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Hospital for Sick Children

10 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging
Jun Dazai *1, Shoshana Spring *1, Lindsay S. Cahill 1, R. Mark Henkelman 1,2
1Mouse Imaging Centre, Hospital for Sick Children, 2Department of Medical Biophysics and Medical Imaging, University of Toronto

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

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Developmental Biology

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus
Steven J. Deimling 1, Rami R. Halabi 2,3, Stephanie A. Grover 4, Jean H. Wang 2,5, Thomas A. Drysdale 2,4,5
1Developmental and Stem Cell Biology, Hospital for Sick Children, 2Children's Health Research Institute, University of Western Ontario, 3Department of Physiology and Pharmacology, University of Western Ontario, 4Neurosciences and Mental Health, Hospital for Sick Children, 5Department of Paediatrics, University of Western Ontario

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

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Biology

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein
Paul D. W. Eckford 1, Canhui Li 1, Christine E. Bear 1,2,3
1Programme in Molecular Structure and Function, Hospital for Sick Children, 2Department of Biochemistry, University of Toronto, 3Department of Physiology, University of Toronto

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

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Bioengineering

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds
Sharareh Shojaie 1, Joyce Lee 1, Jinxia Wang 1, Cameron Ackerley 1, Martin Post 1
1Department of Physiology and Experimental Medicine, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

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Immunology and Infection

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model
Trevor Beaudoin 1, Sarah Kennedy 2, Yvonne Yau 3, Valerie Waters 4
1Physiology and Experimental Medicine, Hospital for Sick Children, 2Department of Clinical Microbiology, Royal College of Surgeons in Ireland, 3Department of Laboratory Medicine and Pathobiology, University of Toronto, 4Department of Pediatrics, University of Toronto

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

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Immunology and Infection

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients
Lindsay Jackson 1, William DePas 2, Amanda J. Morris 1, Kevin Guttman 1, Yvonne C W Yau 1,3, Valerie Waters 1,4
1Translational Medicine, Hospital for Sick Children, 2Center for Microbial Pathogenesis, Department of Pediatrics, University of Pittsburgh, 3Microbiology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, 4Infectious Diseases, Department of Pediatrics, Hospital for Sick Children

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

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Immunology and Infection

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software
Amanda J. Morris 1, Alvin Li 1, Lindsay Jackson 1, Yvonne C. W. Yau 1,2, Valerie Waters 1,3
1Translational Medicine, Hospital for Sick Children, 2Microbiology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, 3Infectious Diseases, Department of Pediatrics, Hospital for Sick Children

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

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Neuroscience

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations
Samantha Ing-Esteves 1,2, Julie L. Lefebvre 1,2
1Program for Neuroscience and Mental Health, Hospital for Sick Children, 2Department of Molecular Genetics, University of Toronto

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

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Immunology and Infection

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression
Kevin Guttman 1, Pauline Wang 2, Lindsay Jackson 1, Amanda Morris 1, Yvonne Yau 3, Valerie Waters 1,4
1Translational Medicine, Hospital for Sick Children, 2Department of Cell and Systems Biology, University of Toronto, 3Microbiology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, 4Infectious Diseases, Department of Pediatrics, Hospital for Sick Children

This protocol presents a method to isolate RNA from Pseudomonas aeruginosa biofilms grown in chamber slides for high throughput sequencing.

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Biology

A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines
Sunny Xia 1,3, Michelle Di Paola 3, Nicola L. Jones 2,4,5, Christine E. Bear 1,3,5
1Molecular Medicine, Hospital for Sick Children, 2Cell Biology, Hospital for Sick Children, 3Department of Physiology, University of Toronto, 4Department of Paediatrics, University of Toronto, 5Department of Biochemistry, University of Toronto

This protocol describes a method for the study of electrogenic membrane proteins by measuring changes in membrane potential. This assay provides a platform for the functional readout of multiple ion channels endogenously expressed in epithelial cell lines.

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