McGill University View Institution's Website 62 articles published in JoVE Cancer Research Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation Keerthana Harwalkar1,2, Nobuko Yamanaka1,3, Yojiro Yamanaka1,2,3 1Rosalind and Morris Goodman Cancer Research Institute, McGill University, 2Department of Human Genetics, McGill University, 3McGill Integrated Core for Animal Modeling (MICAM), McGill University This protocol describes microinjection and in vivo electroporation for regionally restricted CRISPR-mediated genome editing in the mouse oviduct. Medicine Oxygenation-sensitive Cardiac MRI with Vasoactive Breathing Maneuvers for the Non-invasive Assessment of Coronary Microvascular Dysfunction Elizabeth Hillier1,2, Jason Covone1, Matthias G. Friedrich1,3 1Faculty of Medicine and Health Sciences, McGill University, 2Faulty of Medicine and Dentistry, University of Alberta, 3Departments of Medicine and Diagnostic Radiology, McGill University The assessment of microvascular function by oxygenation-sensitive cardiac magnetic resonance imaging in combination with vasoactive breathing maneuvers is unique in its ability to assess rapid dynamic changes in myocardial oxygenation in vivo and, thus, may serve as a critically important diagnostic technique for coronary vascular function. Neuroscience Reliable Acquisition of Electroencephalography Data during Simultaneous Electroencephalography and Functional MRI Hui Ming Khoo1, Yuya Fujita1, Naoki Tani1, Tetsuya Shimokawa2, Natalja Zazubovits3, Satoru Oshino1, Jean Gotman3, Haruhiko Kishima1 1Department of Neurosurgery, Osaka University Graduate School of Medicine, 2Center for Information and Neural Networks, National Institute of Information and Communications Technology, 3Montreal Neurological Institute and Hospital, McGill University This article provides a straightforward protocol for acquiring good quality electroencephalography (EEG) data during simultaneous EEG and functional magnetic resonance imaging by utilizing readily available medical products. Biology Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer Martijn van de Locht1, Josine M. de Winter1, Dilson E. Rassier2, Michiel H.B. Helmes1,3, Coen A.C. Ottenheijm1 1Department of Physiology, Amsterdam UMC, 2Department of Kinesiology and Physical Education, Faculty of Education, McGill University, 3IONOptix BV Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils. Biochemistry A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes Yeganeh Habibi1, Christopher J. Thibodeaux1 1Department of Chemistry, McGill University Lanthipeptide synthetases catalyze multistep reactions during the biosynthesis of peptide natural products. Here, we describe a continuous, bottom-up, hydrogen-deuterium exchange mass spectrometry (HDX-MS) workflow that can be employed to study the conformational dynamics of lanthipeptide synthetases, as well as other similar enzymes involved in peptide natural product biosynthesis. Cancer Research Detection of Lung Tumor Progression in Mice by Ultrasound Imaging Nour Ghaddar1,2, Shuo Wang1, Véronique Michaud1, Urszula Kazimierczak1,3, Nicolas Ah-son1, Antonis E. Koromilas1,4 1Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 2Division of Experimental Medicine, Faculty of Medicine, McGill University, 3Department of Cancer Immunology, Chair of Medical Biotechnology, Poznan University of Medical Sciences, 4Gerald Bronfman Department of Oncology, Faculty of Medicine, McGill University This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software. Immunology and Infection Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain Fernando González Ibanez1,2, Katherine Picard1,2, Maude Bordeleau1,3, Kaushik Sharma1,2,4, Kanchan Bisht1,2,4, Marie-Ève Tremblay1,2 1Axe Neurosciences, Centre de Recherche du CHU de Québec-Université Laval, 2Département de Médecine Moléculaire, Faculté de Médecine, Université Laval, 3Integrated Program in Neuroscience, McGill University, 4Center for Brain Immunology and Glia (BIG), University of Virginia This protocol describes a step-by-step workflow for immunofluorescent costaining of IBA1 and TMEM119, in addition to analysis of microglial density, distribution, and morphology, as well as peripheral myeloid cell infiltration in mouse brain tissue. Chemistry Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers Thomas A. Singleton*1, Mehdi Boudjemeline*1, Robert Hopewell1, Dean Jolly1, Hussein Bdair1, Alexey Kostikov1,2 1McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, 2Department of Neurology and Neurosurgery, McGill University We report an efficient carbon-11 radiolabeling technique to produce clinically relevant tracers for Positron Emission Tomography (PET) using solid phase extraction cartridges. 