CNRS 22 articles published in JoVE Biochemistry Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence Christine Terryn1, Anouck Habrant2, Gabriel Paës2, Corentin Spriet3 1PICT Platform, Université de Reims Champagne-Ardenne, 2Fractionation of AgroResources and Environment (FARE) Laboratory, INRA, Université de Reims Champagne-Ardenne, 3Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate Förster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections. Neuroscience In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices Louis Richevaux1,2, Louise Schenberg1,2, Mathieu Beraneck1,2, Desdemona Fricker1,2 1CNRS (Integrative Neuroscience and Cognition Center, UMR 8002), 2Université Paris Descartes, Sorbonne Paris Cité This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices. Developmental Biology Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle Wenyue Guan1, Lalanti Venkatasubramanian2, Myungin Baek3, Richard S. Mann2, Jonathan Enriquez1 1Institut de Génomique Fonctionnelle de Lyon, ENS de Lyon, CNRS, 2Departments of Biochemistry and Molecular Biophysics, and Neuroscience, Mortimer B. Zuckerman Mind Brain Behavior Institute, Columbia University, 3Neuroscience Program, NYU School of Medicine Here we describe a protocol to visualize the axonal targeting with a florescent protein in adult legs of Drosophila by fixation, mounting, imaging, and post-imaging steps. Chemistry In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis Giovanna Muggiolu*1,2, Marina Simon*1,2, Nathanael Lampe1,2, Guillaume Devès1,2, Philippe Barberet1,2, Claire Michelet1,2, Marie-Hélène Delville3,4, Hervé Seznec1,2 1Centre d'Etudes Nucléaires Bordeaux Gradignan (CENBG), Université de Bordeaux, 2Centre d'Etudes Nucléaires Bordeaux Gradignan (CENBG), CNRS, 3Institut de Chimie de la Matière Condens é e de Bordeaux (ICMCB), CNRS, 4Institut de Chimie de la Matière Condens é e de Bordeaux (ICMCB), Université de Bordeaux We describe a procedure for the detection of chemical elements present in situ in human cells as well as their in vitro quantification. The method is well-suited to any cell type and is particularly useful for quantitative chemical analyses in single cells following in vitro metal oxide nanoparticles exposure. Bioengineering Ligand Nano-cluster Arrays in a Supported Lipid Bilayer Emmanuelle Benard1, Fuwei Pi1,2, Igor Ozerov1, Anne Charrier1, Kheya Sengupta1 1Aix-Marseille Université, CNRS, UMR 7325, CINaM, 2State Key Laboratory of Food Science and Technology, School of Food Science of Jiangnan University We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies. Medicine Techniques of Sleeve Gastrectomy and Modified Roux-en-Y Gastric Bypass in Mice Audrey Ayer1, Frederic Borel1, Francois Moreau1, Xavier Prieur1, Michel Neunlist2, Bertrand Cariou3, Claire Blanchard*1,4, Cedric Le May*1 1l'Institut du Thorax, INSERM, CNRS, UNIV Nantes, 2CHU Nantes, Institut des Maladies de l'Appareil Digestif, INSERM U913, 3l'Institut du Thorax, INSERM, CNRS, UNIV Nantes, CHU Nantes, 4Service de Clinique Chirurgicale Digestive et Endocrinienne, CHU de Nantes Bariatric surgery is the most efficient way to reduce body weight and the deadly metabolic complications (diabetes, obesity, and dyslipidemia) frequently associated with morbid obesity. Mouse models of bariatric surgery represent a unique asset for deciphering molecular mechanisms behind the beneficial effects of these surgeries on diabetes, hypertension, and dyslipidemia. Immunology and Infection Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus Fabio Cafini*1,2, Nguyen Thi Le Thuy*3, Federico Román4, José Prieto5, Sarah Dubrac6,7, Tarek Msadek6,7, Kazuya Morikawa1 1Division of Biomedical Science, Faculty of Medicine, University of Tsukuba, 2Department of Basic Biomedical Science, Universidad Europea de Madrid, 3Human Biology Program, School of Integrative and Global Majors, University of Tsukuba, 4Laboratory of Nosocomial