Teratogenicity Testing: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Updating an Overview of Teratology
	1 Definition of Teratology
	2 Etiological Agents
		2.1 Genetic Factors
		2.2 Maternal Conditions
			2.2.1 Phenylketonuria (PKU)
			2.2.2 Diabetes Mellitus
			2.2.3 Hypothyroidism and Hyperthyroidism
			2.2.4 Hypoparathyroidism and Hyperparathyroidism
		2.3 Physical Agents
			2.3.1 Ionizing Radiation
			2.3.2 Hyperthermia
			2.3.3 Mechanical Forces
		2.4 Chemical Agents
			2.4.1 Anticonvulsants
			2.4.2 Fungicides or Antifungal Agents
			2.4.3 Antimicrobial Agents
			2.4.4 Steroids and Nonsteroids
			2.4.5 Sedatives/Narcotics
			2.4.6 Retinoids
			2.4.7 Heavy Metals
			2.4.8 Other Drugs
		2.5 Biological Agents
			2.5.1 Toxic Plants
			2.5.2 Infectious Agents
	3 Conclusion
	References
Chapter 2: Guidelines on Developmental Toxicity Tests: Brief Insights
	1 Introduction
	2 Legislative Environment and Guidelines
	3 Methods Used in Teratology
		3.1 Alternative Methods
			3.1.1 In Vitro Methodologies
			3.1.2 Nonmammalian Animal Models
		3.2 The Traditional In Vivo Mammalian Tests
	4 Conclusion
	References
Chapter 3: Development Features on the Selection of Animal Models for Teratogenic Testing
	1 Introduction
	2 Biological and Ethical Concerns on the Selection of Animal Models
		2.1 Invertebrate Species
			2.1.1 Eisenia fetida/Eisenia andrei
			2.1.2 Caenorhabditis elegans
			2.1.3 Drosophila melanogaster
		2.2 Vertebrate Species
			2.2.1 Nonmammalian Species
				Danio rerio
				Xenopus laevis
			2.2.2 Mammalian Species
				Rattus norvegicus
				Mus musculus
				Oryctolagus cuniculus
	3 Embryologic Overview of the Models
		3.1 Early Embryonic Development
			3.1.1 Fertilization
			3.1.2 Cleavage
			3.1.3 Gastrulation
			3.1.4 Axis Formation
		3.2 Late Embryonic Development
			3.2.1 Organogenesis
			3.2.2 Comparative Mammal´s Implantation and Placentation
		3.3 After Birth/Hatching Development
			3.3.1 Larval Period
			3.3.2 Sexual Maturity and Gametogenesis
	4 Specific Sensitivities for Teratogenic Tests
		4.1 Invertebrate Species
		4.2 Nonmammalian Vertebrate Species
		4.3 Mammalian Vertebrate Species
	5 Conclusion
	References
Chapter 4: Virus as Teratogenic Agents
	1 Introduction
	2 Zika Virus
		2.1 Zika Fetal Pathogenicity
		2.2 ZIKV Teratogenicity
	3 Virus as Teratogenic Agents in Animals
	4 Bovine Herpesvirus-1 (BHV-1)
		4.1 BHV-1 Teratogenicity
	5 Bovine Viral Diarrhea Virus
		5.1 BVDV Teratogenicity
	6 Schmallenberg Virus (SBV)
	7 Akabane and Aino Viruses
	8 Conclusions
	References
Chapter 5: The Future of the Teratogenicity Testing
	1 Introduction
		1.1 Teratology
		1.2 Principles of Teratology
		1.3 Teratology Mechanisms
		1.4 Comparison of In Vitro and In Vivo Teratological Methods
		1.5 The Importance and Future of Teratology
	2 Conclusion
	References
Chapter 6: The First Steps on AOPs´ Concepts, Development, and Applications in Teratology
	1 AOP Concepts
	2 Relevance of AOPs in Teratology
	3 Knowledge Needed to Build AOPs
	4 Development of AOPs
	5 Perspectives and Conclusion
	References
Chapter 7: Machine Learning to Predict Teratogenicity: Theory and Practice
	1 Introduction
	2 Materials
		2.1 Problem Definition
			2.1.1 Defining the Determinants of Teratogenicity
			2.1.2 Determining the Teratogenic Potential of a Drug
			2.1.3 Determining Whether a Chemical Is Teratogenic or Non-teratogenic
		2.2 Collecting Data
			2.2.1 Preparing Data
			2.2.2 Cleaning Data