11C-methylating agent is passed through a cartridge preloaded with precursor and successive elution with aqueous ethanol provides chemically and radiochemically pure PET tracers in high radiochemical yields. Genetics Microsatellite DNA Genotyping and Flow Cytometry Ploidy Analyses of Formalin-fixed Paraffin-embedded Hydatidiform Molar Tissues Yassemine Khawajkie1, Nawel Mechtouf2, Phuong Nguyen2, Rima Slim1,2,3 1Department of Experimental Medicine, McGill University, 2Department of Human Genetics, McGill University, 3Department of Obstetrics and Gynecology, McGill University Hydatidiform moles are abnormal human pregnancies with heterogeneous aetiologies that can be classified according to their morphological features and parental contribution to the molar genomes. Here, protocols of multiplex microsatellite DNA genotyping and flow cytometry of formalin-fixed paraffin-embedded molar tissues are described in detail, together with results’ interpretation and integration. Bioengineering Experimental Methods to Study Human Postural Control Pouya Amiri1, Abolfazl Mohebbi1, Robert Kearney1 1Department of Biomedical Engineering, McGill University This article presents an experimental/analytic framework to study human postural control. The protocol provides step-by-step procedures for performing standing experiments, measuring body kinematics and kinetics signals, and analyzing the results to provide insight into the mechanisms underlying human postural control. Biochemistry Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry Igor Cestari1 1Institute of Parasitology, McGill University This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry. Immunology and Infection Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment Syim Salahuddin*1,2, Elaine Thomson*1,2,3, Oussama Méziane1,2, Omar Farnos2, Amélie Pagliuzza5, Nicolas Chomont5,6, Ron Olivenstein1, Cecilia Costiniuk1,3,4, Mohammad-Ali Jenabian2,3,6 1Research Institute McGill University Health Centre, 2Department of Biological Sciences, Université de Québec à Montréal, 3Department of Microbiology & Immunology, McGill University, 4Department of Medicine, Division of Infectious Diseases, McGill University, 5 We describe a method for processing bronchoalveolar lavage fluid and matched peripheral blood from chronically HIV-infected individuals on antiretroviral therapy to assess pulmonary HIV reservoirs. These methods result in the acquisition of highly pure CD4 T cells and alveolar macrophages that may subsequently be used for immunophenotyping and HIV DNA/RNA quantifications by ultrasensitive polymerase chain reaction. Bioengineering High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-β on the Epithelial-to-Mesenchymal Transition Haruka Yoshie1, Newsha Koushki1, Clayton Molter1, Peter M. Siegel2,3, Ramaswamy Krishnan4, Allen J. Ehrlicher1,2 1Department of Bioengineering, McGill University, 2Goodman Cancer Research Centre, McGill University, 3Department of Medicine, McGill University, 4Department of Emergency Medicine, Beth Israel Deaconess Medical Center We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-β dependent increase in contractility during the epithelial-to-mesenchymal transition. Immunology and Infection Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy Carlos Oscar S. Sorzano*1,2, Laura Martínez-Muñoz*3,4, Graciela Cascio3,5, Eva M. García-Cuesta3, J. Vargas1,6, Mario Mellado3, Jose Miguel Rodriguez Frade3 1Department of Macromolecular Structures, Centro Nacional de Biotecnología, Campus Univ. Autónoma de Madrid, 2Campus Urb. Montepríncipe s/n, Univ. San Pablo CEU, 3Department of Immunology and Oncology, Centro Nacional de Biotecnología, Campus Univ. Autónoma de Madrid, 4Department of Cell Signaling, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CSIC), 5Meyer Cancer Center, 6Department of Anatomy and Cell Biology, McGill Univ. Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy. Environment Detection of Viruses from Bioaerosols Using Anion Exchange Resin Joshua W. Schaeffer1, Jeffrey C. Chandler2, Margaret Davidson1,3, Sheryl L. Magzamen1, Alma Pérez-Méndez4, Stephen J. Reynolds1, Lawrence D. Goodridge5, John Volckens6, Alan B. Franklin2, Susan A. Shriner2, Bledar Bisha7 1High Plains Intermountain Center for Agricultural Health and Safety, Department of Environmental and Radiological Health Sciences, Colorado State University, 2National Wildlife Research Center, Wildlife Services, Animal and Plant Health Inspection Service, United States Department of Agriculture, 3Western Sydney University, 4Leprino Foods, Inc, 5Department of Food Science and Agricultural Chemistry, McGill University, 6Department of Mechanical Engineering, Colorado State University, 7Department of Animal Science, University of Wyoming An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols. Bioengineering Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation Tao Jiang*1, Jose Munguia-Lopez*2,3, Salvador Flores-Torres2, Joel Grant4, Sanahan Vijayakumar4, Antonio De Leon-Rodriguez3, Joseph M. Kinsella2,5 1Department of Mechanical Engineering, McGill University Montreal, 2Department of Bioengineering, McGill University Montreal, 3Department of Molecular Biology, Instituto Potosino de Investigación Científica y Tecnológica, A.C. (IPICyT), 4Department of Mining and Materials Engineering, McGill University Montreal, 5Department of Biomedical Engineering, McGill University Montreal We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis. Neuroscience How to Find Effects of Stimulus Processing on Event Related Brain Potentials of Close Others when Hyperscanning Partners Amanda Tardif1,3, Ashley Chau-Morris1,2, Zi Yue Wang1, Ehime Takahara1, Tim Hadjis1,2, Jean Debruille1, J. Bruno Debruille1,2,3 1Douglas Mental Health University Institute, 2Department of Psychiatry, McGill University, 3Department of Neurology and Neurosurgery, McGill University This protocol describes key steps involved in assessing the sensitivity of the brain of one person to the stimulus processing of a close other by selecting pairs of partners, recording their electroencephalogram (EEG) simultaneously and computing their event-related brain potentials (ERPs). Immunology and Infection Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography Ryan N. Rys1, Mark D. Blostein1,2, Catherine A. Lemarié1,2 1Lady Davis Institute for Medical Research, Jewish General Hospital, McGill University, 2Department of Medicine, Jewish General Hospital, McGill University The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time. Biochemistry Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions Robert W. Harkness V1, Philip E. Johnson2, Anthony K. Mittermaier1 1Department of Chemistry, McGill University, 2Department of Chemistry, York University We present a protocol for rapid characterization of biomolecular folding and binding interactions with thermolabile ligands using differential scanning calorimetry. Bioengineering Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays Huiyan Li1,2, Sebastien Bergeron3, Heidi Larkin1,2,3, David Juncker1,2 1McGill University and Génome Québec Innovation Centre, 2Biomedical Engineering Department, McGill University, 3Parallex BioAssays Inc. We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination. Neuroscience Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons Matthew Laaper1,2, Takrima Haque1, Ruth S. Slack3, Arezu Jahani-Asl1,2,4 1Lady Davis Institute for Medical Research, Jewish General Hospital, 2Integrated Program in Neuroscience, McGill University, 3Department of Cellular and Molecular Medicine, University of Ottawa, 4Department of Oncology, Faculty of Medicine, McGill University This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model. Chemistry Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI) Huiyan Li1, Robert Popp1, Bjorn Frohlich1, Michael X. Chen2, Christoph H. Borchers1,3,4,5 1University of Victoria-Genome BC Proteomics Centre, 2Jewish General Hospital, McGill University, 3Dept of Biochemistry and Microbiology, University of Victoria, 4Proteomics Centre, Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University, 5Gerald Bronfman Department of Oncology, Jewish General Hospital A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented. Neuroscience Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit Frédéric Manseau1, Sylvain Williams1 1Department of Psychiatry, Douglas Mental Health University Institute, McGill University Here, we present a protocol for recording rhythmic neuronal network theta and gamma oscillations from an isolated whole hippocampal preparation. We describe the experimental steps from extraction of the hippocampus to details of field, unitary and whole-cell patch clamp recordings as well as optogenetic pacing of the theta