University of Montreal View Institution's Website 40 articles published in JoVE Medicine Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model Pierre-Emmanuel Noly1,2, Wei Huang1, Suyash Naik1, Paul Tang1, Ienglam Lei1 1University of Michigan, Ann Arbor, 2University of Montreal Presented here is a protocol to reliably quantify the right and left ventricular function of donor hearts after cold preservation using an ex vivo perfusion system. Neuroscience Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice Julien Dufort-Gervais1, Robbert Havekes2, Valérie Mongrain1,3 1Center for Advanced Research in Sleep Medicine, Recherche CIUSSS-NIM, 2GELIFES, University of Groningen, 3Department of Neuroscience, Université de Montréal This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings. Behavior Measuring the Switch Cost of Smartphone Use While Walking Gabrielle Naïmé Mourra1, David Brieugne1, Emma Rucco1, Élise Labonté-Lemoyne1, François Courtemanche1, Sylvain Sénécal1, Marc Fredette1, Ann-Frances Cameron1, Jocelyn Faubert2, Franco Lepore2, François Bellavance1, Pierre-Majorique Léger1 1HEC Montréal, 2Université de Montréal This study design measures the task-switching cost of using a smartphone while walking. Participants undergo two experimental conditions: a control condition (walking) and a multitasking condition (texting while walking). Participants switch between these tasks and a direction determining task. EEG data as well as behavioral measures are recorded. Immunology and Infection Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment Manuel Flores Molina1,2, Thomas Fabre1,2, Aurélie Cleret-Buhot1, Geneviève Soucy1,3, Liliane Meunier1,4, Mohamed N. Abdelnabi1,2, Nicolas Belforte1,5, Simon Turcotte1,4,6, Naglaa H. Shoukry1,4,7 1 Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays. Genetics Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in Primary Alveolar Epithelial Cells Francis Migneault1,2,3, Frédéric Gagnon2,3, Emmanuelle Brochiero1,3, Yves Berthiaume2,3, André Dagenais2,3 1 Here, we present a tool that can be used to study the posttranscriptional modulation of a transcript in primary alveolar epithelial cells by using an inducible expression system coupled to a pipette electroporation technique. Medicine Pre-clinical Model of Cardiac Donation after Circulatory Death Henry Aceros*1, Leyla Joulali*2, Mélanie Borie1, Roberto Vanin Pinto Ribeiro3, Mitesh Vallabh Badiwala3, Shant Der Sarkissian1,4, Nicolas Noiseux1,4 1 This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death. Biochemistry In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones Louis Masclef*1, Uriarte Maxime*1, Oumaima Ahmed1, Nadine Sen Nkwe1, Haithem Barbour1, Nicholas V.G. Iannantuono1, Amina Boubekeur1, Salima Daou1,2, El Bachir Affar1,2 1Maisonneuve-Rosemont Hospital Research Center and Department of Medicine, University of Montréal, 2Lunenfeld-Tanenbaum Research Institute, Sinai Health System Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components. Neuroscience Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat Andrew R. Brown1,2, Marina Martinez1,2,3 1Department of Neurosciences, Faculté de Médecine, Université de Montréal, 2Hôpital du Sacré-Cœur de Montréal, 3Groupe de Recherche sur le Système Nerveux Central (GRSNC), Université de Montréal The rat thoracic spinal hemisection is a valuable and reproducible model of unilateral spinal cord injury to investigate the neural mechanisms of locomotor recovery and treatment efficacy. This article includes a detailed step-by-step guide to perform the hemisection procedure and to assess locomotor performance in an open-field arena. 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. Developmental Biology Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells Amanda B.T. Hill1,2, Jonathan E.B.T. Hill1, Fabiana F. Bressan3, Maria Angélica Miglino4, Joaquim M. Garcia1 1The School of Agricultural and Veterinary Studies (FCAV), Jaboticabal, São Paulo State University (UNESP), 2CRRF, Université de Montréal, 3FZEA, University of São Paulo, 4FMVZ, University of São Paulo Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue. Medicine A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence Eric Parent1, Patrick Burns2, André Desrochers1, Martine Boulianne1 1Department of Clinical Sciences, Faculty of Veterinary Medicine, Université de Montréal, 2University of Bristol Here we present a protocol to surgically create 'intestinal ligated loops' in chicken small intestines. This procedure allows for the comparison of multiple Clostridium perfringens strains' virulence in situ in a single host. This method markedly decreases the number of chickens usually necessary for similar in vivo experiments. Neuroscience Analyzing the Size, Shape, and Directionality of Networks of Coupled Astrocytes Steven Condamine1, Dorly Verdier1, Arlette Kolta1,2 1Groupe de Recherche sur le Système Nerveux Central, and Département de Neurosciences, Université de Montréal, 2Faculté de Médecine Dentaire, Université de Montréal Here we present a protocol to assess the organization of astrocytic networks. The described method minimizes bias to provide descriptive measures