examining Galectin Gene Regulation by Reporter Assays
	1 Introduction
	2 Materials
		2.1 RACE (Rapid Amplification of cDNA Ends): Determination of Transcription Start Points (TSPs) at the 5′-Ends of Chicken Gale...
		2.2 Generation of Vectors for Analyzing Regulatory DNA Sequences in Eukaryotic Cells
			2.2.1 Cloning of DNA Segments of the (Putative) Regulatory Region of Interest into Firefly Reporter Vector pGL4.20
			2.2.2 Cloning of Transcription Factor-Encoding cDNA into Eukaryotic Expression Vector pcDNA3.1(+)
		2.3 Transfection of Adherent Eukaryotic Cells with Luciferase Reporter Vectors and Transcription Factor-Encoding Vector
		2.4 Luciferase Assay of Galectin Promoter Activity
	3 Methods
		3.1 RACE (Rapid Amplification of cDNA Ends): Determination of TSPs at the 5′-Ends of Chicken Galectin Gene Sequences
		3.2 Generation of Vectors for Analyzing Regulatory DNA Sequences in Eukaryotic Cells
			3.2.1 Cloning of DNA Segments of the (Putative) Regulatory Region of Interest into Firefly Reporter Vector pGL4.20
			3.2.2 Cloning of Transcription Factor-Encoding cDNA into Eukaryotic Expression Vector pcDNA3.1(+)
		3.3 Transfection of Adherent Eukaryotic Cells with Luciferase Reporter Vectors and Transcription Factor-Encoding Vector
		3.4 Luciferase Assay of Galectin Promoter Activity
	4 Notes
	References
Chapter 25: Examining the Impact of Galectin-9 on Latent HIV Transcription
	1 Introduction
	2 Materials
		2.1 Cell Lines
		2.2 General Reagents
		2.3 Reagents for Testing the Impact of Gal-9 on Latent HIV Transcription In Vitro
		2.4 Reagents for Testing the Impact of Gal-9 on Latent HIV Transcription Ex Vivo Using CD4+ T Cells Isolated from HIV-Infected...
	3 Methods
		3.1 Testing the Impact of Gal-9 on Latent HIV Transcription In Vitro Using the J-Lat HIV Latency Model
		3.2 Testing the Impact of Gal-9 on Latent HIV Transcription Ex Vivo Using CD4+ T Cells Isolated from HIV-Infected ART-Suppress...
			3.2.1 Isolation and Treatment of Primary CD4+ T Cells (See Note 2)
			3.2.2 Isolation of RNA from CD4+ T Cells
			3.2.3 Measuring Levels of CD4+ T Cell-Associated HIV RNA (See Notes 4 and 5)
	4 Notes
	References
Chapter 26: Evaluation of the Role of Galectins in Parasite Immunity
	1 Introduction
	2 Materials
		2.1 Cultivation of Parasites
			2.1.1 Hatching H. contortus Eggs from Infected Sheep to the Third Larval Stage (L3)
			2.1.2 Fecal Egg Count for Determining H. contortus Infection
			2.1.3 Storage and Monitoring of H. contortus L3 Larvae
			2.1.4 Exsheathment of H. contortus Third Larval Stage (L3) Using Sodium Hypochlorite
			2.1.5 Exsheathment of H. contortus L3 Using CO2
			2.1.6 Culturing of H. contortus L3 to the Fourth Larval Stage (L4)
			2.1.7 Collection of Adult H. contortus
			2.1.8 Infection of Sheep with Liver Fluke (F. hepatica)
		2.2 RNA Isolation, cDNA Synthesis, and Cloning of LGALS-11 and LGALS-14
		2.3 Recombinant Protein Production
			2.3.1 Protein Expression
			2.3.2 Immobilized Metal Affinity Chromatography (IMAC) Purification of LGALS-11 and LGALS-14
			2.3.3 Removal of Hexahistidine from Recombinant LGALS-11 and LGALS-14
			2.3.4 Size-Exclusion Chromatography (SEC)
		2.4 Recombinant LGALS-11 and LGALS-14 Characterization
			2.4.1 SDS-PAGE Analysis
			2.4.2 Western Blot Analysis
		2.5 Preparation of Parasite-Infected Gastrointestinal Tissues (H. contortus-Infected Abomasum) from Primary and Primed Infecti...