rhythm. Bioengineering Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel Corinne A. Hoesli1, Roger L. J. Kiang2, Kamini Raghuram2, René G. Pedroza3, Karen E. Markwick1, Antonio M. R. Colantuoni1, James M. Piret2 1Department of Chemical Engineering, McGill University, 2Michael Smith Laboratories & Department of Chemical and Biological Engineering, University of British Columbia, 3Michael Smith Laboratories & Department of Pharmaceutical Sciences, University of British Columbia This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications. Neuroscience Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection Margaret H. Magdesian1,2,3, Madeleine Anthonisen1, G. Monserratt Lopez-Ayon1, Xue Ying Chua1, Matthew Rigby1, Peter Grütter1 1Department of Physics, McGill University, 2Department of Neurology and Neurosurgery, Montreal Neurological Institute, 3Ananda Devices This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation. Genetics siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts Adjimon Gatien Lokossou1, Chirine Toufaily2, Amandine Vargas3, Benoit Barbeau1 1Department of Biological Sciences, University of Quebec in Montreal, 2Department of Pharmacology and Therapeutics, McGill University, 3Department of Clinical Sciences, University of Montreal This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time. Immunology and Infection Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy Robert J. Scarborough1,2, Kelsey L. Adams1,2, Olivier Del Corpo1,2, Aïcha Daher1, Anne Gatignol1,2,3 1Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, 2Department of Microbiology & Immunology, McGill University, 3Department of Medicine, McGill University Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones. Biology Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction Palak G. Patel1,2, Shamini Selvarajah1,2, Suzanne Boursalie1,2, Nathan E. How1,2, Joshua Ejdelman3, Karl-Philippe Guerard3, John M. Bartlett4, Jacques Lapointe3, Paul C. Park1, John B. A. Okello1,2, David M. Berman1,2 1 This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks. Chemistry Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology Amir Sheikhi1,2,3, Han Yang1,2,3, Md. Nur Alam1,2,3, Theo G. M. van de Ven1,2,3 1Department of Chemistry, McGill University, 2Center for Self-Assembled Chemical Structures (CSACS), McGill University, 3Pulp and Paper Research Center, McGill University Synthesis schemes to prepare highly stable wood fiber-based hairy nanoparticles and functional cellulose-based biopolymers have been detailed. Behavior An Experimental Analysis of Children's Ability to Provide a False Report about a Crime Joshua Wyman1, Ida Foster1, Victoria Talwar1 1Department of Educational and Counselling Psychology, McGill University The current methodology is designed to provide an ecologically relevant approach for measuring the veracity, length and quality of children's true and false testimonies. Implications of the current methodology for future research and professionals who interview children will also be discussed. Neuroscience Somatosensory Event-related Potentials from Orofacial Skin Stretch Stimulation Takayuki Ito1,2,3, David J. Ostry1,4, Vincent L. Gracco1,5 1Haskins Laboratories, 2Speech and Cognition Department, Gipsa-lab, CNRS, 3Univ. Grenoble-Alpes, 4Department of Psychology, McGill University, 5School of Communication Science and Disorders, McGill University This paper introduces a method for obtaining somatosensory event-related potentials following orofacial skin stretch stimulation. The current method can be used to evaluate the contribution of somatosensory afferents to both speech production and speech perception. Immunology and Infection Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data George Ashdown1, Elvis Pandžić3, Andrew Cope2, Paul Wiseman4, Dylan Owen1 1Academic Department of Rheumatology, Centre for Molecular and Cellular Biology of Inflammation, Division of Immunology, Infection and Inflammatory Disease, King's College London, 3ARC Centre for Advanced Molecular Imaging, Australian Centre for NanoMedicine, University of New South Wales Australia, 4Departments of Chemistry and Physic, McGill University To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy. Neuroscience High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging Julie Winterburn1,2, Jens C. Pruessner3, Chavez Sofia4,5, Mark M. Schira6,7, Nancy J. Lobaugh4,8, Aristotle N. Voineskos5,9, M. Mallar Chakravarty1,2 1Institute of Biomaterials and Biomedical Engineering, University