			2.2.3 Preprocessing Data
		2.3 Adopting an Algorithm
		2.4 Building a ML Model
		2.5 Interpreting the Results
		2.6 Presenting the Model
	3 Methods
		3.1 Problem Definition
		3.2 Collecting Data
		3.3 Adopting an Algorithm
		3.4 Building a ML Model
		3.5 Typical Results
	4 Notes
	References
Chapter 8: Human Pluripotent Stem Cell-Based Assays to Predict Developmental Toxicity
	1 Introduction
	2 Materials
		2.1 Maintenance
			2.1.1 For Human Pluripotent Stem Cells (hPSC)
			2.1.2 For Human Foreskin Fibroblast (hFF)
		2.2 Thawing
		2.3 Passaging
		2.4 Checking Confluence
		2.5 Cryopreservation
		2.6 For Differentiation
		2.7 For 3D Biomass Formation
		2.8 For Confirmatory Test (Immunohistochemistry)
		2.9 For Toxicity Test
		2.10 For Cell Specificity Test
			2.10.1 Calcium Assay
			2.10.2 Spontaneous Beating
			2.10.3 Neurite Outgrowth Assay
		2.11 Cell Viability Test (MTT Assay)
	3 Methods
		3.1 Maintenance of Human Pluripotent Stem Cells and Human Foreskin Fibroblast Cells
		3.2 Thawing of Embryonic Stem Cells
		3.3 Passaging
		3.4 Counting of Cells
		3.5 Cryopreservation
		3.6 Differentiation of Human Embryonic Cells
		3.7 3D Biomass Formation
		3.8 Confirmatory Test (Immunohistochemistry)
		3.9 Testing for Toxicity
		3.10 Cell Specificity Test
			3.10.1 Calcium Assay
			3.10.2 Spontaneous Beating
			3.10.3 Neurite Outgrowth Assay
		3.11 Cell Viability Test (MTT Assay)
	4 Notes
	References
Chapter 9: Developmental Toxicity Using the Rat Whole Embryo Culture
	1 Introduction
	2 Materials
	3 Methods
		3.1 Animal Maintenance and Mating Procedure
		3.2 Preparation of Serum
		3.3 Explantation of Rat Embryos
		3.4 Culture of Rat Embryos
		3.5 Morphological Evaluation
			3.5.1 Yolk Sac Circulatory
			3.5.2 Allantois
			3.5.3 Flexion
			3.5.4 Heart
			3.5.5 Caudal Neural  Tube
			3.5.6 Hindbrain
			3.5.7 Midbrain
			3.5.8 Forebrain
			3.5.9 Otic System
			3.5.10 Optic System
			3.5.11 Olfactory System
			3.5.12 Branchial  Bars
			3.5.13 Maxillary Process
			3.5.14 Mandibular Process
			3.5.15 Forelimb
			3.5.16 Hindlimb
			3.5.17 Somites
	4 Notes
	References
Chapter 10: A Modified Murine Embryonic Stem Cell Test for Evaluating the Teratogenic Effects of Drugs
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Cell Types
		2.3 Maintenance, Culture, and Differentiation Media
		2.4 Expanding and Thawing the Cells
		2.5 Cell Freezing
		2.6 Cytotoxicity Endpoint Assessment (MTT Assay)
		2.7 Gene Expression Analysis (Quantitative Real-Time PCR)
	3 Methods
		3.1 To Culture, Murine  ESC
			3.1.1 Cell Thawing
			3.1.2 3T3 and D3 Cell Expansion
			3.1.3 Cell Freezing
		3.2 Cellular Exposure to Chemicals
			3.2.1 Cellular Exposures to Chemicals for Cell Viability Assessment
			3.2.2 Cellular Exposures to Chemicals for Cell Differentiation Inhibition Assessment
		3.3 Cellular Viability Tests
		3.4 Quantification of Gene Expression
			3.4.1 RNA Extraction
			3.4.2 RNA Retrotranscription
			3.4.3 Quantitative Real-Time  PCR
		3.5 Classification of the Embryotoxic Potential of the Test Compounds
	4 Notes
	References
Chapter 11: Teratogenic Effects of Drugs on Primary Lymphocytes Assessed by Flow Cytometry
	1 Introduction
	2 Materials
		2.1 Common Reagents and Consumables
		2.2 Isolation of Lymphocytes
			2.2.1 Equipment
			2.2.2 Buffers and Solutions
		2.3 Isolation of Mitochondria from Lymphocytes and Measurement of Mitochondrial Membrane Potential Collapse and ROS Production...