		2.6 Detection of Endogenous Galectins in Tissues (Immunohistochemistry in OCT-Embedded Frozen Tissues)
		2.7 Recombinant Galectin Binding to Frozen Tissue Sections
			2.7.1 Fluorescent Labeling of Galectin Probes, Assessment of Activity by Hemagglutination Assay
			2.7.2 Binding of Fluorescent Galectin to Frozen Tissue Sections
			2.7.3 Binding of Non-fluorescent Galectin to Frozen Tissue Sections
		2.8 Galectin Binding to Parasites (H. contortus and F. hepatica)
			2.8.1 Fluorescent Staining of H. contortus L3, L4, and Adult Stage Parasites
			2.8.2 Binding of Non-fluorescent Galectin Probes to F. hepatica
		2.9 Identification of Galectin Ligands Using a Pull-Down Assay
			2.9.1 Preparation of Parasite Lysate
			2.9.2 Conjugation of Galectin to NHS-Activated Sepharose
			2.9.3 Mass Spectrometry Preparation and Analysis
		2.10 Functional Assays with H. contortus
			2.10.1 Larvae Moulting Assay Using H. contortus L3
			2.10.2 Larvae Growth Assay Using H. contortus L4
			2.10.3 Larvae Feeding Assay Using H. contortus L4
	3 Methods
		3.1 Cultivation of the Larval Stages of H. contortus
			3.1.1 Hatching H. contortus Eggs from Infected Sheep to the Third Larval Stage (L3)
			3.1.2 Fecal Egg Count for Determining H. contortus Infection
			3.1.3 Storage and Monitoring of H. contortus L3
			3.1.4 Exsheathment of H. contortus Third Larval Stage (L3) Using Sodium Hypochlorite
			3.1.5 Exsheathment of H. contortus L3 Using CO2
			3.1.6 Culturing H. contortus L3 to the Fourth Larval Stage (L4)
			3.1.7 Collection of Adult H. contortus
			3.1.8 Infection of Sheep with Liver Fluke (F. hepatica)
		3.2 RNA Isolation from Sheep Abomasum, cDNA Synthesis, and Cloning of LGALS-11 and LGALS-14
		3.3 Recombinant Protein Production
			3.3.1 Protein Expression
			3.3.2 Purification of Recombinant LGALS-11 or LGALS-14 by Immobilized Metal Affinity chromatography (IMAC)
			3.3.3 Removal of Hexahistidine Tag from Recombinant LGALS11 or LGALS-14
			3.3.4 Purification of Recombinant LGALS-11 or LGALS-14 by Size-Exclusion Chromatography (SEC)
		3.4 Characterization of Recombinant LGALS-11 and LGALS-14
			3.4.1 SDS-PAGE Analysis
			3.4.2 Western Blot
		3.5 Preparation of Parasite-Infected Gastrointestinal Tissues (H. contortus-Infected Abomasum)
			3.5.1 Preparation of Parasite-Infected Abomasal Tissue in Primed Sheep
			3.5.2 Preparation of Abomasal Tissue from Sheep with a Primary Infection
		3.6 Detection of Endogenous Galectins in Tissues (Immunohistochemistry in OCT-Embedded Frozen Tissues)
		3.7 Recombinant Galectin Binding to Frozen Tissue Sections
			3.7.1 Fluorescent Labeling of Galectin Probes and Assessment of Activity by Hemagglutination Assay
			3.7.2 Binding of Fluorescent Galectin to Frozen Tissues Sections
			3.7.3 Binding of Non-fluorescent Galectins to Frozen Tissue Sections
		3.8 Galectin Binding to Parasites (H. contortus and F. hepatica)
			3.8.1 Fluorescent Staining of H. contortus L3, L4, and Adult Stage Parasites
			3.8.2 Binding of Non-fluorescent Galectin Probes to F. hepatica
		3.9 Galectin Pull-Down Assay
			3.9.1 Preparation of Parasite Lysate
			3.9.2 Conjugation of Galectin to NHS-Activated Sepharose and Capture of Galectin-Specific Parasite Ligands
			3.9.3 Mass Spectrometry Preparation and Analysis
