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63 ARTICLES PUBLISHED IN JoVE

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Biology

Proboscis Extension Response (PER) Assay in Drosophila
Takashi Shiraiwa 1, John R. Carlson 1
1Department of Molecular, Cellular, and Developmental Biology, Yale University

Proboscis extension response or PER is a taste behavior assay that has been used in flies as well as in honeybees. When the proboscis makes contact with an attractive substance, the fly extends its proboscis to consume the substance. Solutions of various sugars are very attractive to the fly.

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Biology

In situ Protocol for Butterfly Pupal Wings Using Riboprobes
Diane Ramos 1, Antonia Monteiro 2
1Department of Biological Sciences, SUNY-University at Buffalo, 2Dept. Ecology and Evolutionary Biology, Yale University

In order to examine gene expression in the pupal wing tissue of Bicyclus anynana, we present an optimized protocol for in situ hybridizations using riboprobes. We also provide guidelines for the further optimization of this protocol for use in pupal wings of other Lepidopteran species.

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Biology

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization
Tim Brend 1, Scott A. Holley 1
1Department of Molecular, Cellular and Developmental Biology, Yale University

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

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Biology

In vivo Imaging of Deep Cortical Layers using a Microprism
Thomas H. Chia 1, Michael J. Levene 1
1Department of Biomedical Engineering, Yale University

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 μm) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

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Biology

Protein Crystallization for X-ray Crystallography
Moshe A. Dessau 1, Yorgo Modis 1
1Molecular Biochemistry and Biophysics, Yale University

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

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Neuroscience

Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition
Gloria Wong 1,2, Sanda Dolcos 1,3, Ekaterina Denkova 1, Rajendra Morey 4,5,6, Lihong Wang 4,5, Gregory McCarthy 6,7, Florin Dolcos 1,2,3,8,9
1Department of Psychiatry, University of Alberta, 2Centre for Neuroscience, University of Alberta, 3Department of Psychology, University of Illinois, 4Brain Imaging and Analysis Center, Duke University , 5Department of Psychiatry and Behavioral Sciences, Duke University , 6Mid-Atlantic Mental Illness Research Education and Clinical Center, VA Medical Center, 7Department of Psychology, Yale University, 8Neuroscience Program, University of Illinois, 9Beckman Institute for Advanced Science & Technology, University of Illinois

We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

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Biology

Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method
Xueqi Liu 1, Hong-Wei Wang 1
1Molecular Biophysics and Biochemistry, Yale University

This article describes a standard method to get a three-dimensional (3D) reconstruction of biological macromolecules using negative staining electron microscopy (EM). In this protocol, we explain how to get the 3D structure of the Saccharomyces cerevisiae exosome complex at medium resolution using the random conical tilt reconstruction method (RCT).

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Bioengineering

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor
Angela H. Huang 1, Laura E. Niklason 1,2
1Department of Biomedical Engineering, Yale University, 2Department of Anesthesiology, Yale University School of Medicine

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

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Bioengineering

Procedure for Lung Engineering
Elizabeth A. Calle *1, Thomas H. Petersen *2, Laura E. Niklason 1,3
1Department of Biomedical Engineering, Yale University, 2Department of Biomedical Engineering, School of Medicine, Duke University, 3Department of Anesthesia, Yale University

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

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Neuroscience

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation
Onkar S. Dhande 1,2, Michael C. Crair 1
1Department of Neurobiology, Yale University, 2Program in Developmental Biology, Baylor College of Medicine

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

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Immunology and Infection

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging
Joel R. Meyerson 1,2, Tommi A. White 1, Donald Bliss 3, Amy Moran 3, Alberto Bartesaghi 1, Mario J. Borgnia 1, M. Jason V. de la Cruz 1, David Schauder 1, Lisa M. Hartnell 1, Rachna Nandwani 1,4, Moez Dawood 5, Brianna Kim 6, Jun Hong Kim 7, John Sununu 8, Lisa Yang 9, Siddhant Bhatia 10, Carolyn Subramaniam 1, Darrell E. Hurt 11, Laurent Gaudreault 12, Sriram Subramaniam 1
1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2The Medical Research Council Mitochondrial Biology Unit, University of Cambridge , 3National Library of Medicine, National Institutes of Health, 4Massachusetts Institute of Technology, 5William Fremd High School, 6University of Virginia , 7Duke University , 8Yale University, 9University of Notre Dame , 10Washington University in St. Louis , 11Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12Thomas Jefferson High School for Science and Technology