			2.3.1 Equipment
			2.3.2 Buffers and Solutions
		2.4 Measurement of Mitochondrial Membrane Potential Collapse, ROS Production, and Lysosomal Membrane Destabilization in Isolat...
			2.4.1 Equipment
			2.4.2 Buffers and Solutions
	3 Methods
		3.1 Isolation Lymphocytes
		3.2 Counting Cells Using a Hemocytometer
			3.2.1 Preparing Hemocytometer
			3.2.2 Preparing Cell Suspension
			3.2.3 Counting
			3.2.4 Viability
		3.3 Flow Cytometric Detection of Mitochondrial Membrane Potential in Intact Cells
		3.4 Flow Cytometric Detection of Cellular ROS Formation in Intact Cells
		3.5 Flow Cytometric Detection of Lysosomal Membrane Integrity in Intact Cells
		3.6 Mitochondria Isolation Form Isolated Lymphocytes
		3.7 Bradford Protein Assay
		3.8 Flow Cytometric Detection of Mitochondrial Membrane Potential in Isolated Mitochondria
		3.9 Flow Cytometric Detection of Mitochondrial ROS Formation in Isolated Mitochondria
	4 Notes
	References
Chapter 12: Assessment of the Teratogenic Effect of Drugs on the Chicken Embryo
	1 Introduction
	2 Materials
		2.1 Receipt and Incubation of Chicken Eggs
		2.2 General Purpose Solutions
		2.3 Drug Application
		2.4 Collection/Harvesting of Chicken Embryos from the Eggs
		2.5 Fixation and Staining
	3 Methods
		3.1 Preparation of Drugs
		3.2 General Instructions for Handling Chicken Eggs
		3.3 Incubation of Chicken Eggs
		3.4 Embryo Manipulation and Drug Application
		3.5 Harvesting the Embryo from the Egg
		3.6 Evaluation of Outcomes
		3.7 Cartilage Staining
	4 Notes
	References
Chapter 13: Daphnia magna as a Model Organism to Predict the Teratogenic Effect of Different Compounds
	1 Introduction
		1.1 What Makes Daphnia as a Model Organism?
		1.2 Nanoparticles as a Teratogen Using D. magna
		1.3 Drugs and Pollutant as Teratogen Using D. magna as a Model Organism
		1.4 Endpoints Taken for Teratogen Testing Study
	2 Materials
		2.1 Preparation of Culture Media to Grow D. magna
		2.2 Egg Collection
		2.3 Egg Measurement
		2.4 Embryo Fixation for Immunostaining
		2.5 Embryo Fixation for Scanning Electron Microscope
		2.6 Embryo Staining with Congo  Red
		2.7 Reproductive and Developmental Defect
		2.8 Morphological Abnormality of Adult Daphnia
		2.9 Growth  Rate
		2.10 Postembryonic Developmental Cycle
		2.11 Behavioral Assays
			2.11.1 Swimming Speed
			2.11.2 Hopping Frequency and Propelling Efficiency Index (PEI)
	3 Methods
		3.1 Culture Media to Grow D. magna
		3.2 Egg Collection
		3.3 Egg Measurement
		3.4 Embryo Fixation for Immunostaining
		3.5 Embryo Fixation for Scanning Electron Microscope (SEM)
		3.6 Embryo Staining with Congo  Red
		3.7 Reproductive and Developmental Defect
		3.8 Morphological Abnormality for Teratogenic Assay
		3.9 Growth  Rate
		3.10 Daphnia Postembryonic Developmental Cycle
		3.11 Behavioral Assays
			3.11.1 Swimming Speed
			3.11.2 Hopping Frequency
			3.11.3 Propelling Efficiency Index (PEI)
	4 Notes
	References
Chapter 14: Caenorhabditis elegans as an In Vivo Model Organism to Elucidate Teratogenic Effects
	1 Introduction
	2 Materials
		2.1 Requesting Strains
		2.2 Bacterial Food
		2.3 Experimental Materials and Software
		2.4 Media and Solution
	3 Methods
		3.1 Caenorhabditis elegans Maintenance
		3.2 Caenorhabditis elegans Synchronization
		3.3 Exposure Protocol
			3.3.1 Solid Medium Exposure
			3.3.2 Liquid Medium Exposure
			3.3.3 Long-Term Exposure
			3.3.4 Multiple Generations (Four) Exposure
			3.3.5 Physical Factors
		3.4 Transgene and Integration