		3.10 Functional Assays Using H. contortus
			3.10.1 Larvae Moulting Assay
			3.10.2 Larvae Growth Assay Using H. contortus L4
			3.10.3 Larvae Feeding Assay Using H. contortus L4
	4 Notes
	References
Chapter 27: Evaluation of the Bactericidal Activity of Galectins
	1 Introduction
	2 Materials
		2.1 Bacteria Preparation
		2.2 Galectin Preparation
		2.3 Assessing Antimicrobial Activity
	3 Methods
		3.1 Bacteria Preparation
			3.1.1 Preparing E. coli O86 for Bacteria Killing Assay
		3.2 Galectin Preparation
		3.3 Assessing Antimicrobial Activity
	4 Notes
	References
Chapter 28: Detection of Phosphatidylserine Exposure on Leukocytes Following Treatment with Human Galectins
	1 Introduction
	2 Materials
		2.1 Neutrophil Isolation
		2.2 Cell Culture
		2.3 Galectin Preparation and Incubation with Cells
		2.4 Annexin V Staining of Galectin-Treated Cells
	3 Methods
		3.1 Isolation of Human Neutrophils from Whole Blood
		3.2 Maintaining Cell Culture of Nonadherent HL60 Cells
		3.3 Galectin Preparation and Incubation with Cells
		3.4 Staining Cells with Annexin V
	4 Notes
	References
Chapter 29: Detection of Reactive Oxygen Species in Human Neutrophils Under Various Conditions of Exposure to Galectin
	1 Introduction
	2 Materials
		2.1 Neutrophil Isolation
		2.2 Galectin Preparation
		2.3 Additional Reagents
	3 Methods
		3.1 Isolation of Human Neutrophils from Whole Blood
		3.2 Galectin Preparation
		3.3 ROS Production by Neutrophils Followed Galectin Treatment
	4 Notes
	References
Chapter 30: Analysis of Galectin-Binding Receptors on B Cells
	1 Introduction
	2 Materials
		2.1 Processing of Tonsil to Isolate Tonsillar Mononuclear Cells
			2.1.1 Magnetic Cell Sort of Tonsillar GC and Naive B-Cell Subsets from Tonsillar Mononuclear Cells Using Miltenyi MACS Beads
		2.2 Assaying Galectin-Binding Activities on Tonsillar B-Cell Subsets Using Recombinant Human Galectin-3 and -9 (rhGal-3 and rh...
		2.3 Identifying Galectin-Binding Proteins on B Cells
	3 Methods
		3.1 Isolation Human B-Cell Subsets from Human Tonsillar Tissue
			3.1.1 Process Tonsil to Isolate Tonsillar Mononuclear Cells (Modified from)
			3.1.2 Magnetic Cell Sorting of Tonsillar GC and Naive B Cells from Tonsillar Mononuclear Cells Using Miltenyi MACS Beads
				Cell Preparation (See Note 2)
				Isolation of Tonsillar GC B Cells by Positive Selection
				Isolation of Tonsillar Naive B Cells by Negative Selection (See Note 3)
				Analysis of Cell Sort Purity
		3.2 Assaying Galectin-Binding Activities on Tonsillar B Cells Using rhGal-3 or rhGal-9 (See Note 4)
			3.2.1 Thawing and Preparation of Cells
			3.2.2 Viability Stain
			3.2.3 Galectin-Binding (Ligand) Staining
			3.2.4 Secondary Reagent Stain
		3.3 Identifying Galectin-Binding Proteins on Naïve B Cells (Fig. 2) (See Note 5)
			3.3.1 B Cell-Galectin Incubation and Cell Lysis
			3.3.2 Preparation of Recombinant Protein G (rProtein G) Agarose Beads
			3.3.3 Incubate Lysate with Antibody
			3.3.4 Elution of Sample (Eluate or Immunoprecipitate (IP))
			3.3.5 Detection of Galectin-Binding Receptors (See Note 7)
	4 Notes
	References
Chapter 31: Methods for Assessing the Effects of Galectins on Leukocyte Trafficking and Clearance
	1 Introduction
	2 Materials
