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20 ARTICLES PUBLISHED IN JoVE

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Biology

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons
Van B. Lu 1, Damian J. Williams 1, Yu-Jin Won 1, Stephen R. Ikeda 1
1Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism (NIAAA), National Institutes of Health (NIH)

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

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Neuroscience

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology
Arvydas Maminishkis 1, Sheldon S. Miller 1
1National Eye Institute, National Institutes of Health

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

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Behavior

How to Detect Amygdala Activity with Magnetoencephalography using Source Imaging
Nicholas L. Balderston 1, Douglas H. Schultz 1, Sylvain Baillet 2,3, Fred J. Helmstetter 1,3
1Department of Psychology, University of Wisconsin-Milwaukee, 2McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, 3Department of Neurology, Medical College of Wisconsin

This article describes how to record amygdala activity with magnetoencephalography (MEG). In addition this article will describe how to conduct trace fear conditioning without awareness, a task that activates the amygdala. It will cover 3 topics: 1) Designing a trace conditioning paradigm using backward masking to manipulate awareness. 2) Recording brain activity during the task using magnetoencephalography. 3) Using source imaging to recover signal from subcortical structures.

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Immunology and Infection

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures
Krisztina Janosko 1, Michael R. Holbrook 1, Ricky Adams 1, Jason Barr 1, Laura Bollinger 1, Je T'aime Newton 2, Corrie Ntiforo 2, Linda Coe 1, Jiro Wada 1, Daniela Pusl 1, Peter B. Jahrling 1, Jens H. Kuhn 1, Matthew G. Lackemeyer 1
1Integrated Research Facility at Frederick, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), 2Environmental Health and Safety, Biological and Chemical Safety Program, University of Texas Medical Branch

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

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Immunology and Infection

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices
Steven Mazur 1, Michael R. Holbrook 1, Tracey Burdette 1, Nicole Josleyn 1, Jason Barr 1, Daniela Pusl 1, Laura Bollinger 1, Linda Coe 1, Peter B. Jahrling 1, Matthew G. Lackemeyer 1, Jiro Wada 1, Jens H. Kuhn 1, Krisztina Janosko 1
1Integrated Research Facility at Frederick, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH)

Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal.

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Immunology and Infection

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 4. Medical Imaging Procedures
Russell Byrum 1, Lauren Keith 1, Christopher Bartos 1, Marisa St. Claire 1, Matthew G. Lackemeyer 1, Michael R. Holbrook 1, Krisztina Janosko 1, Jason Barr 1, Daniela Pusl 1, Laura Bollinger 1, Jiro Wada 1, Linda Coe 1, Lisa E. Hensley 1, Peter B. Jahrling 1, Jens H. Kuhn 1, Margaret R. Lentz 1
1Integrated Research Facility at Frederick, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH)

Here, we present an overview of the preparation and animal handling procedures required to safely perform medical imaging in an animal biosafety level 4 laboratory. Computed tomography of a mock-infected guinea pig illustrates these procedures that may be used to evaluate the disease caused by a high consequence pathogen.

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Immunology and Infection

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology
J. Kyle Bohannon 1, Krisztina Janosko 1, Michael R. Holbrook 1, Jason Barr 1, Daniela Pusl 1, Laura Bollinger 1, Linda Coe 1, Lisa E. Hensley 1, Peter B. Jahrling 1, Jiro Wada 1, Jens H. Kuhn 1, Matthew G. Lackemeyer 1
1Integrated Research Facility at Frederick, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH)

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

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Medicine

A Step by Step Protocol for Subretinal Surgery in Rabbits
Sami Al-Nawaiseh 1, Fabian Thieltges 1, Zengping Liu 1,2, Claudine Strack 1, Ralf Brinken 1, Norbert Braun 3, Marc Wolschendorf 3, Arvydas Maminishkis 5, Nicole Eter 4, Boris V. Stanzel 1,6
1Department of Ophthalmology, University of Bonn, 2Department of Ophthalmology, National University of Singapore, 3Geuder AG, 4Department of Ophthalmology, University of Münster, 5Section on Epithelial and Retinal Physiology and Disease, National Eye Institute/National Institutes of Health, 6Surgical Retina Department, Singapore National Eye Centre

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

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Neuroscience

Analyzing Dendritic Morphology in Columns and Layers
Chun-Yuan Ting 1, Philip G. McQueen 2, Nishith Pandya 3, Evan S. McCreedy 3, Matthew McAuliffe 3, Chi-Hon Lee 1
1Section on Neuronal Connectivity, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (NIH), 2Mathematical and Statistical Computing Laboratory, Center for Information Technology, National Institutes of Health (NIH), 3Biomedical Imaging Research Services Section, Center for Information Technology, National Institutes of Health (NIH)

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

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Behavior

Reducing State Anxiety Using Working Memory Maintenance
Nicholas L. Balderston 1, Abigail Hsiung 1, Jeffrey Liu 1, Monique Ernst 1, Christian Grillon 1
1Section on Neurobiology of Fear and Anxiety, National Institute of Mental Health, National Institutes of Health (NIH)

This protocol demonstrates how to measure anxiety-potentiated startle during the Sternberg Working Memory paradigm.

