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27 ARTICLES PUBLISHED IN JoVE

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Neuroscience

In vivo Neuronal Calcium Imaging in C. elegans
Samuel H. Chung *1,2, Lin Sun *1,2, Christopher V. Gabel 1,2
1Department of Physiology and Biophysics, Boston University School of Medicine, 2Boston University Photonics Center

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

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Medicine

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye
Larry Kagemann 1,2, Bo Wang 2, Gadi Wollstein 1, Hiroshi Ishikawa 1,2, Brandon Mentley 1, Ian Sigal 1,2,3, Richard A Bilonick 1,4, Joel S Schuman 1,2,3
1Department of Ophthalmology, UPMC Eye Center, Eye and Ear Institute, Ophthalmology and Visual Science Research Center, University of Pittsburgh School of Medicine, 2Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, 3The McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, 4Deptartment of Biostatistics, Graduate School of Public Health, University of Pittsburgh

Trabecular meshwork (TM) migration into Schlemm’s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

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JoVE Core

Polygraphic Recording Procedure for Measuring Sleep in Mice
Yo Oishi 1, Yohko Takata 1, Yujiro Taguchi 2, Sayaka Kohtoh 2, Yoshihiro Urade 1, Michael Lazarus 1
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.

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Engineering

High-resolution Thermal Micro-imaging Using Europium Chelate Luminescent Coatings
Timothy M. Benseman 1,2,3, Yang Hao 1,2, Vitalii K. Vlasko-Vlasov 1, Ulrich Welp 1, Alexei E. Koshelev 1, Wai-Kwong Kwok 1, Ralu Divan 4, Courtney Keiser 5, Chiharu Watanabe 6, Kazuo Kadowaki 6
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.

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Medicine

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side
Stefanie Dinner 1, Julia Borkowski 1, Carolin Stump-Guthier 1, Hiroshi Ishikawa 2, Tobias Tenenbaum 1, Horst Schroten 1, Christian Schwerk 1
1Department of Pediatrics, Medical Faculty Mannheim, Heidelberg University, 2Department of NDU Life Sciences, Nippon Dental University

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

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Medicine

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells
Ran-Ran Zhang *1, Yun-Wen Zheng *1,2,3, Hideki Taniguchi 1
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.

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Chemistry

Low-energy Cathodoluminescence for (Oxy)Nitride Phosphors
Yujin Cho 1,3, Benjamin Dierre 2, Takashi Sekiguchi 3, Takayuki Suehiro 4, Kohsei Takahashi 4, Takashi Takeda 4, Rong-Jun Xie 4, Yoshinobu Yamamoto 4, Naoto Hirosaki 4
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).

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Immunology and Infection

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus
Fabio Cafini *1,2, Nguyen Thi Le Thuy *3, Federico Román 4, José Prieto 5, Sarah Dubrac 6,7, Tarek Msadek 6,7, Kazuya Morikawa 1
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.

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Neuroscience

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes
Fuminori Tsuruta 1,2,3, Tomomi Okajima 1, Sarasa Yano 1, Tomoki Chiba 1,2,3
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.

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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 Niwa 3
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.

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Engineering

Fabrication of Polymer Microspheres for Optical Resonator and Laser Applications
Yohei Yamamoto 1,2,3, Daichi Okada 1, Soh Kushida 1, Zakarias Seba Ngara 1, Osamu Oki 1
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.

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Medicine

Application of Granger Causality Analysis of the Directed Functional Connection in Alzheimer's Disease and Mild Cognitive Impairment
Mei Wang 1, Zhengluan Liao 2, Dewang Mao 3, Qi Zhang 1, Yumei Li 3, Enyan Yu 2, Zhongxiang Ding 3
1Zhejiang Chinese Medical University, 2Department of Psychiatry, Zhejiang Provincial People's Hospital, 3Department of Radiology, Zhejiang Provincial People's Hospital

Based on resting-state functional magnetic resonance imaging with Granger causality analysis, we investigated the alterations in the directed functional connectivity between the posterior cingulate cortex and whole brain in patients with Alzheimer's Disease (AD), patients with Mild Cognitive Impairment (MCI), and healthy controls.

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Biology

Precise, High-throughput Analysis of Bacterial Growth
Masaomi Kurokawa 1, Bei-Wen Ying 1,2
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.

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Chemistry

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals
Masaki Hada 1, Shohei Saito 2, Ryuma Sato 3, Kiyoshi Miyata 4, Yasuhiko Hayashi 1, Yasuteru Shigeta 3, Ken Onda 4
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.

