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50 ARTICLES PUBLISHED IN JoVE

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Biology

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy
Akihiro Asai 1, Nita Sahani 1, Yasuyoshi Ouchi 2, Jeevendra Martyn 1, Shingo Yasuhara 1
1Department of Anesthesiology and Critical Care, Shriners Hospital for Children, Massachusetts General Hospital, and Harvard Medical School, 2Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

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Medicine

Technical Aspects of the Mouse Aortocaval Fistula
Kota Yamamoto 1,2, Xin Li 1,3, Chang Shu 3, Tetsuro Miyata 2, Alan Dardik 1,4
1The Department of Surgery and the Interdepartmental Program in Vascular Biology and Therapeutics, Yale University, 2Department of Vascular Surgery, The University of Tokyo, 3Department of Vascular Surgery, Central South University, 4VA Connecticut Healthcare Systems

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

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Neuroscience

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion
Teruyuki Matsunaga 1, Akira Fushiki 1, Akinao Nose 1,2, Hiroshi Kohsaka 1
1Department of Complexity Science and Engineering, Graduate School of Frontier Sciences, The University of Tokyo, 2Department of Physics, Graduate School of Science, The University of Tokyo

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

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Bioengineering

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation
Keiji Itaka 1, Satoshi Uchida 1, Akitsugu Matsui 1, Kayoko Yanagihara 1, Masaru Ikegami 1, Taisuke Endo 2, Takehiko Ishii 3, Kazunori Kataoka 2
1Graduate School of Medicine, Laboratory of Clinical Biotechnology, The University of Tokyo, 2Graduate School of Engineering, Department of Materials Engineering, The University of Tokyo, 3Graduate School of Engineering, Department of Bioengineering, The University of Tokyo

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

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Medicine

Three-dimensional Co-culture Model for Tumor-stromal Interaction
Masafumi Horie 1,2, Akira Saito 1,3, Yoko Yamaguchi 4, Mitsuhiro Ohshima 5, Takahide Nagase 1
1Department of Respiratory Medicine, Graduate School of Medicine, The University of Tokyo, 2Department of Clinical Laboratory, Graduate School of Medicine, The University of Tokyo, 3Division for Health Service Promotion, The University of Tokyo, 4Department of Biochemistry, Nihon University School of Dentistry, 5Department of Biochemistry, Ohu University School of Pharmaceutical Sciences

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

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Developmental Biology

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle
Ikki Horiguchi 1, Yasuyuki Sakai 1
1Center for International Research on Integrative Biomedical System, Institute of Industrial Science, The University of Tokyo

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

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Biology

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy
Takumi Higaki 1
1Graduate School of Frontier Sciences, The University of Tokyo

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

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Engineering

Quantification of Hydrogen Concentrations in Surface and Interface Layers and Bulk Materials through Depth Profiling with Nuclear Reaction Analysis
Markus Wilde 1, Satoshi Ohno 1, Shohei Ogura 1, Katsuyuki Fukutani 1, Hiroyuki Matsuzaki 2
1Institute of Industrial Science, The University of Tokyo, 2Micro Analysis Laboratory, Tandem accelerator, The University Museum, The University of Tokyo

We illustrate the application of 1H(15N,αγ)12C resonant nuclear reaction analysis (NRA) to quantitatively evaluate the density of hydrogen atoms on the surface, in the volume, and at an interfacial layer of solid materials. The near-surface hydrogen depth profiling of a Pd(110) single crystal and of SiO2/Si(100) stacks is described.