of these networks such as cell count, size, area, and position within a nucleus. Anisotropy is assessed with a vectorial analysis. Neuroscience Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons Lara Eid1,2, Mathieu Lachance1, Gilles Hickson1,3, Elsa Rossignol1,2,4 1Centre de recherche du CHU Sainte-Justine, 2Department of Neuroscience, Université de Montréal, 3Department of pathology and cellular biology, Université de Montréal, 4Department of Pediatrics, Université de Montréal Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques. Biochemistry Mouse Adipose Tissue Collection and Processing for RNA Analysis Paul Tan1,2, Émilie Pepin1,4, Julie L. Lavoie1,3,4 1Centre de Recherche du Centre Hospitalier, Université de Montréal, 2Department of Biochemistry and Molecular Medicine, Université de Montréal, 3Department of Kinesiology, Université de Montréal, 4Montreal Diabetes Research Center The purpose of this paper is to present a step-by-step procedure to collect different white adipose tissues from mice, process the fat samples and extract RNA. Biochemistry Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids Tanja Kalstrup1, Rikard Blunck1,2 1Department of Pharmacology and Physiology, University of Montréal, 2Department of Physics, University of Montréal This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements. Immunology and Infection A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules Ahmed Fouda1, Mahasti Tahsini2, Fatemeh Khodayarian2, Fatimah Al-nafisah2, Moutih Rafei1,2,3 1Department of Microbiology, Infectiology and Immunology, Université de Montréal, 2Department of Pharmacology and Physiology, Université de Montréal, 3Molecular Biology Program, Université de Montréal We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation. 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. Medicine Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps Ahmadali Sajadi1,3, Chloé Provost3, Brendon Pham2, Jonathan Brouillette1,3 1Department of Pharmacology, Université de Montréal, 2Department of Neuroscience, Université de Montréal, 3Hôpital du Sacré-coeur de Montréal Research Center, Université de Montréal To study the effects of Aβo in vivo, we developed a model based on repeated hippocampal infusions of soluble Aβo coupled with continuous infusion of Aβo antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of Aβo. Immunology and Infection Preparation and Applications of Organotypic Thymic Slice Cultures Aditi Sood*1,2, Mengqi Dong*1,2, Heather J. Melichar1,3 1Centre de Recherche, Hôpital Maisonneuve-Rosemont, 2Department of Microbiology, Infectiology and Immunology, Université de Montréal, 3Department of Medicine, Université de Montréal We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy. Developmental Biology Instrumentation of Near-term Fetal Sheep for Multivariate Chronic Non-anesthetized Recordings Patrick Burns1, Hai Lun Liu2, Shikha Kuthiala2, Gilles Fecteau1, André Desrochers1, Lucien Daniel Durosier2, Mingju Cao2, Martin G. Frasch2,3,4 1Département de sciences cliniques, CHUV, Université de Montréal, St-Hyacinthe, QC, 2Département d'obstetriques et de gynécologie, CHU Ste-Justine Research Centre, Université de Montréal, 3Département de neurosciences, CHU Ste-Justine Centre de recherche, Université de Montréal, 4Centre de recherche en reproduction animale (CRRA), Université de Montréal, St-Hyacinthe, QC The chronically instrumented non-anesthetized fetal sheep model is used to study human fetal development in health and disease, because it permits surgical placement and maintenance of catheters and electrodes, repetitive blood sampling, substance injection, recording of bioelectrical activity, and in vivo imaging. We describe the procedures required to establish this model. Neuroscience Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain Jimmy Lai1, Marc-André Legault1, Sébastien Thomas1, Christian Casanova1 1École d'optométrie, Université de Montréal Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings. Immunology and Infection Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation Antonio Layoun1, Macha Samba1, Manuela M. Santos1,2 1Centre de recherche du Centre hospitalier de l'Université de Montréal, Institut du cancer de Montréal, 2Département de Médecine, Université de Montréal We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation. Immunology and Infection Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells Chun Yang*1, Wei Xiong*2, Qian Qiu2, Houda Tahiri1, Carmen Gagnon1, Guoxiang Liu2, Pierre Hardy1 1Departments of Pediatrics and Pharmacology, University of Montréal, 2Department of Pulmonology, Chongqing Southwest Hospital, Third Military Medical University Cell membrane–shed microparticles (MPs) are active biological vesicles that can be isolated and their pathophysiological effects investigated in various models. Here we describe a method for generating MPs derived from T lymphocytes (LMPs) and for demonstrating their proapoptotic effect on airway epithelial cells. 