Paris Diderot University 7 articles published in JoVE Biology Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes Natacha Raich1, Vincent Contremoulins1, Roger E. Karess1 1CNRS, Institut Jacques Monod, Université de Paris, F 75006, Paris, France We present here an improved protocol for the whole-mount preparation and immunostaining of Drosophila testes, suitable for confocal microscopy and also allowing reproducible and reliable labeling. We illustrate this protocol by 3D representation and quantification of colocalization experiments on the large S4-S5 spermatocytes. Medicine One-anastomosis Gastric Bypass (OAGB) in Rats Matthieu A. Siebert1, Jean-Marc Chevallier2, Diego D'Aurelle de Paladines1, Yohann Pottier1, Simon Msika1,3, Maude Le Gall1, André Bado1, Lara Ribeiro-Parenti1,3 1Institut national de la santé et de la recherche médicale (Inserm) unité mixte de recherche (UMR) 1149, Unité de formation et de recherche (UFR) de Médecine Paris Diderot, Universit é Paris Diderot, Assistance Publique-Hôpitaux de Paris (AP-HP), 2Department of digestive Surgery, AP-HP Hôpital Européen Georges Pompidou, 3Department of General and Visceral Surgery, Bichat-Claude Bernard University Hospital This protocol is for the performance of one-anastomosis gastric bypass (OAGB) on rat. The operator performs a long and tubular-stapled gastric pouch followed by hand-sewn anastomosis. For this model, the operator reproduces the same ratio between biliopancreatic and common limb in humans; therefore, the biliopancreatic limb measures 35 cm. Medicine Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants Sophia Houari1,2, Sylvie Babajko1,2, Sophia Loiodice1,2, Ariane Berdal1,2, Katia Jedeon1,2 1Institut National de la Santé et Recherche Médicale (INSERM) UMRS 1138, Paris-Diderot University, Pierre & Marie Curie University, Paris-Descartes University, Laboratory of Molecular Oral Pathophysiology, Cordeliers Research Centre, 2Unit of Formation and Research (UFR) of Odontology, Paris-Diderot University Understanding enamel formation and possible alterations requires the study of ameloblast activity. Here, we describe a reliable and consistent method to micro-dissect enamel organs containing secretion- and maturation-stage ameloblasts that may be used for further quantitative and qualitative experimental procedures. Cancer Research Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells Clémence Granier1, Emeline Vinatier2,3,4, Elia Colin2,3, Marion Mandavit2,3, Charles Dariane2,3,5, Virginie Verkarre6, Lucie Biard7, Rami El Zein2, Corinne Lesaffre2, Isabelle Galy-Fauroux2, Hélène Roussel2,3,6, Eléonore De Guillebon8, Charlotte Blanc2,3, Antonin Saldmann4, Cécile Badoual2,6, Alain Gey*4, Éric Tartour*2,4 1PARCC-INSERM U970, Université Paris Descartes, 2INSERM U970, Université Paris Descartes, 3Equipe Labellisée Ligue Contre le Cancer, 4Department of Immunology, Hopital Européen Georges Pompidou, 5Department of Medical Urology, Hopital Européen Georges Pompidou, 6Department of Pathology, Hopital Européen Georges Pompidou, 7Université Paris Diderot Paris 7, 8Department of medical oncology, Hopital Européen Georges Pompidou Studying tumor microenvironment may identify prognostic or predictive biomarkers of clinical response to immunotherapy. Presented here, is an innovative method based on in situ fluorescence multispectral imaging to analyze and count automatically various subpopulations of CD8+ T cells. This reproducible and reliable technique is suitable for large cohort analyses. Bioengineering 3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration Aurore Van de Walle1, Claire Wilhelm1, Nathalie Luciani1 1Laboratoire Matière et Systèmes Complexes (MSC), UMR 7057 CNRS and University Paris Diderot Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production. Medicine Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload Bérengère Koehl1, Florence Missud1, Laurent Holvoet1, Ghislaine Ithier1, Oliver Sakalian-Black2, Zinedine Haouari1, Emmanuelle Lesprit3, André Baruchel4, Malika Benkerrou1 1Reference Centre of Sickle Cell Disease, Hematology Unit, Robert Debré Hospital, AP-HP, 2School of Pharmacy, Université Paris Descartes, 3Etablissement Français du Sang, Robert Debré Hospital, AP-HP, 4Hematology Unit,, Robert Debré Hospital, AP-HP, Univsité Paris Diderot We have outlined a method of continuous manual exchange transfusion for the treatment of sickle cell disease in patients. This safe protocol was designed to effectively limit iron overload in patients in need of chronic transfusions and can be used extensively without any special equipment. Medicine Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury Arnaud Dubory1,2, Elisabeth Laemmel1, Anna Badner3, Jacques Duranteau1,4, Eric Vicaut1, Charles Court2, Marc Soubeyrand1,2 1Laboratoire d'étude de la microcirculation, Faculté de Médecine Paris Diderot Paris VII, U942, 2Department of orthopaedic surgery, Bicetre Universitary Hospital, Public Assistance of Paris Hospital, 3Institute of Medical Science, Faculty of Medicine, University of Toronto, 4Department of Intensive care and Anesthesiology, Bicetre Universitary Hospital, Public Assistance of Paris Hospital Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.