of Toronto, 2Computational Brain Anatomy Laboratory, Douglas Institute, McGill University, 3McGill Centre for Studies in Aging, McGill University, 4MRI Unit, Research Imaging Centre, Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, 5Department of Psychiatry, University of Toronto, 6School of Psychology, University of Wollongong, 7Neuroscience Research Australia, 8Department of Medicine, University of Toronto, 9Kimel Family Translational Imaging Genetics Research Laboratory, Research Imaging Centre, Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum. Neuroscience Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders Surya T. Madabattula1, Joel C. Strautman1, Andrew M. Bysice1, Julia A. O’Sullivan1, Alaura Androschuk1, Cory Rosenfelt1, Kacy Doucet1, Guy Rouleau2, Francois Bolduc1 1Department of Pediatrics, University of Alberta, 2Montreal Neurological Institute and Hospital, McGill University We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets. Biology Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates Elite Possik1,2, Arnim Pause1,2 1Goodman Cancer Research Center, McGill University, 2Department of Biochemistry, McGill University C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates. Behavior Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test R. Brian Roome1,2, Jacqueline L. Vanderluit1 1BioMedical Sciences, Faculty of Medicine, Memorial University of Newfoundland, 2Neurosciences, Faculty of Medicine, McGill University Classical forelimb asymmetry analysis of the cylinder test is routinely used to assess behavioural deficits in rats following brain injury or stroke; however, it fails to detect consistent deficits in mice. This study demonstrates that quantifying paw-dragging behaviour is a more sensitive analysis of brain injury in mice. Immunology and Infection Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling Sara Najmeh*1, Jonathan Cools-Lartigue*1, Betty Giannias1, Jonathan Spicer1, Lorenzo E. Ferri1 1LD MacLean Surgical Research Laboratories, Department of Surgery, McGill University In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells. Biology Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example Carine Fillebeen1,2, Nicole Wilkinson1,2, Kostas Pantopoulos1,2 1Lady Davis Institute for Medical Research, Jewish General Hospital, 2Department of Medicine, McGill University Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography. Behavior Investigating the Effects of Antipsychotics and Schizotypy on the N400 Using Event-Related Potentials and Semantic Categorization Vivian Gu1, Ola Mohamed Ali1, Katherine L'Abbée Lacas2, J. Bruno Debruille3,4 1Department of Psychology, McGill University, 2Department of Cognitive Science, McGill University, 3Douglas Institute, Department of Psychiatry, McGill University, 4Neurology and Neurosurgery Department, McGill University Using event-related EEG potentials (ERPs), we investigate the effects of antipsychotic medications on abnormal semantic brain activations in healthy individuals with schizotypal traits. We use ERPs to track distinct changes in brain activity, shedding insight into the cognitive processes associated with semantic categorization. Neuroscience The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism Sara Tremblay1, Vincent Beaulé1, Sébastien Proulx2, Louis-Philippe Lafleur1, Julien Doyon1, Małgorzata Marjańska3, Hugo Théoret1 1Department of Psychology, University of Montréal, 2Montreal Neurological Institute, McGill University, 3Center for Magnetic Resonance Research and Department of Radiology, University of Minnesota This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (1H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism. Biology RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes Houtan Moshiri*1,2, Vaibhav Mehta*1,2, Reza Salavati1,2,3 1Department of Biochemistry, McGill University, 2Institute of Parasitology, McGill University, 3McGill Centre for Bioinformatics, McGill University A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics. Neuroscience Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters Nicolas Unsain1, Kristen N. Heard1, Julia M. Higgins1, Philip A. Barker1 1Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration. Neuroscience Assessment of Vascular Regeneration in the CNS Using the Mouse Retina Khalil Miloudi1, Agnieszka Dejda2, François Binet3, Eric Lapalme2, Agustin Cerani2, Przemyslaw Sapieha1,2,3 1Department of Neurology-Neurosurgery, McGill University, 2Department of