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

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Medicine

Skeletal Muscle Gender Dimorphism from Proteomics
Kalina Dimova 1, Lauren Ann Metskas 2, Mohini Kulp 3, Stylianos P. Scordilis 4
1Center for Proteomics, Smith College, 2Department of Molecular Biophysics and Biochemistry, Yale University, 3Department of Chemistry, Smith College, 4Department of Biological Sciences and Center for Proteomics, Smith College

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

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Neuroscience

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP
Sonia G. Parra *1, Sam S. Vesuna *1, Teresa A. Murray 1,2, Michael J. Levene 1
1Department of Biomedical Engineering, Yale University, 2Department of Biomedical Engineering, Louisiana Tech University

Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.

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Biology

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes
Yanning Wu 1, Shuo Wang 1, Chunying Li 1,2,3
1Department of Biochemistry & Molecular Biology, Wayne State University School of Medicine, 2Cardiovascular Research Institute, Wayne State University School of Medicine, 3Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

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Neuroscience

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes
Silvio Sacchetti 1, Kambiz N. Alavian 1, Emma Lazrove 1, Elizabeth A. Jonas 1
1Department of Internal Medicine, Yale University

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

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Biology

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter
Oswald J. Schmitz 1, Mark A. Bradford 1, Michael S. Strickland 1,2, Dror Hawlena 3
1School of Forestry and Environmental Studies, Yale University, 2Department of Biological Sciences, Virginia Tech, 3Department of Ecology, Evolution and Behavior, The Hebrew University of Jerusalem

We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.

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Behavior

Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET: Dopamine Movies of Cigarette Smoking
Evan D. Morris 1,2,3,4, Su Jin Kim 1,3, Jenna M. Sullivan 1,3,4, Shuo Wang 3,4, Marc D. Normandin 5, Cristian C. Constantinescu 6, Kelly P. Cosgrove 1,2,3
1Diagnostic Radiology, Yale University, 2Psychiatry, Yale University, 3Yale PET Center, Yale University, 4Biomedical Engineering, Yale University, 5Nuclear Medicine, Massachusetts General Hospital, 6Radiological Sciences, University of California, Irvine

We present a novel PET imaging approach for capturing dopamine fluctuations induced by cigarette smoking. Subjects smoke in the PET scanner. Dynamic PET images are modeled voxel-by-voxel in time by lp-ntPET, which includes a time-varying dopamine term. The results are 'movies' of dopamine fluctuations in the striatum during smoking.

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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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JoVE Journal

Fast Imaging Technique to Study Drop Impact Dynamics of Non-Newtonian Fluids
Qin Xu 1,2, Ivo Peters 2, Sam Wilken 1,2, Eric Brown 3, Heinrich Jaeger 1,2
1Department of Physics, The University of Chicago, 2James Franck Institute, The University of Chicago, 3Department of Mechanical Engineering and Materials Science, Yale University

Drop impact of non-Newtonian fluids is a complex process since different physical parameters influence the dynamics over a very short time (less than one tenth of a millisecond). A fast imaging technique is introduced in order to characterize the impact behaviors of different non-Newtonian fluids.

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Neuroscience

Electrophysiological Recording From Drosophila Labellar Taste Sensilla
Rebecca Delventhal 1, Aidan Kiely 1, John R. Carlson 1
1MCDB, Yale University

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

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Neuroscience

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster
Robert W. Fernandez 1, Marat Nurilov 2, Omar Feliciano 2, Ian S. McDonald 3, Anne F. Simon 3
1Department of Molecular Biophysics and Biochemistry, Yale University, 2Department of Biology, York College/CUNY, 3Department of Biology, Western Ontario University