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Biology

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions
Gen Kaneko 1,4, Tatsuki Yoshinaga 2, Kristin E. Gribble 3, David M. Welch 3, Hideki Ushio 1
1Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 2School of Marine Biosciences, Department of Marine Biosciences, Kitasato University, 3Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, 4School of Arts and Sciences, University of Houston-Victoria

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

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JoVE Journal

Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System
Takayuki Ueno 1,2, Kiyoshi Kawasaki 2, Takeo Kubo 1
1Department of Biological Sciences, Graduate School of Sciences, The University of Tokyo, 2Faculty of Pharmaceutical Sciences, Doshisha Women's College

Here we describe our detailed protocol for the preparation of single-cohort honeybee colonies – a useful tool for analyzing the role-associated worker physiology. We also describe detailed protocols for treating workers with juvenile hormone and ecdysone to evaluate the involvement of these hormones in the regulation of worker behavior and/or physiology.

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Neuroscience

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation
Daisuke Ino 1,2, Masamitsu Iino 1
1Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, 2Laboratory for Cell Polarity Regulation, Quantitative Biology Center, RIKEN

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

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Neuroscience

Insect-controlled Robot: A Mobile Robot Platform to Evaluate the Odor-tracking Capability of an Insect
Noriyasu Ando 1, Shuhei Emoto 1, Ryohei Kanzaki 1
1Research Center for Advanced Science and Technology, The University of Tokyo

The capability to localize an odor source is necessary for insect survival and is expected to be applicable to artificial odor-tracking. The insect-controlled robot is driven by an actual silkmoth and enables us to evaluate the odor-tracking capability of insects through a robotic platform.

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Medicine

2-Methacryloyloxyethyl Phosphorylcholine Polymer Treatment of Complete Dentures to Inhibit Denture Plaque Deposition
Kenji Ikeya 1, Miya Fukunishi 1, Fuminori Iwasa 1, Yuuki Inoue 2, Kazuhiko Ishihara 2, Kazuyoshi Baba 1
1Department of Prosthodontics, School of Dentistry, Showa University, 2Department of Materials Engineering, School of Engineering, The University of Tokyo

Removable poly(methyl methacrylate) (PMMA) dentures are prone to bacterial adherence and plaque formation. Denture plaque-associated infection is a source of serious dental and medical complications in the elderly. This paper introduces a novel protocol to treat PMMA dentures with 2-methacryloyloxyethyl phosphorylcholine polymer, poly(MPC-co-BMA-co-MPAz), to suppress plaque deposition on PMMA dentures.

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Bioengineering

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface
Kosuke Okeyoshi 1, Kensuke Osada 2, Maiko K. Okajima 1, Tatsuo Kaneko 1
1Japan Advanced Institute of Science and Technology, 2Department of Bioengineering, School of Engineering, The University of Tokyo

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

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Biochemistry

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion
Yuno Natsume 1, Hsin-i Wen 2, Tong Zhu 2, Kazumi Itoh 1, Li Sheng 2, Kensuke Kurihara 2,3,4
1Department of Mathematical and Physical Sciences, Faculty of Science, Japan Women's University, 2Department of Bioorganization Research, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 3Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, National Institutes of Natural Sciences, 4Research Center for Complex Systems Biology, The University of Tokyo

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

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Bioengineering

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles
Usein Ismailov 1, Esma Ismailova 1, Seiichi Takamatsu 2
1Department of Bioelectronics, Ecole Nationale Supérieure des Mines de Saint-Étienne, CMP-EMSE, MOC, 2Graduate School of Frontier Sciences, The University of Tokyo

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

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Neuroscience

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes
Naoki Ihara 1, Yuji Ikegaya 1,2, Haruki Takeuchi 1,3
1Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 2Center for Information and Neural Networks, National Institute of Information and Communications Technology, 3PRESTO, Japan Science and Technology Agency (JST)

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

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Biology

Establishment of an Extracellular Acidic pH Culture System
Ayano Kondo 1, Tsuyoshi Osawa 2
1Innovative Technology Laboratories, Kyowa Hakko Kirin Co., Ltd, 2Laboratory for Systems Biology and Medicine, RCAST, The University of Tokyo

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

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Biochemistry

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag
Genta Ito 1, Taisuke Tomita 1,2
1Laboratory of Brain and Neurological Disorders, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 2Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

