Tsukuba University View Institution's Website 21 articles published in JoVE Developmental Biology Electroporation-mediated RNA Interference Method in Odonata Genta Okude1,2, Takema Fukatsu1,2,3, Ryo Futahashi2 1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 3Graduate School of Life and Environmental Sciences, University of Tsukuba We provide a detailed protocol for electroporation-mediated RNA interference in insects of the order Odonata (dragonflies and damselflies) using the blue-tailed damselfly (Ischnura senegalensis: Coenagironidae: Zygoptera) and the pied skimmer dragonfly (Pseudothemis zonata: Libellulidae: Anisoptera). Biology Reconstruction of Single-Cell Innate Fluorescence Signatures by Confocal Microscopy Tomohiro Hirayama*1, Kyosuke Takabe*2, Tatsunori Kiyokawa1, Nobuhiko Nomura2,3, Yutaka Yawata*2,3 1Graduate School of Life and Environmental Sciences, University of Tsukuba, 2Faculty of Life and Environmental Sciences, University of Tsukuba, 3Microbiology Research Center for Sustainability, University of Tsukuba Here, a protocol is presented for optically extracting and cataloging innate cellular fluorescence signatures (i.e., cellular autofluorescence) from every individual live cell distributed in a three-dimensional space. This method is suitable for studying the innate fluorescence signature of diverse biological systems at a single-cell resolution, including cells from bacteria, fungi, yeasts, plants, and animals. Bioengineering Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior Francesco Carrara1, Douglas R. Brumley2, Andrew M. Hein3, Yutaka Yawata4,5, M. Mehdi Salek1, Kang Soo Lee1, Elzbieta Sliwerska1, Simon A. Levin6, Roman Stocker1 1Institute of Environmental Engineering, Department of Civil, Environmental and Geomatic Engineering, 2School of Mathematics and Statistics, University of Melbourne, 3Institute of Marine Sciences, University of California, Santa Cruz, 4Faculty of Life and Environmental Sciences, University of Tsukuba, 5Microbiology Research Center for Sustainability, University of Tsukuba, 6Department of Ecology and Evolutionary Biology, Princeton University A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level. Biology A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency Marianne Theile1, Linus Wiora1, Dominik Russ1, Jonas Reuter1, Hiroshi Ishikawa2, Christian Schwerk3, Horst Schroten3, Stefan Mogk1 1Interfaculty Institute of Biochemistry, University of Tübingen, 2Laboratory of Clinical Regenerative Medicine, Department of Neurosurgery, Faculty of Medicine, University of Tsukuba, 3Department of Pediatrics, Medical Faculty Mannheim, Heidelberg University Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements. Environment Microfocus X-ray CT (microCT) Imaging of Actinia equina (Cnidaria), Harmothoe sp. (Annelida), and Xenoturbella japonica (Xenacoelomorpha) Akiteru Maeno1, Hisanori Kohtsuka2, Kensuke Takatani3, Hiroaki Nakano3 1Mammalian Genetics Laboratory, National Institute of Genetics, 2Misaki Marine Biological Station, The University of Tokyo, 3Shimoda Marine Research Center, University of Tsukuba Here, protocols for performing microfocus X-ray computed tomography (microCT) imaging of three marine invertebrate animals are explained in detail. This study describes steps such as sample fixation, staining, mounting, scanning, image reconstruction, and data analyses. Suggestions on how the protocol can be adjusted for different samples are also provided. Neuroscience Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice Shota Kodani*1, Shingo Soya*2, Takeshi Sakurai2,3 1Department of Molecular Neuroscience and Integrative Physiology, Faculty of Medicine, Kanazawa University, 2International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, 3Faculty of Medicine, University of Tsukuba Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example. Immunology and Infection Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing Lin Sun1, Naoko Kono2, Hiroyuki Toh3, Hanbing Xue1, Kaori Sano4,5, Tadaki Suzuki4, Akira Ainai4, Yasuko Orba6, Junya Yamagishi7,8, Hideki