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

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Neuroscience

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus
Tharakeswari Selvakumar 1, Kambiz N. Alavian 2, Travis Tierney 1
1Department of Neurosurgery, Brigham & Women's Hospital, Harvard Medical School, 2Division of Brain Sciences, Department of Medicine, Imperial College London

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

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Behavior

Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats
Robert J. Wickham 1, Jinwoo Park 2, Eric J. Nunes 3, Nii A. Addy 1,3,4
1Interdepartmental Neuroscience Program, Yale University, 2Department of Biotechnical and Clinical Laboratory Sciences, School of Medicine and Biomedical Sciences, University at Buffalo, 3Department of Psychiatry, Yale School of Medicine, 4Department of Cellular and Molecular Physiology, Yale School of Medicine

Rapid fluctuations in extracellular dopamine (DA) mediate both reward processing and motivated behavior in mammals. This manuscript describes the combined use of fast scan cyclic voltammetry (FSCV) and intra-oral tastant administration to determine how tastants alter rapid dopamine release in awake, freely moving rats.

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Neuroscience

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains
Maria Weinert 1, Tharakeswari Selvakumar 2, Travis S. Tierney 2, Kambiz N. Alavian 1,3
1Division of Brain Sciences, Department of Medicine, Imperial College London, 2Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, 3Department of Internal Medicine, Endocrinology, Yale University School of Medicine

The causes of degeneration of midbrain dopaminergic neurons during Parkinson’s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

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Neuroscience

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy
Magalie Bénard 1, Alexis Lebon 1, Hitoshi Komuro 2, David Vaudry 1, Ludovic Galas 1
1PRIMACEN, Cell Imaging Platform of Normandy, Inserm, IRIB, University of Rouen, 2Department of Neurobiology, School of Medicine, Yale University

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

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Stimulus-Independent Analysis of Affective Touch Using fMRI
Molly Lucas 1,2, Laura Anderson 1,3, Danielle Bolling 1, Kevin A Pelphrey 1, Martha D Kaiser 1
1Yale Child Study Center, Yale University, 2School of Continuing Education, Columbia University, 3Clinical / Developmental Psychology, University of Maryland

C-tactile (CT) afferents respond to caress-like touch, which has been found to activate “social brain” regions 1. Using fMRI, we compared neural responses of experiencing and imagining CT-targeted vs. non-CT-targeted touch to explore how the affective component of social touch is imbued.

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Bioengineering

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography
Dustin R. Morado 1, Bo Hu 1, Jun Liu 1
1Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

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Developmental Biology

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres
Miguel A. Lopez-Ramirez *1,2, Charles-Félix Calvo *3, Emma Ristori 1, Jean-Léon Thomas 1,3, Stefania Nicoli 1
1Yale Cardiovascular Research Center, Internal Medicine, Yale University, 2Department of Medicine, University of California, San Diego, 3APHP Groupe Hospitalier Pitié-Salpètrière, Université Pierre and Marie Curie

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

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Medicine

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium
Takuya Hashimoto 1,2,3, Kota Yamamoto 1,2,3, Trenton Foster 1, Hualong Bai 1, Kunihiro Shigematsu 4, Alan Dardik 1,3
1Department of Surgery and the Vascular Biology and Therapeutics Program, Yale University, 2Department of Vascular Surgery, University of Tokyo, 3Department of Vascular Surgery, VA Connecticut Healthcare Systems, 4Department of Vascular Surgery, International University of Health and Welfare Mita Hospital

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

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Neuroscience

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice
Melanie Maya Kaelberer 1,2, Sven-Eric Jordt 2
1Department of Cellular & Molecular Physiology, Yale University, 2Department of Anesthesiology, Duke University Medical Center

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

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JoVE Core

Knowledge Based Cloud FE Simulation of Sheet Metal Forming Processes
Du Zhou 1, Xi Yuan 1, Haoxiang Gao 1, Ailing Wang 1, Jun Liu 1, Omer El Fakir 1, Denis J. Politis 1, Liliang Wang 1, Jianguo Lin 1
1Department of Mechanical Engineering, Imperial College London

The following paper presents a novel FE simulation technique (KBC-FE), which reduces computational cost by performing simulations on a cloud computing environment, through the application of individual modules. Moreover, it establishes a seamless collaborative network between world leading scientists, enabling the integration of cutting edge knowledge modules into FE simulations.