		2.1 In Vitro Flow Chamber Assay
			2.1.1 Isolation and Culture of Human Umbilical Vein Endothelial Cells (HUVEC)
			2.1.2 Subculturing of HUVEC
			2.1.3 Seeding Cells into Ibidi Slides
			2.1.4 Coating Chambers with Recombinant Proteins
			2.1.5 Isolation of Neutrophils from Human Blood
			2.1.6 Preparation of Whole Blood for Flow Assays
			2.1.7 In Vitro Flow Chamber Assay
		2.2 Zymosan-Induced Peritonitis
			2.2.1 Peritoneal Inflammation and Lavage
			2.2.2 Leukocyte Recruitment
			2.2.3 Leukocyte Clearance
	3 Methods
		3.1 In Vitro Flow Chamber Adhesion Assay
			3.1.1 Isolation of Human Umbilical Vein Endothelial Cells
			3.1.2 Subculturing of HUVEC
			3.1.3 Seeding Cells into Ibidi Slides
			3.1.4 Coating Chambers with Recombinant Proteins
			3.1.5 PMN Isolation
			3.1.6 Flow Chamber Assay (PMN)
			3.1.7 Flow Chamber Assay (Whole Blood)
			3.1.8 Data Analysis
		3.2 Zymosan-Induced Peritonitis
			3.2.1 Peritoneal Inflammation and Lavage
			3.2.2 Leukocyte Recruitment
			3.2.3 Leukocyte Clearance
			3.2.4 Data Analysis
			3.2.5 Additional Analysis
	4 Notes
	References
Chapter 32: Examination of the Contributions of Maternal/Placental-Derived Galectin-1 to Pregnancy Outcome
	1 Introduction
	2 Materials
		2.1 Superovulation and In Vitro Fertilization in Mice
		2.2 Determination of Placental Weight, Pregnancy Outcome, Embryo Development
		2.3 Quantitative Analyses of Circulating Gal-1 Levels in Pregnant Mice
		2.4 Tissue Preparation for Immunohistochemistry (Cryo and Paraffin Sectioning)
		2.5 Immunofluorescent Determination of Gal-1 Expression at the Feto-Maternal Interface
		2.6 Lectin Binding to Whole Implantation Tissue
	3 Methods
		3.1 Superovulation
		3.2 In Vitro Fertilization for the Production of Maternal or Fetal-Placental Gal-1-Deficient Mice (Fig. 1a)
		3.3 Determination of Placental Weight, Pregnancy Outcome, Embryo Development (Fig. 1b, c) and Tissue Preparation for Cryo and ...
		3.4 Quantitative Analysis of Circulating Gal-1 Levels in Pregnant Mice (Fig. 1d)
		3.5 Cryo and Paraffin Sectioning for Immunohistochemistry
		3.6 Immunofluorescent Determination of Gal-1 Expression at the Feto-Maternal Interface Using Tissue Cryosections
		3.7 Lectin Binding to Whole Implantation Tissue
	4 Notes
	References
Chapter 33: Method to Study the Role of Galectins in Angiogenesis In Vivo Using the Chick Chorioallantoic Membrane Assay
	1 Introduction
	2 Materials
		2.1 Incubation of the Chicken Eggs
		2.2 Application of Galectins/Galectin Inhibitors onto the CAM
		2.3 Data Acquisition and Analysis
		2.4 Data Acquisition and Analysis
		2.5 Grafting Tumor Cells onto the CAM
		2.6 Intravenous Injection of Labeling Agents or Cancer Cells
		2.7 Special Equipment
	3 Methods
		3.1 Incubation of the Chicken Eggs
		3.2 Application of Galectins/Galectin Inhibitors onto the CAM
		3.3 Data Acquisition
		3.4 Data Analysis
		3.5 Grafting Tumor Cells onto the CAM
		3.6 Intravenous Injection of Cancer Cells or Labeling Agents
	4 Notes
	References
Chapter 34: Untangling Galectin-Mediated Circuits that Control Hypoxia-Driven Angiogenesis
	1 Introduction
	2 Materials
		2.1 Hypoxia Induction
		2.2 HIF-1α Detection
		2.3 Detection of Soluble VEGF by ELISA
		2.4 Isolation and Purification of Human Umbilical Vein Endothelial Cells (HUVECs)