Infections, Department of Bacteriology, Centro Nacional de MicrobiologÍa, Instituto de Salud Carlos III, 5Division of Microbiology, Department of Medicine, School of Medicine, Universidad Complutense, 6Biology of Gram-Positive Pathogens, Department of Microbiology, Institut Pasteur, Paris, France, 7ERL3526, CNRS, Paris, France We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus. Medicine Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases Margarita Arango-Lievano1,2,3, Patrizia Giannoni1,2,3, Sylvie Claeysen1,2,3, Nicola Marchi1,2,3, Freddy Jeanneteau1,2,3 1Inserm, U1191, Institute of Functional Genomics, 2CNRS, UMR-5203, 3Université de Montpellier This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer's disease. Cancer Research Operating Procedures of the Electrochemotherapy for Treatment of Tumor in Dogs and Cats Natasa Tozon1, Ursa Lampreht Tratar2, Katarina Znidar3, Gregor Sersa2, Justin Teissie4,5, Maja Cemazar2,3 1Clinic for Surgery and Small Animals, Veterinary Faculty, University of Ljubljana, 2Department of Experimental Oncology, Institute of Oncology Ljubljana, 3Faculty of Health Sciences, University of Primorska, 4IPBS (Institut de Pharmacologie et de Biologie Structurale), CNRS, 5IPBS (Institut de Pharmacologie et de Biologie Structurale), Université de Toulouse Electrochemotherapy is the local treatment of solid tumors. The main mechanism is electroporation-mediated permeabilization of the tumor cells' membrane which enables increased internalization of cytostatics like bleomycin and cisplatin. Thus, antitumor effectiveness is potentiated at the site of the application of electric pulses to the tumors. Immunology and Infection Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1 Gabrielle Lê-Bury1, Chantal Deschamps1, Audrey Dumas1, Florence Niedergang1 1Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes. Biology An Easy Method for Plant Polysome Profiling Cécile Lecampion1,2,3, Maïna Floris1,2,3,4, Jean Raphaël Fantino5,6, Christophe Robaglia1,2,3, Christophe Laloi1,2,3 1Laboratoire de Génétique et Biophysique des Plantes, Aix-Marseille Université, 2UMR 7265 Biologie Végétale & Microbiologie Environnementales, CNRS, 3BIAM, CEA, 4Department of Biology, Biocenter, University of Copenhagen, 5Laboratoire de Chimie Bactérienne, 6CNRS, LCB UMR 7283, Aix Marseille Université This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs. Immunology and Infection Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells Alain Casanova*1, Shyan H. Low*1, Mario Emmenlauer*1,4, Raquel Conde-Alvarez*1,3, Suzana P. Salcedo2, Jean-Pierre Gorvel2, Christoph Dehio1 1Focal Area Infection Biology, Biozentrum, University of Basel, 2Centre d’Immunologie de Marseille-Luminy, Université de la Méditérannée UM2, INSERM U1104 CNRS UM7280, 3Departmento de Microbiologìa and Instituto de Salud Tropical, Universidad de Navarra, 4BioDataAnalysis GmbH Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols. Biology Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope Vasyl Mytskaniuk*1, Fabrice Bardin*2,3, Hassan Boukhaddaoui1,5, Herve Rigneault4, Nicolas Tricaud1 1INSERM U1051, Institut des Neurosciences de Montpellier (INM), Université de Montpellier, 2Université de Nîmes, 3CNRS, IES, UMR 5214, 4Aix-Marseille Université, CNRS, École Centrale Marseille, Institut Fresnel, UMR 7249, 5Montpellier RIO Imaging (MRI) Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser. Developmental Biology Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo Beatrice C. Durand1,2,3,4 1Institut Curie, 2UMR 3387, CNRS, 3PSL Research University, 4Université Paris-Sud In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. Biology Xenopus laevis as a Model to Identify Translation Impairment Amélie de Broucker1, Pierre Semaille1, Katia Cailliau2, Alain Martoriati2, Thomas Comptdaer1, Jean-François Bodart2, Alain Destée1, Marie-Christine Chartier-Harlin1 1Team "Early Stages of Parkinson's