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Neuroscience

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy
Joerg Nikolaus 1,2, Erdem Karatekin 1,2,3,4
1Department of Cellular and Molecular Physiology, Yale University School of Medicine, 2Nanobiology Institute, Yale University, 3Department of Molecular Biophysics and Biochemistry, Yale University, 4Laboratoire de Neurophotonique, Université Paris Descartes, Faculté des Sciences Fondamentales et Biomédicales, Centre National de la Recherche Scientifique (CNRS)

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

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JoVE Core

Ultrasound Images of the Tongue: A Tutorial for Assessment and Remediation of Speech Sound Errors
Jonathan L. Preston 1,2, Tara McAllister Byun 3, Suzanne E. Boyce 2,4, Sarah Hamilton 4, Mark Tiede 2, Emily Phillips 2, Ahmed Rivera-Campos 4, Douglas H. Whalen 2,5,6
1Department of Communication Sciences and Disorders, Syracuse University, 2Haskins Laboratories, 3Department of Communicative Sciences and Disorders, New York University, 4Department of Communication Sciences and Disorders, University of Cincinnati, 5Program in Speech-Language-Hearing Sciences, City University of New York Graduate Center, 6Department of Linguistics, Yale University

Ultrasound imaging can be used to display the shape and movements of the tongue in real time during speech. The images can be used to determine the nature of speech sound errors. Visual feedback of the tongue can be used to facilitate improvements in speech sound production in clinical populations.

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Medicine

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava
Hualong Bai 1,2,3,4,5, Xin Li 6, Takuya Hashimoto 1,2,5, Haidi Hu 1,2,5, Trenton R. Foster 1,2,5, Jesse J. Hanisch 1,2,5, Jeans M. Santana 1,2,5, Alan Dardik 1,2,5
1Department of Surgery, Yale University, 2Vascular Biology and Therapeutics Program, Yale University, 3Department of Vascular Surgery, First Affiliated Hospital of Zhengzhou University, 4Basic Medical College of Zhengzhou University, 5VA Connecticut Healthcare Systems, West Haven, CT, 6Department of Vascular Surgery, Xiangya Second Hospital of Central South University, Changsha, China

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

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Developmental Biology

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity
Shen Zhang 1, Yonggen Wu 1, Yimin Weng 2, Zhihui Xu 1, Wenmin Chen 3, Dahan Zheng 4, Wei Lin 5, Jun Liu 6, Ying Zhou 1,7
1Reproductive Medicine Center, The First Affiliated Hospital of Wenzhou Medical University, 2Department of Orthopaedics, The Second Affiliated Hospital of Wenzhou Medical University, 3Department of Obstetrics, The First Affiliated Hospital of Wenzhou Medical University, 4School of Laboratory Medicine and Life Science, Wenzhou Medical University, 5School of Pharmaceutical Science, Wenzhou Medical University, 6Stem Cells and Genetic Engineering Group, AgriBioscience Research Centre, Department of Economic Development, Jobs, Transport and Resources, 7Department of Histology and Embryology, Wenzhou Medical University

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

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Neuroscience

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons
Seth Villarreal 1, Sung Hoon Lee 1,2, Ling-Gang Wu 1
1National Institute of Neurological Disorders and Stroke, 2College of Pharmacy, Chung-ang University

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

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Biochemistry

Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall
Xun Zhang 1, Sheng Chen 1, Feng Xu 1
1Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University

This protocol aims to present a general method to visualize lignin, cellulose, and hemicellulose in plant cell walls using Raman imaging and multivariate analysis.