Biochemistry, Maisonneuve-Rosemont Hospital Research Centre, University of Montréal, 3Department of Ophthalmology, Maisonneuve-Rosemont Hospital Research Centre, University of Montréal The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS. Environment Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water Bledar Bisha1, Jaclyn A. Adkins2, Jana C. Jokerst3, Jeffrey C. Chandler1, Alma Pérez-Méndez4, Shannon M. Coleman4, Adrian O. Sbodio5, Trevor V. Suslow5, Michelle D. Danyluk6, Charles S. Henry2, Lawrence D. Goodridge7 1Department of Animal Science, University of Wyoming, 2Department of Chemistry, Colorado State University, 3Department of Environmental and Radiological Health Sciences, Colorado State University, 4Department of Animal Sciences, Colorado State University, 5Department of Plant Sciences, University of California, Davis, 6Department of Food Science and Human Nutrition, Citrus Research and Education Center, University of Florida, 7Department of Food Science and Agricultural Chemistry, McGill University A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (µPAD) imbedded with bacterial-indicative colorimetric substrates. Biology Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale Valentina Gandin1, Kristina Sikström2, Tommy Alain3, Masahiro Morita3, Shannon McLaughlan1, Ola Larsson2, Ivan Topisirovic1 1Lady Davis Institute and Department of Oncology, McGill University, 2Department of Oncology-Pathology, Karolinska Institutet, 3Goodman Cancer Centre and Department of Biochemistry, McGill University Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules. Biology Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy Eva C. Arnspang1, Jennifer S. Koffman1, Saw Marlar1, Paul W. Wiseman2, Lene N. Nejsum1 1Institute of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center, Aarhus University, 2Departments of Chemistry and Physics, McGill University This paper provides a step by step guide to the fluctuation analysis technique k-Space Image Correlation Spectroscopy (kICS) for measuring diffusion coefficients of fluorescently labeled plasma membrane proteins in live mammalian cells. Behavior Measuring Sensitivity to Viewpoint Change with and without Stereoscopic Cues Jason Bell1,2, Edwin Dickinson2, David R. Badcock2, Frederick A. A. Kingdom3 1Research School of Psychology, Australian National University, 2School of Psychology, University of Western Australia, 3McGill Vision Research, Department of Ophthalmology, McGill University We discuss a novel method forviewpoint-rotation of visual stimuli, and demonstrate using a mirror stereoscopethe three-dimensional percept of rotation-in-depth. The technique can be used to investigate the role of stereoscopic cues in encoding viewpoint-rotated figures. Bioengineering Permeabilization of Adhered Cells Using an Inert Gas Jet Scott Cooper1, Paul Jonak1, Guillaume Chouinard-Pelletier1, Sylvain Coulombe1, Elizabeth Jones1, Richard L. Leask1,2 1Chemical Engineering, McGill University, 2Montreal Heart Institute This protocol describes a method for the temporary permeabilization of adherent cells using an inert gas jet. This technique facilitates the transfer of genetic material and biomolecules into adherent mammalian cells by the utilization of mechanical forces to disrupt the plasma membrane. Behavior How to Detect Amygdala Activity with Magnetoencephalography using Source Imaging Nicholas L. Balderston1, Douglas H. Schultz1, Sylvain Baillet2,3, Fred J. Helmstetter1,3 1Department of Psychology, University of Wisconsin-Milwaukee, 2McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, 3Department of Neurology, Medical College of Wisconsin This article describes how to record amygdala activity with magnetoencephalography (MEG). In addition this article will describe how to conduct trace fear conditioning without awareness, a task that activates the amygdala. It will cover 3 topics: 1) Designing a trace conditioning paradigm using backward masking to manipulate awareness. 2) Recording brain activity during the task using magnetoencephalography. 