			3.4.1 Agarose Plate Preparation
			3.4.2 Preparation Before Microinjection
			3.4.3 Microinjection
			3.4.4 Postoperative Recovery
			3.4.5 Gene Integration
		3.5 Physiological Indicator Assay
			3.5.1 Livability
			3.5.2 Body Index Test
			3.5.3 Growth Curve
			3.5.4 Reproduction Phenotype Test: Fecundity and Population Quantity
			3.5.5 Behavioral Function Assay
		3.6 Biochemical Indicator Assay
			3.6.1 Reactive Oxygen Species (ROS) Assay
			3.6.2 The Contents of the Enzymes
		3.7 Morphology Assay
			3.7.1 Apoptosis
			3.7.2 Morphology of Vulva
		3.8 Oil Red O Lipid Staining
		3.9 Statistical Analysis
	4 Notes
	References
Chapter 15: Enduring Ethanol-Induced Behavioral Alterations in Caenorhabditis elegans After Developmental Lead Exposure
	1 Introduction
	2 Materials
		2.1 Nematode Maintenance
			2.1.1 General Material
			2.1.2 Nematode Culture Media
			2.1.3 Bacterial Lysogeny Broth Medium
			2.1.4 Nematode Synchronization
		2.2 Developmental Pb Exposure
		2.3 Acute Functional Tolerance
	3 Methods
		3.1 Nematode Maintenance
			3.1.1 E. coli OP50 Bacteria Growth Preparation
			3.1.2 NGM Plates Seeded with E. coli OP50
			3.1.3 Nematode Synchronization
		3.2 Developmental Pb Exposure
			3.2.1 Preliminary Exposure to Pb(C2H3O2)2 in Solid Medium
			3.2.2 Preliminary Exposure to (PbNO3)2 in Solid Medium
			3.2.3 Preliminary Exposure to Pb(NO3)2 Added to the to the E. coli OP50 Graze
			3.2.4 Definitive Procedure
		3.3 Acute Functional Tolerance Assay in Response to Ethanol Exposure
		3.4 Video Analysis
		3.5 Statistical Analysis
	4 Notes
	References
Chapter 16: Exploration of Teratogenic and Genotoxic Effects on Model Organism Drosophila melanogaster
	1 Introduction
		1.1 Comet Assay
		1.2 SMART Assays of Drosophila (Wing and Eye Spot Assessment)
		1.3 Micronuclei Detection in Drosophila
		1.4 Teratogen Toxicity Assessment
	2 Materials
		2.1 Comet Assay
			2.1.1 Chemicals Required
			2.1.2 Equipment and Laboratory Consumables
		2.2 SMART Assay
			2.2.1 Chemicals Required
			2.2.2 Equipment and Laboratory Consumables
		2.3 Micronuclei Detection
			2.3.1 Chemicals Required
			2.3.2 Equipment and Laboratory Consumables
		2.4 Embryo Assessment
			2.4.1 Chemicals Required
			2.4.2 Equipment and Laboratory Consumables
	3 Methods
		3.1 Comet Assay Methods
			3.1.1 Hemolymph Extraction
			3.1.2 Suspension of Hemolymph on the Slide
			3.1.3 Electrophoresis and Lysis
			3.1.4 Slide Staining and Analysis
		3.2 SMART Assay Methods
			3.2.1 Preparation of Normal Fly  Food
			3.2.2 Preparation of Wing for Identifying the Wing  Spot
			3.2.3 Methods to Identify the Eye  Spot
		3.3 Micronuclei Detection Method
			3.3.1 Dissection of Larva
			3.3.2 Fixation and Staining
			3.3.3 Slide Preparation for Microscopy
		3.4 Methodology to Check the Defect in the Embryo
	4 Notes
	References
Chapter 17: Ecotoxicological Assessment of Seaweed-Based Crop Biostimulant on Earthworm Eudrilus eugeniae Kinb
	1 Introduction
	2 Materials
		2.1 Seaweed Collection and Extract Preparation
		2.2 Earthworm Culture and Assessment
	3 Methods
		3.1 Seaweed Extraction and Preparation
			3.1.1 Collection and Pre-preparation
			3.1.2 Extraction
			3.1.3 Preparation of SWE for Ecotoxicological Assessment
		3.2 Earthworm Culture
		3.3 Ecotoxicological Assessment
		3.4 Teratogenic Evaluation
	4 Notes
	References
Chapter 18: Evaluation of the Ecotoxicology of Seaweed-Based Biopesticide Used in Combat of the Polyphagous Pest Using Eudrilu...