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JoVE Core

Electric-field Control of Electronic States in WS2 Nanodevices by Electrolyte Gating
Feng Qin 1, Toshiya Ideue 1, Wu Shi 2, Yijin Zhang 3,4, Ryuji Suzuki 1, Masaro Yoshida 5, Yu Saito 1, Yoshihiro Iwasa 1,5
1Quantum-Phase Electronics Center (QPEC) and Department of Applied Physics, The University of Tokyo, 2Materials Sciences Division, Lawrence Berkeley National Laboratory, 3Institute of Scientific and Industrial Research, Osaka University, 4Max Planck Institute for Solid State Research, 5RIKEN Center for Emergent Matter Science (CEMS)

Here, we present a protocol to control the carrier number in solids by using the electrolyte.

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Cancer Research

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology
Kenji Amemiya 1,2, Yosuke Hirotsu 1, Toshio Oyama 2, Masao Omata 1,3
1Genome Analysis Center, Yamanashi Central Hospital, 2Pathology Division, Laboratory Department, Yamanashi Central Hospital, 3The University of Tokyo

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

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Neuroscience

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat
Yu Shikano 1, Takuya Sasaki 1, Yuji Ikegaya 1,2
1Graduate School of Pharmaceutical Sciences, The University of Tokyo, 2Center for Information and Neural Networks

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

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Biology

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-β Signaling
Hiroshi I. Suzuki 1, Masafumi Horie 2,3, Hajime Mihira 4, Akira Saito 2
1David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 2Department of Respiratory Medicine, Graduate School of Medicine, The University of Tokyo, 3Hastings Center for Pulmonary Research, Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Keck School of Medicine, University of Southern California, 4Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-β in MS-1 endothelial cells.

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Environment

Self-standing Electrochemical Set-up to Enrich Anode-respiring Bacteria On-site
Akihiro Okamoto 1, Annette Rowe 2, Xiao Deng 3, Kenneth H. Nealson 2
1International Center for Materials Nanoarchitectonics, National Institute for Materials Science, 2Department of Earth Sciences, University of Southern California, 3Department of Applied Chemistry, The University of Tokyo

On-site microbial enrichment or in situ cultivation techniques can facilitate the isolation of difficult-to-culture microbial taxa, especially from low-biomass or geochemically extreme environments. Here, we describe an electrochemical set-up without using an external power source to enrich microbial strains that are capable of extracellular electron transport (EET).

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Chemistry

Preparation of Polyoxometalate-based Photo-responsive Membranes for the Photo-activation of Manganese Oxide Catalysts
Akira Yamaguchi 1,5, Toshihiro Takashima 2, Kazuhito Hashimoto 1,6, Ryuhei Nakamura 3,4
1Department of Applied Chemistry, The University of Tokyo, 2Clean Energy Research Center, University of Yamanashi, 3Biofunctional Catalyst Research Team, RIKEN Center for Sustainable Resource Science, 4Earth-Life Science Institute (ELSI), Tokyo Institute of Technology, 5Department of Materials Science and Engineering, Tokyo Institute of Technology, 6National Institute for Materials Science

Here, we present a protocol to prepare charge transfer chromophores based on a polyoxometalate/polymer composite membrane.

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Biochemistry

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee
Shota Suenami 1,2, Ryo Miyazaki 1, Takeo Kubo 2
1Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, 2Department of Biological Sciences, Graduate School of Science, The University of Tokyo

To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors.

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Engineering

Theoretical Calculation and Experimental Verification for Dislocation Reduction in Germanium Epitaxial Layers with Semicylindrical Voids on Silicon
Motoki Yako 1, Yasuhiko Ishikawa 2, Eiji Abe 1, Kazumi Wada 1,3
1Department of Materials Engineering, The University of Tokyo, 2Department of Electrical and Electronic Information Engineering, Toyohashi University of Technology, 3Department of Materials Science and Engineering, Massachusetts Institute of Technology

Theoretical calculation and experimental verification are proposed for a reduction of threading dislocation (TD) density in germanium epitaxial layers with semicylindrical voids on silicon. Calculations based on the interaction of TDs and surface via image force, TD measurements, and transmission electron microscope observations of TDs are presented.