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. Immunology and Infection Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay Maxime Veillette1, Mathieu Coutu*1, Jonathan Richard*1, Laurie-Anne Batraville*1, Anik Désormeaux1, Michel Roger1, Andrés Finzi1 1Centre de recherche du CHUM, Department of Microbiology, Infectiology and Immunology, Université de Montréal Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies. Immunology and Infection Development of an IFN-γ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation Insaf Salem Fourati1, Anne-Julie Grenier1, Élyse Jolette1, Natacha Merindol1, Philippe Ovetchkine2,3, Hugo Soudeyns1,3 1Unité d'Immunopathologie Virale, Centre de Recherche du CHU Sainte-Justine, Department of Microbiology, Infectiology & Immunology, Faculty of Medicine, Université de Montréal, 2Infectious Diseases Service, CHU Sainte-Justine, Faculty of Medicine, Université de Montréal, 3Department of Paediatrics, Université de Montréal Novel generations of functional assays such as gamma interferon (IFN-γ) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV). 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. Medicine Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy Marie-Yvonne Akoume1, Anita Franco1, Alain Moreau1,2 1Viscogliosi Laboratory in Molecular Genetics of Musculoskeletal Diseases, Sainte-Justine University Hospital Research Center, 2Department of Stomatology, Faculty of Dentistry and Department of Biochemistry, Faculty of Medicine, Université de Montréal A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days. Medicine A Model of Chronic Nutrient Infusion in the Rat Grace Fergusson1, Mélanie Ethier1, Bader Zarrouki1,2, Ghislaine Fontés1, Vincent Poitout1,2 1Montreal Diabetes Research Center, CRCHUM, 2Department of Medicine, University of Montreal A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters. Biology Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos Félix Legendre*1,2, Neal Cody*1, Carole Iampietro1, Julie Bergalet1, Fabio Alexis Lefebvre1,2, Gaël Moquin-Beaudry1,2, Olivia Zhang1, Xiaofeng Wang1, Eric Lécuyer1,2 1Institut de Recherches Cliniques de Montréal (IRCM), 2Department of Biochemistry, Université de Montréal Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster. Immunology and Infection Murine Superficial Lymph Node Surgery Mélissa Mathieu1,2, Nathalie Labrecque1,2,3 1Maisonneuve-Rosemont Hospital Research Center, 2Department of Microbiology and Immunology, University of Montreal, 3Department of Medicine, University of Montreal To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed. Neuroscience Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice Sandeep Sharma1, Cecile Hryhorczuk1, Stephanie Fulton1 1CRCHUM and the Montreal Diabetes Research Center, University of Montreal The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task. Medicine A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia Gauthier Julie1, Fadi F. Hamdan2, Guy A. Rouleau3 1Centre of Excellence in Neuromics, CHUM Research Center and the Department of Medicine, Universite de Montreal, 2Center of Excellence in Neuromics, CHU Sainte Justine and CHUM Notre-Dame Research Centers, Universite de Montreal, 3Department of Medicine, Universite de Montreal Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia. Immunology and Infection In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells Jason Letourneau1, Cynthia Levesque1, Frederic Berthiaume1, Mario Jacques1, Michael Mourez1 1Universite de Montreal, Groupe de Recherche sur les Maladies Infectieuses du Porc GREMIP, Faculte de medecine veterinaire This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis. 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 Brain Banking: Making the Most of your Research Specimens Mark W. Burke1, Shahin Zangenehpour2, Maurice Ptito2 1Department of Physiology, University of Montreal, 2School of Optometry, University of Montreal Brain banking and systematic sampling of biological material provides the basis for unbiased stereology and maximizes the potential data obtained from each specimen. Biology Dissecting the Non-human Primate Brain in Stereotaxic Space Mark W. Burke1, Shahin Zangenehpour2, Denis Boire3, Maurice Ptito2 1Department of Physiology, University of Montreal, 2School of Optometry, University of Montreal, 3Département de chimie-biologie , Université du Québes à Trois-Rivières The non-human primate is an important translational species for our understanding of the normal processing of the brain. The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans. Biology The Gateway to the Brain: Dissecting the Primate Eye Mark Burke1, Shahin Zangenehpour2, Joseph Bouskila2, Denis Boire3, Maurice Ptito2 1Department of Physiology, University of Montreal, 2School of Optometry, University of Montreal, 3Departement de chimie-biologie, Universite du Quebec a Trois-Rivieres The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans. Biology Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain Mark Burke1, Shahin Zangenehpour2, Peter R. Mouton3, Maurice Ptito2 1Department of Physiology, University of Montreal, 2Ecole d’optometrie, University of Montreal, 3Stereology Resource Center The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans. Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given reference space1.