Microcephaly: Methods and Protocols

Description

This volume discusses the latest imaging and molecular techniques for studying microcephaly through neural progenitor proliferation, survival, and gene expression. All of the methods covered in this book use the mouse as a model system, and include cellular, metabolic, transcriptomic studies of neural progenitors, MR imaging studies of brain growth in 3-dimensions and developmentally-adapted studies of behavior in neonatal mice. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Preface References Contents Contributors Part I: Cellular Analyses of Neural Progenitors Chapter 1: Dissociation of Cerebellar Granule Neuron Progenitors for Culture, FACS, Transcriptomics, and Molecular Biology 1 Introduction 2 Materials 3 Methods 4 Notes References Chapter 2: Maintaining Cerebellar Granule Neuron Progenitors in Cell Culture 1 Introduction 2 Materials 3 Methods 4 Notes References Chapter 3: Proliferation Analysis of Cerebellar Granule Neuron Progenitors for Microcephaly Research, Using Immunofluorescent ... 1 Introduction 2 Materials 2.1 Thymidine Analog Labeling 2.2 Immunofluorescence 2.3 Flow Cytometry 3 Methods 3.1 Thymidine Analog Labeling 3.2 Immunofluorescent Staining for BrdU/EdU 3.3 Measuring Proliferation and Cell Cycle Exit Fraction in Tissue Sections on Slides 3.4 CGNP Preparation for Flow Cytometry 3.5 Quantifying Neural Progenitor Proliferation by Flow Cytometry 3.6 Quantifying Replication Stress Using pH2A.X 4 Notes References Chapter 4: Conventional and Spectral Karyotyping of Murine Cerebellar Granule Neuron Progenitors 1 Introduction 2 Materials 2.1 Slide Preparation 2.2 DAPI-Banding 2.3 Spectral Karyotyping 2.3.1 Probe Denaturation 2.3.2 Slide Pretreatment 2.3.3 Slide Denaturation and Hybridization 2.3.4 Post-hybridization Wash and Detection 2.4 Image Acquisition and Analysis 2.4.1 DAPI-Banding 2.4.2 Spectral Karyotyping (SKY) 3 Methods 3.1 Slide Preparation 3.2 DAPI-Banding 3.3 Spectral Karyotyping 3.3.1 Probe Denaturation 3.3.2 Slide Pretreatment 3.3.3 Slide Denaturation and Hybridization 3.3.4 Post-hybridization Wash and Detection 3.4 Image Acquisition and Analysis 3.4.1 DAPI-Banding 3.4.2 Spectral Karyotyping (SKY) 4 Notes References Part II: Analyses of Neural Progenitors In Situ Chapter 5: Processing Neonatal Mouse Brains for Immunohistochemical Analysis: As Required for Including Spindle Orientation An... 1 Introduction 2 Materials 2.1 Tissue Preparation and Embedding 3 Methods 3.1 Tissue Isolation and Fixation 3.2 Paraffin Embedding 3.3 Deparaffinization and Antigen Retrieval 4 Notes References Chapter 6: Automated Immunofluorescence Staining for Analysis of DNA Damage and Apoptosis in Brain Sections 1 Introduction 2 Materials 2.1 Immunohistochemistry 3 Methods 3.1 Automated Dual-Color Indirect Fluorescent Immunohistochemistry to Identify DNA Damage and Apoptosis 3.1.1 Overview 3.1.2 Preparing Staining Solutions for Leica Bond III 3.1.3 Setting Up and Running Staining Program 3.2 Imaging and Quantification of pH2AX+ and cC3+ Cells in Brain Section 3.2.1 Image Acquisition 3.2.2 Image Analysis 4 Notes References Chapter 7: Automated Immunofluorescence Staining for Analysis of Mitotic Stages and Division Orientation in Brain Sections 1 Introduction 2 Materials 2.1 Immunohistochemistry 3 Methods 3.1 Automated Dual-Color Indirect Fluorescent Immunohistochemistry to Identify Mitotic Cells 3.1.1 