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Environment

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings
Kae Akita 1, Takumi Higaki 2
1Department of Integrated Frontier Sciences, The University of Tokyo, 2International Research Organization for Advanced Science and Technology, Kumamoto University

The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy.

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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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Chemistry

An Electrochemical Cholesteric Liquid Crystalline Device for Quick and Low-Voltage Color Modulation
Shoichi Tokunaga *1, Mengyan Zeng *1,2, Yoshimitsu Itoh 1, Fumito Araoka 3, Takuzo Aida 3
1Department of Chemistry and Biotechnology, The University of Tokyo, 2Department of Chemistry, Tsinghua University, 3RIKEN Center for Emergent Matter Science

A protocol for the fabrication of a reflective cholesteric liquid crystalline display device containing a redox-responsive chiral dopant allowing quick and low-voltage operation is presented.

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Immunology and Infection

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes
Naoyoshi Sakitani *1, Takahiro Maekawa *1, Kumiko Saitou 1,2, Katsuhiko Suzuki 3, Shuhei Murase 1,4, Masakuni Tokunaga 1, Daisuke Yoshino 5, Keisuke Sawada 6, Atsushi Takashima 7, Motoshi Nagao 1, Toru Ogata 1, Yasuhiro Sawada 1,8
1Department of Rehabilitation for Motor Functions, National Rehabilitation Center for Persons with Disabilities, 2Graduate School of Sport Sciences, Waseda University, 3Faculty of Sport Sciences, Waseda University, 4Department of Orthopaedic Surgery, Graduate School of Medicine, The University of Tokyo, 5Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, 6University of Cincinnati College of Medicine, 7Department of Assistive Technology, National Rehabilitation Center for Persons with Disabilities, 8Department of Clinical Research, National Rehabilitation Center for Persons with Disabilities

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

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Medicine

Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System
Kazuma Sekiba 1,2, Motoyuki Otsuka 1, Kazuhiko Koike 1
1Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, 2Research Fellow of Japan Society for the Promotion of Science

Here, we present a method for screening anti-hepatitis B viral agents that inhibit the HBx-DDB1 interaction using a split luciferase assay system. This system allows easy detection of protein-protein interactions and is suitable for identifying inhibitors of such interactions.

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Neuroscience

Three-Dimensional Motor Nerve Organoid Generation
Tatsuya Osaki 1,2, Siu Yu A. Chow 1,2, Yui Nakanishi 1,2, Joel Hernández 1,3, Jiro Kawada 4, Teruo Fujii 1, Yoshiho Ikeuchi 1,2
1Institute of Industrial Science, The University of Tokyo, 2Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 3Faculty of Science and Engineering, Tecnologico de Monterrey, 4Jiksak Bioengineering, Inc.

This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

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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

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes)
Muhammad Rahmad Royan 1, Shinji Kanda 2, Daichi Kayo 3, Weiyi Song 4, Wei Ge 4, Finn-Arne Weltzien 1, Romain Fontaine 1
1Physiology Unit, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, 2Laboratory of Physiology, Atmosphere and Ocean Research Institute, The University of Tokyo, 3Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 4Centre of Reproduction, Development and Aging (CRDA), Faculty of Health Sciences, University of Macau

The article describes a quick protocol to gonadectomize and sample blood from the small teleost fish, using Japanese medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology.