Hasegawa4,5, Yoshimasa Takahashi9, Shigeyuki Itamura2, Kazuo Ohnishi9,10 1Graduate School of Life and Environmental Sciences, University of Tsukuba, 2Center for Influenza Virus Research, National Institute of Infectious Diseases, 3School of Science and Technology, Kwansei Gakuin University, 4Department of Pathology, National Institute of Infectious Diseases, 5Division of Infectious Diseases Pathology, Department of Global Infectious Diseases, Tohoku University Graduate School of Medicine, 6Division of Molecular Pathobiology, Research Center for Zoonosis Control, Hokkaido University, 7Division of Collaboration and Education, Research Center for Zoonosis Control, Hokkaido University, 8Global Station for Zoonosis Control, GI-CoRE, Hokkaido University, 9Department of Immunology, National Institute of Infectious Diseases, 10Faculty of Life and Environmental Sciences, University of Tsukuba Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing. Biology A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging Shigeyuki Betsuyaku1,2,3,4, Nobuhiko Nomura3,4, Hiroo Fukuda2 1Japan Science and Technology Agency (JST), PRESTO, 2Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 3Faculty of Life and Environmental Sciences, University of Tsukuba, 4Microbiology Research Center for Sustainability, University of Tsukuba We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses. Behavior An Automated T-maze Based Apparatus and Protocol for Analyzing Delay- and Effort-based Decision Making in Free Moving Rodents Qi Zhang1,2, Yuki Kobayashi1, Hiromichi Goto1, Shigeyoshi Itohara1 1Laboratory of Behavioral Genetics, Center for Brain Science, RIKEN, 2Faculty of Human Science, University of Tsukuba This article introduces an automated T-maze apparatus that we invented, and a protocol based on this apparatus for analyzing delay-based decision making and effort-based decision making in free moving rodents. Chemistry Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals Masaki Hada1, Shohei Saito2, Ryuma Sato3, Kiyoshi Miyata4, Yasuhiko Hayashi1, Yasuteru Shigeta3, Ken Onda4 1Graduate School of Natural Science and Technology, Okayama University, 2Graduate School of Science, Kyoto University, 3Center for Computational Sciences, University of Tsukuba, 4Graduate School of Science, Kyushu University Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material. Biology Precise, High-throughput Analysis of Bacterial Growth Masaomi Kurokawa1, Bei-Wen Ying1,2 1Graduate School of Life and Environmental Sciences, University of Tsukuba, 2Institute of Biology and Information Science, East China Normal University Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology. Engineering Fabrication of Polymer Microspheres for Optical Resonator and Laser Applications Yohei Yamamoto1,2,3, Daichi Okada1, Soh Kushida1, Zakarias Seba Ngara1, Osamu Oki1 1Faculty of Pure and Applied Sciences, University of Tsukuba, 2Tsukuba Research Center for Interdisciplinary Materials Science (TIMS), University of Tsukuba, 3Center for Integrated Research in Fundamental Science and Technology (CiRfSE), University of Tsukuba Protocols for the synthesis of microspheres from polymers, the manipulation of microspheres, and micro-photoluminescence measurements are presented. Neuroscience Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes Fuminori Tsuruta1,2,3, Tomomi Okajima1, Sarasa Yano1, Tomoki Chiba1,2,3 1Graduate School of Life and Environmental Sciences, University of Tsukuba, 2PhD Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, 3Life Science Center of Tsukuba Advanced Research Alliance (TARA), University of Tsukuba The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system. Engineering High-resolution Thermal Micro-imaging Using Europium Chelate Luminescent Coatings Timothy M. Benseman1,2,3, Yang Hao1,2, Vitalii K. Vlasko-Vlasov1, Ulrich Welp1, Alexei E. Koshelev1, Wai-Kwong Kwok1, Ralu Divan4, Courtney Keiser5, Chiharu Watanabe6, Kazuo Kadowaki6 1Materials Science Division, Argonne National Laboratory, 2Department of Physics, University of Illinois at