CARDIOVASCULAR DEVELOPMENT methods and protocols.

Preface
Contents
Contributors
Chapter 1: Tissue Clearing and 3-D Visualization of Vasculature with the PEGASOS Method
	1 Introduction
	2 Materials
		2.1 Animals
		2.2 Anesthesia System
		2.3 Retro-Orbital Injection
		2.4 Perfusion and Fixation
		2.5 PEGASOS Passive Immersion Procedure
		2.6 Whole-Mount Immunostaining
		2.7 3-D Imaging Acquisition for Cleared Samples
		2.8 Data Processing
	3 Methods
		3.1 Retro-Orbital Injection of Vasculature Dye (See Note 3)
		3.2 Perfusion (Including Postmortem Dye Perfusion) and Tissue Preparation
		3.3 PEGASOS Passive Immersion Procedure (Fig. 1)
		3.4 Whole-Mount Immunostaining Within PEGASOS Passive Immersion Procedure
		3.5 3-D Imaging Acquisition and Reconstruction
	4 Notes
	References
Chapter 2: Preparation and Identification of Cardiac Myofibrils from Whole Heart Samples
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Preparation of Myofibril Solutions
		2.3 Excision of Heart Tissue
		2.4 Instruments
	3 Methods
		3.1 Tissue Collection and Myofibril Preparation
		3.2 Protein Estimation and SDS-PAGE
		3.3 Imaging and Protein Identification
		3.4 Preparation for MS
	4 Notes
	References
Chapter 3: RNA-Sequencing and Transcriptome Analysis after Fluid Shear Stress Stimulation in Lymphatic Endothelial Cells
	1 Introduction
	2 Materials
		2.1 HDLEC Culture and FSS Stimulation
		2.2 Total RNA Extraction
		2.3 RNA-Seq and Analysis
	3 Methods
		3.1 HDLEC Culture and FSS Stimulation
		3.2 Total RNA Extraction
		3.3 RNA-Seq and Analysis
	4 Notes
	References
Chapter 4: Preparation of Neonatal Rat Papillary Muscles for Contractile Studies
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Surgical Instruments
		2.3 Isolation Procedure Plastic  Ware
		2.4 Preparation of Media and Materials
		2.5 Animals
	3 Methods
		3.1 Preparation of Instrumentation
		3.2 Harvesting Neonatal Rat Hearts
		3.3 Exposing of the Right Ventricular Papillary Muscles
		3.4 Isolation of the Right Ventricular Papillary Muscles
		3.5 Preparation of the Small Intact Muscle Test Apparatus
		3.6 Attachment of Papillary Muscle to the Force Transducer and Fixed Adapter  Arms
		3.7 Preparation of Attached Papillary Muscle for Functional Studies
	4 Notes
	References
Chapter 5: In Vitro Study of Permeability in Lymphatic Endothelial Cells Responding to Histamine
	1 Introduction
	2 Materials
		2.1 HDLEC Culture and Monolayer Formation
		2.2 FITC-Dextran-Based Transwell Assay
		2.3 TEER Assay
	3 Methods
		3.1 HDLEC Culture and Monolayer Formation
		3.2 FITC-Dextran-Based Transwell Assay
		3.3 TEER Assay
	4 Notes
	References
Chapter 6: Optimizing the Differentiation of Cardiomyocytes from Human Induced Pluripotent-Derived Stem Cells
	1 Introduction
	2 Materials
		2.1 Stem Cell Culturing and Differentiation
			2.1.1 Reagents Needed
		2.2 Immunofluorescent Identification of Cardiomyocytes
			2.2.1 Immunofluorescence Reagents
	3 Methods
		3.1 iPSC Culturing and Differentiation Setup
		3.2 Cardiomyocyte Differentiation Process
		3.3 Immunofluorescent Characterization of Cardiomyocytes
	4 Notes
	References
Chapter 7: Analysis of Angiogenesis in Mouse Embryonic Dorsal Skin by Whole-Mount Fluorescent Staining
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Instruments/Equipment
	3 Methods
		3.1 Harvest Anterior Dorsal Skin of Embryos from Time-Crossed Pregnant Animals
		3.2 Whole-Mount Fluorescent Immunohistochemistry
	4 Notes
	References
Chapter 8: Isolation and Culture of Mouse Lymphatic Endothelial Cells from Lung Tissue
	1 Introduction
	2 Materials
		2.1 Digestion of Lung Tissue
		2.2 Magnetic Purification and Culture of LEC
	3 Methods
		3.1 Preparing the Gelatin-Coated Tissue Culture Plate
		3.2 Making a Single-Cell Suspension from Mouse Lung