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Cancer Research

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma
Hiroki Kobayashi 1,2,3,4, Krystyna A. Gieniec 1,2, Jia Q. Ng 1,2, Jarrad Goyne 1,2, Tamsin R. M. Lannagan 1,2, Elaine M. Thomas 1,2, Georgette Radford 1,2, Tongtong Wang 1,2, Nobumi Suzuki 1,2,5, Mari Ichinose 1,2, Josephine A. Wright 2, Laura Vrbanac 1,2, Alastair D. Burt 6, Masahide Takahashi 3,4,7, Atsushi Enomoto 3, Daniel L. Worthley 2, Susan L. Woods 1,2
1Adelaide Medical School, University of Adelaide, 2South Australian Health and Medical Research Institute (SAHMRI), 3Department of Pathology, Nagoya University Graduate School of Medicine, 4Division of Molecular Pathology, Center for Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, 5Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, 6Translational and Clinical Research Institute, Newcastle University, 7International Center for Cell and Gene Therapy, Fujita Health University

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

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Engineering

Electroantennography-based Bio-hybrid Odor-detecting Drone using Silkmoth Antennae for Odor Source Localization
Daigo Terutsuki 1, Tomoya Uchida 2, Chihiro Fukui 3, Yuji Sukekawa 1, Yuki Okamoto 4, Ryohei Kanzaki 1
1Research Center for Advanced Science and Technology, The University of Tokyo, 2Department of Mechano-Informatics, Graduate School of Information Science and Technology, The University of Tokyo, 3Department of Applied Biological Science, Graduate School of Science and Technology, Tokyo University of Science, 4Sensing System Research Center, National Institute of Advanced Industrial Science and Technology

This study introduces experimental protocols for a bio-hybrid odor-detecting drone based on silkmoth antennae. The operation of an experimental electroantennogram device with silkmoth antennae is presented, in addition to the structure of a bio-hybrid drone designed for odor source localization using the spiral-surge algorithm.

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JoVE Journal

Optical Clearing of Plant Tissues for Fluorescence Imaging
Daisuke Kurihara 1, Yoko Mizuta 1,2, Shiori Nagahara 1, Yoshikatsu Sato 1,3, Tetsuya Higashiyama 1,3,4
1Institute of Transformative Bio-Molecules (ITbM), Nagoya University, 2Institute for Advanced Research (IAR), Nagoya University, 3Division of Biological Science, Graduate School of Science, Nagoya University, 4Department of Biological Sciences, Graduate School of Science, The University of Tokyo

Here, a method is described for making plant tissues transparent while maintaining the stability of fluorescent proteins. This technique facilitates deep imaging of cleared plant tissues without physical sectioning.

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Medicine

Whole-Kidney Three-Dimensional Staining with CUBIC
Sho Hasegawa 1, Masaomi Nangaku 1
1Division of Nephrology and Endocrinology, The University of Tokyo

The present protocol describes a tissue clearing method and whole-mount immunofluorescent staining for three-dimensional (3D) kidney imaging. This technique can offer macroscopic perspectives in kidney pathology, leading to new biological discoveries.

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Neuroscience

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus
Ryosuke Kamei 1, Shinji Urata 1, Hisato Maruoka 1, Shigeo Okabe 1
1Department of Cellular Neurobiology, Graduate School of Medicine, The University of Tokyo

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.

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Neuroscience

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice
Hisato Maruoka 1, Ryosuke Kamei 1, Shunsuke Mizutani 1, Qingrui Liu 1, Shigeo Okabe 1
1Department of Cellular Neurobiology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

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Neuroscience

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window
Satoshi Manita 1, Eiji Shigetomi 2,3, Haruhiko Bito 4, Schuichi Koizumi 2,3, Kazuo Kitamura 1
1Department of Neurophysiology, Faculty of Medicine, University of Yamanashi, 2Department of Neuropharmacology, Faculty of Medicine, University of Yamanashi, 3Yamanashi GLIA center, Interdisciplinary Graduate School of Medicine, University of Yamanashi, 4Department of Neurochemistry, Graduate School of Medicine, The University of Tokyo