	1 Introduction
	2 Materials
		2.1 Earthworm Culture and Assessment
		2.2 Seaweed Collection and Extraction
		2.3 Bioassays
			2.3.1 Contact Filter Paper (CFP) Test
			2.3.2 Morphology
			2.3.3 Physiology
			2.3.4 Biochemistry
			2.3.5 Histology
	3 Methods
		3.1 Seaweed Collection, Preparation, and Extraction
		3.2 Active Fraction Isolation-Column Chromatography
		3.3 Contact Filter Paper Test
		3.4 Estimation of Acetylcholinesterase (AChE)
		3.5 Catalase Assay
		3.6 SOD Assay
		3.7 Histology
	4 Notes
	References
Chapter 19: Biochemical Studies to Understand Teratogenicity and Lethality Outcomes in Modified-FETAX
	1 Introduction
	2 Materials
		2.1 Toxicity Tests
		2.2 Biochemical Studies
	3 Methods
		3.1 Test Organisms
		3.2 Modified-FETAX
		3.3 Biochemical Studies
			3.3.1 Exposure
			3.3.2 Preparation of Samples
			3.3.3 Analysis of Biochemical Markers
				Glutathione S-Transferase (GST)
				Glutathione Reductase (GR)
				Glutathione Peroxidase (GPx)
				Catalase (CAT)
				Carboxylesterase (CaE)
				Acetylcholinesterase (AChE)
				Malondialdehyde (MDA)
				Total Protein
	4 Notes
	References
Chapter 20: Bioinformatics Methods for Transcriptome Analysis on Teratogenesis Testing
	1 Introduction
	2 Materials
		2.1 Microarray Analysis
		2.2 RNA-Seq Analysis
		2.3 Functional Enrichment Analysis
	3 Methods
		3.1 Microarray Analysis
		3.2 RNA-Seq Analysis
		3.3 Functional Enrichment Analysis
	4 Notes
	References
Chapter 21: Angiogenesis Assay for Live and Fixed Zebrafish Embryos/Larvae
	1 Introduction
	2 Materials
	3 Methods
		3.1 Zebrafish Embryos Collection
		3.2 Live Embryo/Larval Assay
			3.2.1 Embryos/Larvae Preparation
			3.2.2 Video Acquisition
			3.2.3 Video Processing
			3.2.4 Image Analysis Using  Fiji
			3.2.5 Image Subtraction
			3.2.6 Blood Flow Observation
		3.3 Fixed Larval Staining
			3.3.1 Staining
			3.3.2 Fixation
			3.3.3 Bleaching and Storage
			3.3.4 Observation
	4 Notes
	References
Chapter 22: High-Resolution Respirometry for the Assessment of Teratogenic Chemicals
	1 Introduction
	2 Materials
		2.1 Embryo Rearing Media (ERM)
		2.2 Test Chemicals
		2.3 Seahorse Chemical Stocks
	3 Methods
		3.1 Loading Beaker for the Chemical Exposure of Embryos (24 Hours Before Seahorse Run)
		3.2 Utility Plate (1 Day Before the Run)
		3.3 Seahorse Protocol (Day of)
		3.4 Retrieve and Thaw Stock Solutions (Day of)
		3.5 Washing the Embryos (Day of, 24 Hours After Exposure)
		3.6 Loading the Islet Capture Microplate (Day of)
		3.7 Data Analysis
	4 Notes
	References
Chapter 23: A Stereological Approach to Quantify Immunohistochemical Staining in Zebrafish Larvae
	1 Introduction
	2 Materials
	3 Methods
		3.1 Samples Preparation
		3.2 Point Counting
		3.3 Relative Volume Density
			3.3.1 Example of Point Counting and Relative Volume Density (%) Estimation
	4 Notes
	References
Chapter 24: Whole-Mount Immunohistochemical and Immunofluorescence Assays in Zebrafish Embryos
	1 Introduction
	2 Materials
	3 Methods
		3.1 Larva Preparation
		3.2 Immunohistochemical Analyses
		3.3 Whole-Mount Immunofluorescence Assays for Zebrafish Embryos-Larvae
	4 Notes
	References
Chapter 25: Assessment of Developmental Neurotoxicity Using Semi-automatic Behavior Analysis System for Zebrafish
	1 Introduction
	2 Materials
		2.1 Required Equipment and Solution