		2.5 Assessment of Angiogenesis In Vitro
			2.5.1 Endothelial Cell Migration
			2.5.2 Endothelial Cell Tubulogenesis
			2.5.3 Endothelial Spheroid Sprout Assay
		2.6 Assessment of Angiogenesis In  Vivo
			2.6.1 Matrigel Plug Assay
			2.6.2 Inoculation of Matrigel Plugs to Evaluate Angiogenesis In  Vivo
			2.6.3 Determination of Angiogenesis In Vivo in Matrigel Plugs
		2.7 Special Equipment
	3 Methods
		3.1 Hypoxia Induction
			3.1.1 Induction of Hypoxia in Modular Incubator Chamber
			3.1.2 Evaluation of Hypoxia: HIF-1α Detection by Western Blot Analysis
			3.1.3 Evaluation of Hypoxia: HIF-1α Detection by Flow Cytometry
			3.1.4 Evaluation of Hypoxia: Detection of Soluble VEGF by ELISA
		3.2 Isolation and Purification of Human Umbilical Vein Endothelial Cells (HUVECs)
		3.3 Assessment of Angiogenesis In Vitro
			3.3.1 Endothelial Cell Migration
			3.3.2 Endothelial Cell Tubulogenesis
			3.3.3 Spheroid Sprout Assays
				Spheroid Formation
				Sprouting Assay
				Sprouting Staining and Quantification
		3.4 Methods to Study Angiogenesis In  Vivo
			3.4.1 Matrigel Plug Assay
				Matrigel Preparation
				Inoculation of Matrigel Plugs to Evaluate Angiogenesis In  Vivo
				Determination of Angiogenesis In Vivo in Matrigel Plugs
					Hemoglobin Content
					Number of Endothelial Cells by Flow Cytometry
					Analysis of Microvascular Density (MVD)
	4 Notes
	References
Chapter 35: Examination of the Role of Galectins and Galectin Inhibitors in Endothelial Cell Biology
	1 Introduction
	2 Materials
		2.1 Migration
		2.2 Network Formation
		2.3 Sprouting
		2.4 Special Equipment
	3 Methods
		3.1 Migration
		3.2 Network Formation
		3.3 Sprouting
	4 Notes
	References
36: Evaluating Therapeutic Activity of Galectin-1 in Sarcolemma Repair of Skeletal Muscle
	1 Introduction
	2 Materials
		2.1 Production and Purification of Recombinant Human Galectin-1 (Gal-1)
			2.1.1 Special Equipment
			2.1.2 Buffers
			2.1.3 Separation
		2.2 H2K A/J-/- and H2K A/J+/+ Cell Culture
		2.3 Calcium Dependency on Membrane Repair
		2.4 Activity of Carbohydrate Recognition Domain in Membrane Repair
		2.5 Labeling Galectin-1 Through Primary Amines
		2.6 Confocal Visualization of Labeled Galectin-1
		2.7 Muscle Fiber Isolation
	3 Methods
		3.1 Production of Recombinant Human Galectin-1
		3.2 Purification of Recombinant Human Galectin-1
		3.3 H2K A/J-/- (A/J-/-) and A/J+/+ (WT) Cell Culture
		3.4 Laser Injury Assay and Quantification
			3.4.1 Activity of Carbohydrate Recognition Domain in Membrane Repair
			3.4.2 Examination of Galetin-1 Calcium Dependency in Membrane Repair
		3.5 Labeling Galectin-1 Amines and Confocal Visualization
			3.5.1 Labeling Galectin-1 Amines
			3.5.2 In Vitro Confocal Immunofluorescence Using Alexa Fluor 647 Labeled Gal-1
		3.6 Muscle Fiber Isolation
	4 Notes
	References
Chapter 37: Exploring the Role of Galectins in Cancer: In Vitro and In Vivo Approaches
	1 Introduction
	2 Materials
		2.1 Galectin Immunohistochemistry in Paraffin-Embedded Tissue Sections
		2.2 Gal-1 Downregulation and Overexpression Strategies
			2.2.1 Gal-1 Transient Downregulation in Adherent Human Cancer Cell Lines
			2.2.2 Gal-1 Stable Downregulation in Adherent Human Cancer Cell Lines