Overview 3.1.2 Preparing Staining Solutions for Leica Bond III 3.1.3 Setting Up and Running Staining Program 3.2 Imaging and Analysis of Mitotic Stages and Division Orientation 3.2.1 Image Acquisition 3.2.2 Quantifying Division Orientation by Categorical Metrics 3.2.3 Measuring Division Orientation Angles 4 Notes References Part III: RNA-seq Analysis with Single-Cell Resolution (scRNA-seq) Chapter 8: scRNA-seq for Microcephaly Research [I]: Single-Cell Droplet Encapsulation, mRNA Capture, and cDNA Synthesis 1 Introduction 2 Materials 2.1 DNA Oligos 2.2 Beads 2.3 Chemical Reagents 2.4 Enzymes and Kits 2.5 Equipment 2.6 Buffers to Prepare in Advance 3 Methods 3.1 Preparing the Equipment 3.2 Preparing the Beads 3.3 Preparing for Your First Run 3.4 Executing the Drop-Seq Run 3.5 Post-run Bead Processing (Washing and Reverse Transcription) 3.6 Post-run Bead Processing (Exonuclease Treatment and Template Switch PCR) 3.7 Post-PCR Cleanup and cDNA QC 3.8 Post PCR Cleanup for Larger Amounts of cDNA Samples 4 Notes References Chapter 9: scRNA-seq for Microcephaly Research [II]: Preparation of Single-Cell cDNA Libraries 1 Introduction 2 Materials 2.1 DNA Oligos 2.2 Chemical Reagents 2.3 Enzymes and Kits 2.4 Equipment 2.5 Buffers to Prepare in Advance 3 Methods 3.1 Preparation of Illumina Library for Sequencing (Tagmentation) 3.2 Sequencing of the Drop-Seq Library 3.3 Finding the Final Concentration of Pooled Libraries by Qubit 3.4 Pooling Libraries for Sequencing 4 Notes References Chapter 10: scRNA-seq for Microcephaly Research [III]: Computational Analysis of scRNA-seq Data 1 Introduction 2 Required Software and Hardware 3 Method 3.1 Constructing the Reference 3.2 Raw Data Processing 3.3 Data Filtering 3.4 Data Normalization and Scaling 3.5 Dimensional Reduction: PCA, tSNE, and Clusters 3.6 Differential Gene Analysis: Identifying Cells 4 Further Analysis 5 Notes References Chapter 11: scRNA-seq for Microcephaly Research [IV]: Dirichlet Regression for Single-Cell Population Differences 1 Introduction 2 Required Software 3 Data Analysis Reference Part IV: MRI Analyses of the Mouse Brain Chapter 12: Mouse Brain MRI: Including In Vivo, Ex Vivo, and fcMRI for the Study of Microcephaly 1 Introduction 2 Materials 2.1 In Vivo Anatomical Imaging 2.2 Whole-Body Mouse Perfusion and Fixation 2.3 Optimization of Scan Parameters for In Vivo and Ex Vivo Imaging 2.4 fcMRI 3 Methods 3.1 In Vivo Imaging 3.2 Perfusion and Ex Vivo Imaging 3.3 Optimization of Scan Parameters for In Vivo and Ex Vivo Imaging 3.4 fcMRI 4 Notes References Chapter 13: Whole-Body Mouse Fluxomic Analysis to Detect Metabolic Disruptions Associated with Microcephaly: Using 13C Isotopes 1 Introduction 2 Materials 3 Methods 3.1 Stable Isotope Labeling 3.2 Tissue Collection 3.3 Extraction 3.4 For LC-MS Analysis 3.5 For NMR Analysis 4 Notes References Part V: Behavioral Phenotyping in Neonatal Mice Chapter 14: Neonatal Behavioral Screen for Mouse Models of Neurodevelopmental Disorders 1 Introduction 2 Materials 2.1 Subjects 2.2 Litter Transport and Holding 2.3 Negative Geotaxis and Righting Reflex 2.4 Open Field 2.5 Acoustic Startle 3 Methods 3.1 PD 6-8 Behavior Screen 3.1.1 Litter Transport and Holding 3.1.2 Negative Geotaxis 3.1.3 Righting Reflex 3.1.4 Open Field (3-min Test) 3.2 Open Field: 10-min Tests 3.3 Acoustic Startle (PD 14-15 and PD 20-21) 3.4 Tail Suspension Test for Limb Clasping 4 Notes References Index