Chicago, 3Department of Physics, CUNY Queens College, 4Center for Nanoscale Materials, Argonne National Laboratory, 5Department of Physics, University of Northern Iowa, 6Institute for Materials Science, University of Tsukuba Europium thenoyltrifluoroacetonate (EuTFC) has an optical luminescence line at 612 nm, whose activation efficiency decreases strongly with temperature. If a sample coated with a thin film of this material is micro-imaged, the 612 nm luminescent response intensity may be converted into a direct map of sample surface temperature. Developmental Biology Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster Eisuke Imura*1, Yuto Yoshinari*1, Yuko Shimada-Niwa*2, Ryusuke Niwa3 1Graduate School of Life and Environmental Sciences, University of Tsukuba, 2Life Science Center of Tsukuba Advanced Research Alliance, University of Tsukuba, 3Faculty of Life and Environmental Sciences, University of Tsukuba We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells. 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. Chemistry Low-energy Cathodoluminescence for (Oxy)Nitride Phosphors Yujin Cho1,3, Benjamin Dierre2, Takashi Sekiguchi3, Takayuki Suehiro4, Kohsei Takahashi4, Takashi Takeda4, Rong-Jun Xie4, Yoshinobu Yamamoto4, Naoto Hirosaki4 1Graduate School of Pure and Applied Science, University of Tsukuba, 2CNRS — Saint-Gobain, UMI 3629, Laboratory for Innovative Key Materials and Structures (LINK), 3Nano Device Characterization Group, National Institute for Materials Science (NIMS), 4Sialon Unit, National Institute for Materials Science (NIMS) An excellent chemical and luminescence stabilities of (oxy)nitride phosphors present it as an promising alternative to currently used sulfide and oxide phosphors. In this paper, we present the way to investigate its local luminescence properties using low-energy cathodoluminescence (CL). Medicine Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells Ran-Ran Zhang*1, Yun-Wen Zheng*1,2,3, Hideki Taniguchi1 1Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, 2Department of Advanced Gastroenterological Surgical Science and Technology, Faculty of Medicine, University of Tsukuba, 3Regenerative Medicine Research Center, Jiangsu University Hospital Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells. Neuroscience Polygraphic Recording Procedure for Measuring Sleep in Mice Yo Oishi1, Yohko Takata1, Yujiro Taguchi2, Sayaka Kohtoh2, Yoshihiro Urade1, Michael Lazarus1 1International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, 2Public Sector/Medical Solutions, Kissei Comtech Co., Ltd The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice. Engineering Detection and Recovery of Palladium, Gold and Cobalt Metals from the Urban Mine Using Novel Sensors/Adsorbents Designated with Nanoscale Wagon-wheel-shaped Pores Sherif A. El-Safty1,2, Mohamed A. Shenashen1, Masaru Sakai3, Emad Elshehy1, Kohmei Halada1 1National Institute for Materials Science, Japan, 2Graduate School for Advanced Science and Engineering, Waseda University, 3Center for Research in Isotopes and Environmental Dynamics, Tsukuba University Because of the importance and extensive use of palladium, gold and cobalt metals in high-technology equipment, their recovery and recycling constitute an important industrial challenge. The metal recovery system described herein is a simple, low-cost means for the effective detection, removal, and recovery of these metals from the urban mine. Neuroscience SDS-PAGE/Immunoblot Detection of Aβ Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval Rebecca F. Rosen1, Yasushi Tomidokoro2, Jorge A. Ghiso3, Lary C. Walker1,4 1Yerkes National Primate Research Center, Emory University, 2Department of Neurology, Institute of Clinical Medicine, Tsukuba University, 3Department of Pathology, New York University School of Medicine, 4Department of Neurology, Emory University We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the Aβ peptide. Using this protocol, we consistently detect monomeric and multimeric Aβ in cortical tissue from humans with Alzheimer's pathology.