		2.2 Required Hardware and Software
	3 Methods
		3.1 Maintenance of Zebrafish
		3.2 Touch Response to Assess Behavior in Zebrafish Embryos
			3.2.1 Processing Videos on Wondershare
			3.2.2 Semi-automatic Tracking in the Kinovea Program
			3.2.3 Performing the Analysis with Toxtrac
		3.3 Behavior Analysis in Adult Zebrafish
	4 Notes
	References
Chapter 26: Behavioral Profiling of Zebrafish (Danio rerio) Larvae: Activity, Anxiety, Avoidance, and Startle Response
	1 Introduction
	2 Materials
		2.1 Zebrafish Reproduction and Larvae Growth
		2.2 Behavioral Assays
	3 Methods
		3.1 Reproduction and Zebrafish Larvae Growth
		3.2 Behavioral Assays
			3.2.1 Locomotor Activity and Thigmotaxis
			3.2.2 Larval Diving Response
			3.2.3 Avoidance Behavior
			3.2.4 Touch-Evoked Response
			3.2.5 Dark/Light Challenge and Startle Reflex
	4 Notes
	References
Chapter 27: Alcian Blue Staining for Chondrocranium Development in Zebrafish
	1 Introduction
	2 Materials
		2.1 Required Equipment
		2.2 Required Software
		2.3 Required Solutions
	3 Methods
		3.1 Protocol Stages
	4 Notes
	References
Chapter 28: Protocol of Geometric Morphometrics for Teratogenicity Testing
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 29: Biochemical Markers for Liver Injury in Zebrafish Larvae
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Sample Processing
		2.3 Protein Determination
		2.4 Aspartate Aminotransferase Determination
		2.5 Alanine Aminotransferase Determination
		2.6 Alkaline Phosphatase Determination
		2.7 Lactate Dehydrogenase Determination
		2.8 Glutathione Peroxidase Determination
	3 Methods
		3.1 Sample Processing
		3.2 Protein Determination
		3.3 Determination of Aminotransferases (ALT and AST)
		3.4 Alkaline Phosphatase (ALP) Determination
		3.5 Lactate Dehydrogenase Determination
		3.6 Glutathione Peroxidase Determination
	4 Notes
	References
Chapter 30: Cortisol Quantification for Assessing Stress-Induced Changes in Zebrafish Larvae
	1 Introduction
	2 Materials
		2.1 Animal Maintenance and Collection
		2.2 Protein Quantification
		2.3 Cortisol Extraction and Quantification
		2.4 Behavior Evaluation
	3 Methods
		3.1 Zebrafish Maintenance and Reproduction
		3.2 Stress Event Assay
		3.3 Behavior Evaluation
		3.4 Sample Processing and Protein Quantification
		3.5 Cortisol Extraction and Quantification
	4 Notes
	References
Chapter 31: Metabolomic Fingerprint Assay in Zebrafish Embryos
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
	3 Methods
		3.1 Preparation of Zebrafish Embryos for Exposure
		3.2 Metabolite Extraction
		3.3 Spectra Recording and Deconvolution
	4 Notes
	References
Chapter 32: Alkaline Comet Assay to Assess Genotoxicity in Zebrafish Larvae
	1 Introduction
	2 Materials
		2.1 Isolation of Zebrafish Larvae Cells
		2.2 Pre-Coating Microscope Slides
		2.3 Embedding Cells in Agarose
		2.4 Lysis
		2.5 Denaturation and Electrophoresis
		2.6 Staining Slides
		2.7 Damage Assessment
	3 Methods
		3.1 Isolation of Zebrafish Larvae Cells
		3.2 Pre-Coating Microscope Slides
		3.3 Embedding Cells in Agarose
		3.4 Lysis
		3.5 Denaturation and Electrophoresis
		3.6 Neutralization
		3.7 Staining Slides
		3.8 Comets Quantification
	4 Notes
	References
Chapter 33: A Chromogenic Quantification of Protein Expression in Zebrafish Larvae
	1 Introduction
	2 Materials