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Bioengineering

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement
Jun Liu 1, John Wilson 2, Claire Davis 1, Anthony Peyton 2
1Advanced Steel Research Centre, Warwick Manufacturing Group, University of Warwick, 2School of Electrical and Electronic Engineering, University of Manchester

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

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Bioengineering

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture
Amanda S. Pellowe 1, Holly M. Lauridsen 1, Rita Matta 1, Anjelica L. Gonzalez 1
1Biomedical Engineering, Yale University

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

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Engineering

Quantitative Analysis of Vacuum Induction Melting by Laser-induced Breakdown Spectroscopy
Tianzhuo Zhao 1,2,3, Xin Li 1,2, Qixiu Zhong 1,2, Hong Xiao 1,3, Shuzhen Nie 1,3, Fuqiang Lian 1,3, Sining Sun 4, Zhongwei Fan 1,2,3
1The Academy of Opto-electronics, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3National Engineering Research Center for DPSSL, 4Beijing GK Laser Technology Co., Ltd.

During vacuum induction melting, laser-induced breakdown spectroscopy is used to perform real-time quantitative analysis of the main-ingredient elements of a molten alloy.

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Developmental Biology

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity
Mengrou Liu *1, Chunsen Zhu *1, Shun Bai *2, Xin Li 1, Kaiqiang Fu 1, Lan Ye 1, Ke Zheng 1
1State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 2Center for Reproductive Medicine, Department of Obstetrics and Gynecology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

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Neuroscience

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum
Regina T. Martuscello 1, Elan D. Louis 2,3,4, Phyllis L. Faust 1
1Department of Pathology and Cell Biology, Columbia University, 2Division of Movement Disorders, Department of Neurology, Yale University, 3Department of Chronic Disease Epidemiology, Yale School of Public Health, Yale University, 4Center for Neuroepidemiology and Clinical Neurological Research, Yale School of Medicine, Yale University

This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.

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Behavior

Simultaneous Eye Tracking and Single-Neuron Recordings in Human Epilepsy Patients
Shuo Wang 1, Nand Chandravadia 2, Adam N. Mamelak 2, Ueli Rutishauser 2,3,4
1Department of Chemical and Biomedical Engineering, and Rockefeller Neuroscience Institute, West Virginia University, 2Departments of Neurosurgery and Neurology, Cedars-Sinai Medical Center, 3Center for Neural Science and Medicine, Department of Biomedical Sciences, Cedars-Sinai Medical Center, 4Division of Biology and Biological Engineering, California Institute of Technology

We describe a method to conduct single-neuron recordings with simultaneous eye tracking in humans. We demonstrate the utility of this method and illustrate how we used this approach to obtain neurons in the human medial temporal lobe that encode targets of a visual search.

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Bioengineering

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients
Tae-Yun Kang 1, David Ellison 2, Sung Hoon Lee 1, Andrew J. Ewald 2,3, Andre Levchenko 1
1Department of Biomedical Engineering and Yale Systems Biology Institute, Yale University, 2Department of Biomedical Engineering, Johns Hopkins University, 3Center for Cell Dynamics and Department of Cell Biology, Johns Hopkins University

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

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Immunology and Infection

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue
Xin Li 1, Zhengmei Mao 2, Li Yang 1, Kai Sun 1
1Center for Metabolic and Degenerative Diseases, the Brown Foundation of Institute of Molecular Medicine, University of Texas Health Science Center at Houston, 2Microscopy Core, the Brown Foundation of Institute of Molecular Medicine, University of Texas Health Science Center at Houston

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

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Biology

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture
Laurel R. Yohe *1,2, Paolo Devanna *3, Kalina T.J. Davies 4, Joshua H.T. Potter 4, Stephen J. Rossiter 4, Emma C. Teeling 5, Sonja C. Vernes *3,6, Liliana M. Dávalos *2,7
1Department of Geology & Geophysics, Yale University, 2Department of Ecology & Evolution, Stony Brook University, 3Neurogenetics of Vocal Communication, Max Planck Institute for Psycholinguistics, 4School of Biological and Chemical Sciences, Queen Mary University of London, 5School of Biology & Environmental Science, University College Dublin, 6Donders Institute for Brain, Cognition and Behavior, 7Consortium for Inter-Disciplinary Environmental Research, Stony Brook University

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

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JoVE Journal

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules
Mohammed Mahamdeh 1,2, Jonathon Howard 1
1Department of Molecular Biophysics and Biochemistry, Yale University, 2Harvard Medical School, Harvard University

This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays.