3) Using source imaging to recover signal from subcortical structures. Biology Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique Toby K. McGovern*1, Annette Robichaud*2, Liah Fereydoonzad2, Thomas F. Schuessler2, James G. Martin1 1Meakins-Christie Laboratories, Department of Medicine, McGill University, 2SCIREQ Scientific Respiratory Equipment Inc. The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). Neuroscience Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents Michael Verwey1, Barry Robinson2, Shimon Amir2 1Douglas Mental Health University Institute, Department of Psychiatry, McGill University, 2Center for Studies in Behavioral Neurobiology, Department of Psychology, Concordia University Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock. Medicine The Measurement and Treatment of Suppression in Amblyopia Joanna M. Black1, Robert F. Hess2, Jeremy R. Cooperstock3, Long To3, Benjamin Thompson1 1Department of Optometry and Vision Science, University of Auckland, 2Department of Ophthalmology, McGill University, 3Centre for Intelligent Machines, McGill University Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device. Neuroscience Methods for Intravenous Self Administration in a Mouse Model Elizabeth K. Kmiotek1, Corey Baimel1, Kathryn J. Gill1 1Addictions Unit, McGill University Health Centre The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted. Biology Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging Jamila Andoh1, Robert J. Zatorre1 1Montreal Neurological Institute and International laboratory for Brain, Music, and Sound (BRAMS), McGill University Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex. Bioengineering Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count Soonhyang Park1, Hicham Chibli1, Jay Nadeau1 1Department of Biomedical Engineering, McGill University, Montreal, QC Canada Nanoparticles such as semiconductor quantum dots (QDs) can be used to create photoactivatable agents for anti-microbial or anti-cancer applications. This technique shows how to water-solubilize cadmium telluride (CdTe) QDs, conjugate them to an antibiotic, and perform a bacterial inhibition assay based upon growth curves and plate count. Biology Detection of Viral RNA by Fluorescence in situ Hybridization (FISH) Kishanda Vyboh1,2, Lara Ajamian1,3, Andrew J. Mouland1,2,3 1Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, 2Department of Microbiology and Immunology, McGill University, 3Department of Medicine, Division of Experimental Medicine, McGill University A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events. Medicine Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct Yusuke Kuriki1, Younan Liu1, Dengsheng Xia1, Eva M. Gjerde1, Saeed Khalili1, Brennan Mui1, Changyu Zheng2, Simon D. Tran1 1Faculty of Dentistry, McGill University, 2National Institutes of Health, Bethesda, MD, USA A protocol for the cannulation of the mouse submandibular salivary gland via the Wharton's duct is described. For this experiment, the trypan blue solution is used as a dyer to demonstrate how this technique effectively delivers infusions into the targeted gland, and to suggest the reliability of this new approach as a potential clinical drug/cell therapy for the regeneration of salivary glands. Biology Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms Lee A. Freiburger1, Anthony K. Mittermaier1, Karine Auclair1 1Department of Chemistry, McGill University ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters. Neuroscience Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons Carole A. Farah1, Wayne S. Sossin1 1Neurology and Neurosurgery, McGill University In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs. Neuroscience Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord Sébastien D. Langlois*1,2, Steves Morin*1, Patricia T. Yam1,3,4, Frédéric Charron1,2,5,6,7 1Molecular Biology of Neural Development, Institut de Recherches Cliniques de Montréal, 2Division of Experimental Medicine and Program in Neuroengineering, McGill University, 3Program in Neuroengineering, McGill University, 4Montreal Neurological Institute, 5Department of Anatomy and Cell Biology, McGill University, 6Department of Biology, McGill University, 7Department of Medicine, Universite de Montreal - University of Montreal This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance. Biology Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain Shahin Zangenehpour1,2, Mark W. Burke2, Avi Chaudhuri3, Maurice Ptito2 1Cognitive Neuroscience Unit, Montreal Neurological Institute, 2Ècole d’Optomètrie, Universitè de Montrèal, 3Department of Psychology, McGill University Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time. Biology The Microfluidic Probe: Operation and Use for Localized Surface Processing Cecile M. Perrault1, Mohammad A. Qasaimeh1, David Juncker1 1Department of Biomedical Engineering, McGill University In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.