Disease" of the Jean-Pierre Aubert Research Center, INSERM UMR-S1172, CHRU Lille, University of Lille 1 and Lille 2, 2Team "Signal Division Regulation", CNRS UMR 8576, University of Lille 1 Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1. Neuroscience Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics Mathieu Daynac*1,2,3,4,5, Lise Morizur*1,2,3,4, Thierry Kortulewski1,2,3,4, Laurent R. Gauthier1,2,3,4, Martial Ruat5, Marc-André Mouthon1,2,3,4, François D. Boussin1,2,3,4 1CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, UMR 967, 2INSERM, UMR 967, 3Université Paris Diderot, Sorbonne Paris Cité, UMR 967, 4Université Paris Sud, UMR 967, 5CNRS, Université Paris Sud, UMR 9197, Neuroscience Paris-Saclay Institute, Molecules Circuits Department We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging. Behavior The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents Robert D. Kirch1, Richard C. Pinnell1, Ulrich G. Hofmann1, Jean-Christophe Cassel2,3 1Section of Neuroelectronic Systems, Dept. of Neurosurgery, University Hospital Freiburg, 2Laboratoire d'Imagerie et de Neurosciences Cognitives, UMR 7364 Université de Strasbourg, CNRS, 3Faculté de Psychologie, Neuropôle de Strasbourg The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems. Medicine Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease Ophélie Vacca1,2,3, Brahim El Mathari1,2,3, Marie Darche1,2,3, José-Alain Sahel1,2,3, Alvaro Rendon1,2,3, Deniz Dalkara1,2,3 1Department of Therapeutics, Institut de la Vision, Sorbonne Universtés, UPMC Univ Paris 06, UMR_S 968, 2INSERM, U968, 3CNRS, UMR_7210 To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability. Developmental Biology Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes Estelle Hirsinger1, João Emanuel Carvalho2, Christine Chevalier1,3, Georges Lutfalla4, Jean-François Nicolas1, Nadine Peyriéras5, Michael Schubert2 1Département de Biologie du Développement et Cellules Souches, Institut Pasteur, 2Laboratoire de Biologie du Développement de Villefranche-sur-Mer (UMR7009 CNRS/UPMC Univ Paris 06), Sorbonne Universités, 3Equipe Epigenetic Control of Normal and Pathological Hematopoiesis, Centre de Recherche en Cancérologie de Marseille, 4Unité de Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS UMR5235/DAA/cc107/Université Montpellier II, 5Plateforme BioEmergences IBiSA FBI, CNRS-NED, Institut de Neurobiologie Alfred Fessard We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations. Immunology and Infection Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling Laura Megrelis1,2,3, Jérôme Delon1,2,3 1Inserm U1016, Institut Cochin, 2Cnrs, UMR8104, 3Université Paris Descartes, Sorbonne Paris Cité Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events. Immunology and Infection Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells Célia Parinot1,2,3, Quentin Rieu1,2,3, Jonathan Chatagnon1,2,3, Silvia C. Finnemann4, Emeline F. Nandrot1,2,3 1INSERM, U968, 2Sorbonne Universités, UPMC Paris 06, UMR_S 968, Institut de la Vision, 3CNRS, UMR_7210, 4Department of Biological Sciences, Center for Cancer, Genetic Diseases and Gene Regulation, Fordham University This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 °C. Engineering Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue Lucie Sancey1, Vincent Motto-Ros2, Shady Kotb1, Xiaochun Wang3, François Lux1, Gérard Panczer3, Jin Yu2, Olivier Tillement1 1ILM-FENNEC UMR 5306, CNRS - Université Lyon 1, 2ILM-PUBLI UMR 5306, CNRS - Université Lyon 1, 3ILM-SOPRANO UMR 5306, CNRS - Université Lyon 1 Laser-induced breakdown spectroscopy performed on thin organ and tumor tissue successfully detected natural elements and artificially injected gadolinium (Gd), issued from Gd-based nanoparticles. Images of chemical elements reached a resolution of 100 μm and quantitative sub-mM sensitivity. The compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of a same biological tissue.