			2.2.3 Stable Overexpression of Gal-1 in Adherent Human Cell Lines
		2.3 In Vitro Functional Analysis
			2.3.1 Cancer Cell Proliferation
				Crystal Violet Staining
				BrdU Incorporation Assay
			2.3.2 Cancer Cell Migration and Invasion
				Cell Migration Using Scratch Assay
				Cell Migration Assay in Transwells
				Cell Invasion Assay in Transwells
			2.3.3 Soft Agar Colony Formation Assay
		2.4 Preclinical Strategies
			2.4.1 Tumor Generation by Cell Line Injection in Mice
			2.4.2 Development of Tissue-Specific Oncogene-Driven Tumors in Galectin KO Background
			2.4.3 Data Collection and Analysis for Preclinical Studies
		2.5 Galectin-1 Determination in Blood Samples
	3 Methods
		3.1 Galectin Immunohistochemistry in Paraffin-Embedded Tissue Sections
		3.2 Gal-1 Downregulation and Overexpression Strategies
			3.2.1 Gal-1 Transient Downregulation in Adherent Human Cancer Cell Lines
			3.2.2 Gal-1 Stable Downregulation in Adherent Human Cancer Cell Lines (See Note 7)
			3.2.3 Stable Overexpression of Gal-1 in Adherent Human Cell Lines
		3.3 In Vitro Functional Assays
			3.3.1 Cancer Cell Proliferation
				Crystal Violet Staining
				BrdU Incorporation
			3.3.2 Cancer Cell Migration and Invasion
				Cell Migration Using Scratch Assay
				Cell Migration Assay in Transwells
				Cell Invasion Assay in Transwells
			3.3.3 Soft Agar Colony Formation Assay
		3.4 Preclinical Strategies
			3.4.1 Tumor Generation by Cell Line Injection in Mice
			3.4.2 Development of Tissue-Specific Oncogene-Driven Tumors in Galectin KO Background
			3.4.3 Data Collection and Analysis for Preclinical Studies
		3.5 Galectin-1 Determination in Blood Samples
	4 Notes
	References
Chapter 38: Galectin-3-U1 snRNP Complexes Initiate Splicing Activity in U1-Depleted Nuclear Extracts
	1 Introduction
	2 Materials
		2.1 Preparation of Nuclear Extract (NE) and the Splicing Substrate
		2.2 Preparation of Nuclear Extract Depleted of U1 snRNP (U1ΔNE)
		2.3 Glycerol Gradient Fractionation of NE
			2.3.1 Preparation of Gradients
			2.3.2 Fractionation of NE on a 12-32% Glycerol Gradient
		2.4 Immunoprecipitation of 10S Fractions of Glycerol Gradient by Anti-Gal3
		2.5 Reconstitution of Splicing Activity in U1ΔNE by Anti-Gal3 Precipitate of 10S Fractions
	3 Methods
		3.1 Preparation of Nuclear Extract Depleted of U1 snRNP (U1ΔNE)
			3.1.1 Preparation of Anti-U1 Beads for Immunoadsorption
			3.1.2 Depletion of U1-snRNP from NE (Fig. 1A)
			3.1.3 Analysis of the RNA and Protein Content of U1ΔNE and Material Bound on Anti-U1 Beads
		3.2 Glycerol Gradient Fractionation of NE
			3.2.1 Preparation of Gradients
			3.2.2 Fractionation of NE on 12-32% Glycerol Gradient (Fig. 1B)
		3.3 Immunoprecipitation of 10S Fractions of Glycerol Gradient by Anti-Gal3
			3.3.1 Preparation of Anti-Gal3 Beads for Immunoadsorption
			3.3.2 Immunoprecipitation of Glycerol Gradient Fractions by Anti-Gal3 (Fig. 1B)
			3.3.3 Analysis of the RNA and Protein Content in the Unbound and Bound Material from the Anti-Gal3 Precipitation of 10S Gradie...
		3.4 Reconstitution of Splicing Activity in U1ΔNE by Anti-Gal3 Precipitate of 10S Fractions
			3.4.1 Reconstitution of Splicing Activity in U1ΔNE (Fig. 1C)
			3.4.2 RNA Extraction, and Analysis of Product
	4 Notes
	References
Index