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Medicine

Murine Model of Central Venous Stenosis using Aortocaval Fistula with an Outflow Stenosis
Toshihiko Isaji 1,2,3, Shun Ono 1,2,4,5, Takuya Hashimoto 1,2,3, Kota Yamamoto 1,2,3, Ryosuke Taniguchi 1,2,3, Haidi Hu 1,2, Tun Wang 1,2, Jun Koizumi 4, Toshiya Nishibe 5, Katsuyuki Hoshina 3, Alan Dardik 1,2,6
1Department of Surgery, Yale University, 2Vascular Biology and Therapeutics Program, Yale University, 3Department of Vascular Surgery, University of Tokyo, 4Department of Diagnostic Radiology, Tokai University School of Medicine, 5Department of Cardiovascular Surgery, Tokyo Medical University, 6Department of Vascular Surgery, VA Connecticut Healthcare Systems

An aortocaval fistula was created by puncturing the murine infra-renal aorta through both walls into the inferior vena cava and was followed by creation of a stenosis in its outflow via partial ligation of the inferior vena cava. This reproducible model can be used to study central venous stenosis.

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Genetics

Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting
Lucas D. Sanor 1, G. Parker Flowers 1, Craig M. Crews 1
1Department of Molecular, Cellular, and Developmental Biology, Yale University

This goal of this protocol is to produce chimeric axolotls with haploid forelimbs derived from Cas9-mutagenized donor tissue using embryonic tissue grafting techniques.

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Cancer Research

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging
Nour Ghaddar 1,2, Shuo Wang 1, Véronique Michaud 1, Urszula Kazimierczak 1,3, Nicolas Ah-son 1, Antonis E. Koromilas 1,4
1Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 2Division of Experimental Medicine, Faculty of Medicine, McGill University, 3Department of Cancer Immunology, Chair of Medical Biotechnology, Poznan University of Medical Sciences, 4Gerald Bronfman Department of Oncology, Faculty of Medicine, McGill University

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

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Immunology and Infection

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System
David Chetrit 1, Donghyun Park 1,2, Bo Hu 3, Jun Liu 1,2, Craig R. Roy 1
1Department of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale University School of Medicine, 2Microbial Sciences Institute, Yale University, 3Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

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Neuroscience

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine
Robert J. Wickham 1, Makenzie Lehr *1, Lauryn Mitchell *1, Nii A. Addy 2,3
1Department of Psychology, Elizabethtown College, 2Department of Psychiatry, Yale University, 3Department of Cellular and Molecular Physiology, Yale University

The goal of this protocol is to directly manipulate ventral tegmental area receptors to study their contribution to subsecond dopamine release.

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Bioengineering

Engineered Lung Tissues Prepared from Decellularized Lung Slices
Katherine L. Leiby 1,2, Ronald Ng 1, Stuart G. Campbell 1,3, Laura E. Niklason 1,4
1Department of Biomedical Engineering, Yale University, 2Yale School of Medicine, 3Department of Cellular and Molecular Physiology, Yale School of Medicine, 4Department of Anesthesiology, Yale School of Medicine

This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells.

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Biology

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy
Nikola Lukic *1, Trishna Saha *1, Stefanie Lapetina 1, Michal Gendler 1, Gilad Lehmann 1, Anthony J. Koleske 2,3, Hava Gil-Henn 1
1The Azrieli Faculty of Medicine, Bar-Ilan University, 2Department of Molecular Biophysics and Biochemistry, Yale University, 3Department of Neuroscience, Yale University

This protocol aims to measure the dynamic parameters (protrusions, retractions, ruffles) of protrusions at the edge of spreading cells.

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Biochemistry

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays
Ruchir C. Bobde *1,2, Ketul Saharan *1,2, Somanath Baral 1,3, Surajit Gandhi 1,2, Archana Samal 1,2, Rajivgandhi Sundaram 1, Ashish Kumar 1,4, Ajit K. Singh 1,5, Aritreyee Datta 1, Dileep Vasudevan 1
1Institute of Life Sciences, 2Regional Centre for Biotechnology, 3School of Biotechnology, KIIT University, 4Department of Molecular Biophysics and Biochemistry, Yale University, 5Department of Pharmacology, University of Vermont College of Medicine

This protocol describes a battery of methods that includes analytical size-exclusion chromatography to study histone chaperone oligomerization and stability, pull-down assay to unravel histone chaperone-histone interactions, AUC to analyze the stoichiometry of the protein complexes, and histone chaperoning assay to functionally characterize a putative histone chaperone in vitro.