		3.3 Magnetic Purification of LECs
		3.4 Culture of LECs
	4 Notes
	References
Chapter 9: Laser-Induced Choroidal Neovascularization in Rats
	1 Introduction
	2 Materials
	3 Methods
		3.1 Prepare
		3.2 Anesthesia and Prelaser Preparation
		3.3 Laser Procedure
	4 Notes
	References
Chapter 10: Detecting Three-Dimensional Vascular Networks in the Mouse Embryonic Hindbrain
	1 Introduction
	2 Material
	3 Method
		3.1 Dissection the Hindbrain
		3.2 Sectioning the Hindbrain with the Vibratome
		3.3 Immunofluorescence Staining
		3.4 Image
	4 Notes
	References
Chapter 11: Visualizing Blood Vessel Development in Cultured Mouse Embryos Using Lightsheet Microscopy
	1 Introduction
	2 Materials
		2.1 Hollow Agarose Cylinders
		2.2 Cell Culture Media
		2.3 Instruments for Dissection
		2.4 Lightsheet Microscope Components
		2.5 Imaging Parameters
		2.6 Transgenic Mice
	3 Methods
		3.1 Pairing of Mice for E8.5 Embryos
		3.2 Preparation of Hollow Agarose Cylinders
		3.3 Mouse Embryo Dissection and Culture
		3.4 Chamber Preparation and Addition of Mouse Embryo to Imaging Chamber
		3.5 Imaging Parameters to Visualize Blood Vessels
	4 Notes
	References
Chapter 12: Laser Capture Microdissection of Vascular Endothelial Cells from Frozen Heart Tissues
	1 Introduction
	2 Materials
		2.1 Immunofluorescence
		2.2 Laser Capture Microdissection
		2.3 RNA Isolation
	3 Methods
		3.1 Immunofluorescence
		3.2 Laser Capture Microdissection
		3.3 RNA Isolation
	4 Notes
	References
Chapter 13: Visualization of Retinal Blood Vessels
	1 Introduction
	2 Materials
	3 Methods
		3.1 Instrument Preparation
		3.2 Animal Preparation
		3.3 cSLO Image Acquisition
		3.4 Fluorescein Angiography
		3.5 OCT Imaging Acquisition
	4 Notes
	References
Chapter 14: Transient Transgenics: An Efficient Method to Identify Gene Regulatory Elements
	1 Introduction
	2 Materials
		2.1 Creating the BAC Reporter
			2.1.1 BAC Clones
			2.1.2 Reporter Genes
			2.1.3 PCR of Reporter to Create BAC Insertion Fragment
			2.1.4 Bacteria for Recombineering Experiments
			2.1.5 Device to Electroporate Bacteria
			2.1.6 Molecular Biology Materials
			2.1.7 Buffers Used in this Section
		2.2 Microinjection of the Engineered BAC DNA
			2.2.1 Core Facility for Embryo Injections
			2.2.2 Preparing BAC DNA for Injection Experiments
			2.2.3 Buffers Used in this Section
		2.3 Reporter Expression Analysis
			2.3.1 Histology Equipment
			2.3.2 Buffers Utilized in this Section
			2.3.3 Molecular Biology and General Reagents
	3 Methods
		3.1 Creating the BAC Reporter by Recombineering
		3.2 Microinjection of the Targeted BAC DNA into One Cell-Staged Mouse Embryos
		3.3 Collecting and Analysis of Reporter Gene Expression
	4 Notes
	References
Chapter 15: Isolation of Lymphatic Muscle Cells (LMCs) from Rat Mesentery
	1 Introduction
	2 Material
		2.1 Mesenteric Lymphatic Vessel Dissection
		2.2 Culture and Split Primary LMC
		2.3 LMCs Characterization and Storage
	3 Methods
		3.1 Dissect Rat Mesentery
		3.2 Culture and Split Primary LMCs
		3.3 LMCs Characterization and Storage
	4 Notes
	References
Chapter 16: Isolation of Adult Mouse Cardiomyocytes Using Langendorff Perfusion Apparatus
	1 Introduction
	2 Materials
		2.1 Heart Perfusion System
		2.2 Heart Cannulation and Cardiomyocytes Isolation System
		2.3 Buffers
			2.3.1 Transfer Solution (See Note 8)
	3 Methods
		3.1 System Setup
		3.2 Heart Cannulation and Cardiomyocytes Isolation
	4 Notes
	References
Chapter 17: Analysis of Lymphatic Vessel Formation by Whole-Mount Immunofluorescence Staining
	1 Introduction
	2 Materials
		2.1 Reagents
	3 Methods
		3.1 Harvest the Mesentery from Time-Crossed Pregnant Female Mice
		3.2 Whole-Mount Immunofluorescence Staining
	4 Notes
	References
Index