		2.1 Equipment and Disposables
		2.2 Sample Preparation
		2.3 Protein Quantification
		2.4 SDS-PAGE and Electroblotting
		2.5 Immunodetection
	3 Methods
		3.1 Zebrafish Embryo Collection
		3.2 Sample Preparation
		3.3 Protein Quantification
		3.4 Gel Electrophoresis
		3.5 Check Protein Running (Optional)
		3.6 Transfer
		3.7 Check Membrane Transfer (Optional)
		3.8 Membrane Storage (Optional)
		3.9 Membrane Fixation (Optional)
		3.10 Immunoblotting
		3.11 Signal Detection
		3.12 Check Total Protein (Optional)
		3.13 Reprobing
		3.14 Quantification
		3.15 Typical Results
	4 Notes
	References
Chapter 34: Confocal Microscopy Technique in Teratogenicity Testing Using Zebrafish (Danio rerio) Embryos as Model
	1 Introduction
	2 Materials
		2.1 General Material
		2.2 TUNEL (Terminal Deoxynucleotide Transferase dUTP Nick End Labeling) Assay
		2.3 Oxidative Stress Determination
		2.4 Immunohistochemistry
	3 Methods
		3.1 TUNEL (Terminal Deoxynucleotide Transferase dUTP Nick End Labeling) Assay
		3.2 Oxidative Stress Determination
		3.3 Immunohistochemistry
	4 Notes
	References
Chapter 35: Whole-Mount RNA In Situ Hybridization of Zebrafish Embryos
	1 Introduction
	2 Materials
		2.1 Required Equipment
		2.2 Required Reagents and Solutions
	3 Methods
		3.1 Making Antisense RNA Probes with DIG Labels
		3.2 Embryo Handling for In Situ Hybridization
			3.2.1 Day 1
			3.2.2 Day 2
			3.2.3 Day 3
	4 Notes
	References
Chapter 36: Quantitative Real-Time PCR Method to Evaluate Gene Expression in Zebrafish Embryos
	1 Introduction
	2 Materials
		2.1 Required Equipment
		2.2 Reagents and Solutions
	3 Methods
		3.1 Preparation of Samples
			3.1.1 Sample Collection
			3.1.2 RNA Extraction
		3.2 cDNA Synthesis
		3.3 Quantitative RT-PCR Protocol
		3.4 Data Analysis
	4 Notes
	References
Chapter 37: Microarray Analysis to Determine Gene Expression Changes in Zebrafish Embryos
	1 Introduction
	2 Materials
		2.1 Required Equipment
		2.2 Reagents and Solutions
		2.3 Required Hardware and Software
	3 Methods
		3.1 Sample Preparation
			3.1.1 Collection of Samples
			3.1.2 Total RNA Extraction
			3.1.3 Prepare Spike a Mix
			3.1.4 Labeling Reaction Preparation
			3.1.5 The Labeled/Amplified RNA Purification
			3.1.6 The cRNA Quantification
		3.2 Hybridization
			3.2.1 Hybridization Samples Preparation
			3.2.2 The Hybridization Assembly Preparation
			3.2.3 Hybridize
		3.3 Microarray Wash
			3.3.1 Add Triton X-102 to Gene Expression Wash Buffers
			3.3.2 Prewarm Gene Expression Wash Buffer 2
			3.3.3 Wash the Microarray Slides
		3.4 Scanning and Feature Extraction
			3.4.1 Scan the Slide
			3.4.2 Agilent C Scanner Setting
			3.4.3 Extract Data Using Agilent Feature Extraction Software
		3.5 The Equipment Cleaning
			3.5.1 Solvent Wash
			3.5.2 Milli-Q Water Wash
	4 Notes
	References
Chapter 38: Cell-Free DNA and Next-Generation Sequencing for Prenatal Diagnosis
	1 Introduction
	2 Materials
		2.1 Reagents and Solutions
		2.2 Instruments
	3 Methods
		3.1 Preanalytical Sample Handling
		3.2 DNA Purification
		3.3 PCR Set Up
		3.4 Thermocycling
		3.5 Purification of Amplicons with AMPure XL Beads and Concentration Measurement
		3.6 Sequencing on the MiSeq Instrument (See Note 15)
		3.7 Data Analysis (See Note 24)
	4 Notes
	References
General Terms Used in Teratology
	Introduction
	Glossary
Index