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Biochemistry

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry
Aniruddha Panda 1,2, Rajan Thakur 1,3, Aastha Kumari 1, Padinjat Raghu 1
1National Centre for Biological Sciences-TIFR, GKVK Campus, 2Department of Cell Biology, Yale University, 3Department of Neuroscience, Brown University

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

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JoVE Journal

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins
Yazgan Tuna 1, Amer Al-Hiyasat 1, Jonathon Howard 1,2
1Department of Molecular Biophysics & Biochemistry, Yale University, 2Department of Physics, Yale University

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins.

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Medicine

Determining Binding Affinity (KD) of Radiolabeled Antibodies to Immobilized Antigens
Erika Belitzky 1, Alessandra Cavaliere 1, Khashayar Rajabimoghadam 1, Bernadette Marquez-Nostra 1
1Department of Radiology and Biomedical Imaging, Yale University

Here, a method is described to determine the binding affinity (KD) of radiolabeled antibodies to immobilized antigens. KD is the equilibrium dissociation constant that can be determined from a saturation binding experiment by measuring the total, specific, and nonspecific binding of a radiolabeled antibody at various concentrations to its antigen.

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Bioengineering

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles
Sheng Chen 1, Zachary Gao Sun 2,3, Michael P. Murrell 1,2,3
1Department of Biomedical Engineering, Yale University, 2Systems Biology Institute, Yale University, 3Department of Physics, Yale University

In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome membrane.

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Neuroscience

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain
Sofia Massaro Tieze 1,2,3, Sreeganga S. Chandra 1,2, D. J. Vidyadhara 1,2
1Department of Neurology, Yale University, 2Department of Neuroscience, Yale University, 3Interdepartmental Neuroscience Program, Yale University

This protocol presents a robust, detailed method to obtain highly pure synaptosomes, synaptic vesicles, and other synaptic fractions from the mouse brain. This method enables the evaluation of synaptic processes, including the biochemical analysis of protein localization and function with compartmental resolution.

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Bioengineering

An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue
Amanda L. Holt 1,3, Yakir Luc Gagnon 2,4, Alison M. Sweeney 1,3
1Department of Physics, Yale University, 2Lund Vision Group, Department of Biology, Lund University, 3Department of Physics and Astronomy, University of Pennsylvania, 4Department of Biology, Duke University

In this paper, a method for measuring radiance in situ in living tissue is described. This work includes details of the construction of micro-scale probes for different measurements of radiance and irradiance, provides guidance for mounting tissue for the characterization of radiance, and outlines computational methods for analyzing the resulting data.

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Neuroscience

Simultaneous Data Collection of fMRI and fNIRS Measurements Using a Whole-Head Optode Array and Short-Distance Channels
Sara Sanchez-Alonso 1, Rebecca R. Canale 2, Isabel F. Nichoson 1, Richard N. Aslin 1,2,3
1Child Study Center, Yale University School of Medicine, 2Department of Psychology, University of Connecticut, 3Department of Psychology, Yale University

We present a method for simultaneously collecting fMRI and fNIRS signals from the same subjects with whole-head fNIRS coverage. The protocol has been tested with three young adults and can be adapted for data collection for developmental studies and clinical populations.

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Neuroscience

Base Recording: A Technique for Analyzing Responses of Taste Neurons in Drosophila
Hany K. M. Dweck 1,2, John R. Carlson 1
1Department of Molecular, Cellular and Developmental Biology, Yale University, 2Department of Entomology, The Connecticut Agricultural Experiment Station

A rarely used method of electrophysiological recording, base recording, allows analysis of features of taste coding that cannot be examined by conventional recording methods. Base recording also allows the analysis of taste responses to hydrophobic stimuli that cannot be studied using traditional electrophysiological methods.

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