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Behavior

An Automated T-maze Based Apparatus and Protocol for Analyzing Delay- and Effort-based Decision Making in Free Moving Rodents
Qi Zhang 1,2, Yuki Kobayashi 1, Hiromichi Goto 1, Shigeyoshi Itohara 1
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.

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Neuroscience

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice
Shota Kodani *1, Shingo Soya *2, Takeshi Sakurai 2,3
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.

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Immunology and Infection

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing
Lin Sun 1, Naoko Kono 2, Hiroyuki Toh 3, Hanbing Xue 1, Kaori Sano 4,5, Tadaki Suzuki 4, Akira Ainai 4, Yasuko Orba 6, Junya Yamagishi 7,8, Hideki Hasegawa 4,5, Yoshimasa Takahashi 9, Shigeyuki Itamura 2, Kazuo Ohnishi 9,10
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.

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Biology

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging
Shigeyuki Betsuyaku 1,2,3,4, Nobuhiko Nomura 3,4, Hiroo Fukuda 2
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.

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Environment

Microfocus X-ray CT (microCT) Imaging of Actinia equina (Cnidaria), Harmothoe sp. (Annelida), and Xenoturbella japonica (Xenacoelomorpha)
Akiteru Maeno 1, Hisanori Kohtsuka 2, Kensuke Takatani 3, Hiroaki Nakano 3
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.

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Education

A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency
Marianne Theile 1, Linus Wiora 1, Dominik Russ 1, Jonas Reuter 1, Hiroshi Ishikawa 2, Christian Schwerk 3, Horst Schroten 3, Stefan Mogk 1
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.

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Bioengineering

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior
Francesco Carrara 1, Douglas R. Brumley 2, Andrew M. Hein 3, Yutaka Yawata 4,5, M. Mehdi Salek 1, Kang Soo Lee 1, Elzbieta Sliwerska 1, Simon A. Levin 6, Roman Stocker 1
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.

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Biology

Reconstruction of Single-Cell Innate Fluorescence Signatures by Confocal Microscopy
Tomohiro Hirayama *1, Kyosuke Takabe *2, Tatsunori Kiyokawa 1, Nobuhiko Nomura 2,3, 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.

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Developmental Biology

Electroporation-mediated RNA Interference Method in Odonata
Genta Okude 1,2, Takema Fukatsu 1,2,3, Ryo Futahashi 2
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).

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Biology

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate
Yen Xuan Ngo 1,2,3, Kenta Haga 4, Ayako Suzuki 4, Hiroko Kato 4, Hiromi Yanagisawa 1,5, Kenji Izumi 4, Aiko Sada 1,3
1Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, 2Ph.D. Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, 3International Research Center for Medical Sciences (IRCMS), Kumamoto University, 4Division of Biomimetics, Faculty of Dentistry and Graduate School of Medical and Dental Sciences, Niigata University, 5Faculty of Medicine, University of Tsukuba

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

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Medicine

Clinical Application of 24 G Cannula Needle and 3-0 Polypropylene Suture in Vas Deferens Exploration
Jin Wang *1, Wenjia Li *2, Yao He 1, Yuanbin Xia 1, Lin Sun 1, Youpeng Zhang 1, Zhaohui Zhu 1
1Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 2Department of Stomatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology

This protocol presents the clinical application of a 24 G cannula and 3-0 polypropylene suture as a simple and effective method for the exploration of the vas deferens.

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Developmental Biology

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice
Yoko Tanimoto *1, Natsuki Mikami *1,2, Miyuki Ishida 1, Natsumi Iki 1, Kanako Kato 1, Fumihiro Sugiyama 1, Satoru Takahashi 1, Seiya Mizuno 1
1Laboratory Animal Resource Center and Trans-Border Medical Research Center, University of Tsukuba, 2Ph.D. Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba

The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.

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Genetics

Primordial Germ Cell Cryopreservation and Revival of Drosophila Strains
Kaori Nishimura *1, Miho Asaoka *2, Yurina Sakamaki *2, Tatsuya Fukumoto *3,4, Daisuke Tanaka 3, Satoru Kobayashi 2, Toshiyuki Takano-Shimizu-Kouno 1
1KYOTO Drosophila Stock Center, Kyoto Institute of Technology, 2Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, 3Research Center of Genetic Resources, National Agriculture and Food Research Organization, 4Shizuoka Prefectural Ogasa High School

A long-term preservation method for Drosophila strains as an alternative to the frequent transfer of adult flies to fresh food vials is highly desirable. This protocol describes the cryopreservation of Drosophila primordial germ cells and strain revival via their transplantation to agametic host embryos.

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