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

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Developmental Biology

Fluorescent In Situ Hybridization and 5-Ethynyl-2'-Deoxyuridine Labeling for Stem-Like Cells in the Hydrozoan Jellyfish Cladonema pacificum
Sosuke Fujita 1, Erina Kuranaga 1, Masayuki Miura 2, Yu-ichiro Nakajima 2
1Graduate School of Life Sciences, Tohoku University, 2Graduate School of Pharmaceutical Sciences, The University of Tokyo

Here, we describe a protocol for visualizing stem-like proliferating cells in the jellyfish Cladonema. Whole-mount fluorescent in situ hybridization with a stem cell marker allows for the detection of stem-like cells, and 5-ethynyl-2'-deoxyuridine labeling enables the identification of proliferating cells. Together, actively proliferating stem-like cells can be detected

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Developmental Biology

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models
Manabu Ozawa *1, Chihiro Emori *2, Masahito Ikawa 1,2
1Laboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, 2Research Institute for Microbial Diseases, Osaka University

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

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Medicine

Modified Heterotopic Abdominal Heart Transplantation and a Novel Aortic Regurgitation Model in Rats
Shigeto Tsuji 1, Shogo Shimada 1, Minoru Ono 1
1Department of Cardiac Surgery, The University of Tokyo

This study demonstrates a reproducible heterotopic abdominal heart transplantation technique in rats that beginners can learn and perform. Additionally, a novel aortic regurgitation model in rats is generated by performing heterotopic abdominal heart transplantation and damaging the donor's aortic valve using a guidewire after harvesting.

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Neuroscience

Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons
Noriko Koganezawa 1, Reiko T. Roppongi 2, Yuko Sekino 3,4, Izuo Tsutsui 3, Ayaka Higa 5, Tomoaki Shirao 1,5
1Department of Pharmacology, Graduate School of Medicine, Gunma University, 2Gunma University Initiative for Advanced Research, Gunma University, 3Department of Veterinary Pathophysiology and Animal Health, Graduate school of Agricultural and Life Sciences, The University of Tokyo, 4Institute for Drug Discovery Innovation, 5AlzMed, Inc.

A ready-to-use frozen stock of neurons is a powerful tool for evaluating synaptic functions. Here, we introduce an easy low-density primary culture from frozen stock using a 96-well plate.

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Biology

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator
Kaede Saito 1,2, Yuta Hiraike 1,2,3, Misato Oguchi 2, Toshimasa Yamauchi 2
1Division for Health Service Promotion, The University of Tokyo, 2Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, 3The University of Tokyo Excellent Young Researcher Program, The University of Tokyo

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

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Biology

A Pacing-Controlled Procedure for the Assessment of Heart Rate-Dependent Diastolic Functions in Murine Heart Failure Models
Genri Numata 1,2, Eiki Takimoto 1,3
1Department of Cardiovascular Medicine, The University of Tokyo Hospital, 2Department of Therapeutic Strategy for Heart Failure, Graduate School of Medicine, The University of Tokyo, 3Division of Cardiology, Department of Medicine, The Johns Hopkins Medical Institutions

The present protocol describes obtaining the pressure-volume relationship through transesophageal pacing, which serves as a valuable tool in evaluating diastolic function in mouse models of heart failure.

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Medicine

Three-Dimensional Morphogenesis in Canine Gut-on-a-Chip Using Intestinal Organoids Derived from Inflammatory Bowel Disease Patients
Itsuma Nagao 1,2, Meg Nakazawa 1, Yoko M. Ambrosini 1
1Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, 2Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo

Integration of canine intestinal organoids and a Gut-on-a-Chip microfluidic system offers relevant translational models for human intestinal diseases. Protocols presented allow for 3D morphogenesis and dynamic in vitro modeling of the gut, aiding in the development of effective treatments for intestinal diseases in dogs and humans with One Health.

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