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JoVE Journal

New Methods to Study Gustatory Coding
Alejandra Boronat-García *1, Sam Reiter *1,2, Kui Sun 1, Mark Stopfer 1
1National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), 2Max Planck Institute for Brain Research

We present three new methods to study gustatory coding. Using a simple animal, the moth Manduca sexta (Manduca), we describe a dissection protocol, the use of extracellular tetrodes to record the activity of multiple gustatory receptor neurons, and a system for delivering and monitoring precisely timed pulses of tastants.

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JoVE Journal

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies
Xuesong Feng *1, Faiza Naz *2, Aster H. Juan 1, Stefania Dell'Orso 2, Vittorio Sartorelli 1
1Laboratory of Muscle Stem Cells and Gene Regulation, National Institutes of Health, 2Office of Science and Technology, National Institute of Arthritis, Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

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Cancer Research

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay
Yukitomo Arao 1, Kenneth S. Korach 1
1Reproductive Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health (NIH)

We present a method for analyzing the 4-hydroxy-tamoxifen-dependent estrogen receptor alpha ligand-binding domain dimerization activity using the mammalian two-hybrid assay.

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Cancer Research

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions
Ayşegül Doğan 1, Selami Demirci 2, Hüseyin Apdik 1, Ezgi Avşar Apdik 1, Fikrettin Şahin 1
1Genetics and Bioengineering, Yeditepe University, 2National Heart, Lung, and Blood Institute (NHLBI), Sickle Cell Branch, National Institutes of Health (NIH)

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

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Biochemistry

A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput
Nasir Malik 1, Rohini Manickam 1, Muznabanu Bachani 1, Joseph P. Steiner 1
1National Institute of Neurological Disorders and Stroke (NINDS), Neurotherapeutic Development Unit (NTDU), National Institutes of Health (NIH)

It is often necessary to assess the potential cytotoxicity of a set of compounds on cultured cells. Here, we describe a strategy to reliably screen for toxic compounds in a 96-well format.

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Chemistry

Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium
Kiyoharu J. Miyagishima 1, Congxiao Zhang *2, Volha V. Malechka *3, Kapil Bharti 2, Wei Li 1
1Retinal Neurophysiology Section, National Eye Institute, National Institutes of Health, 2Ocular and Stem Cell Translational Research Section, National Eye Institute, National Institutes of Health, 3Human Visual Function Core, National Eye Institute, National Institutes of Health

Here, we present a method for recording light-evoked electrical responses of the retinal pigment epithelium (RPE) in mice using a technique known as DC-ERGs first described by Marmorstein, Peachey, and colleagues in the early 2000s.

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Developmental Biology

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle
Giulia Riparini 1, James M. Simone 2, Vittorio Sartorelli 1
1Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis, Musculoskeletal, and Skin Diseases (NIAMS), National Institutes of Health (NIH), 2Flow Cytometry Section, National Institute of Arthritis, Musculoskeletal, and Skin Diseases (NIAMS), National Institutes of Health (NIH)

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

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Biology

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis
Davide Ortolan 1, Andrei Volkov 1, Arvydas Maminishkis 1, Ruchi Sharma 1, Kapil Bharti 1
1Ocular and Stem Cell Translational Research Section, National Eye Institute, National Institutes of Health (NIH)

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

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Biology

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium
Zander Esh *1, Sharanya Suresh *1, Davide Ortolan 1, Mitra Farnoodian 1, Devika Bose 1, Jiwon Ryu 1, Andrei Volkov 1, Kapil Bharti 1, Ruchi Sharma 1
1Ocular and Stem Cell Translational Research Section, National Eye Institute, National Institutes of Health (NIH)

Degenerative eye diseases that affect the retinal pigment epithelium layer of the eye have monogenic and polygenic origins. Several disease models and a software application, LipidUNet, have been developed to study mechanisms of disease, as well as potential therapeutic interventions.

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Immunology and Infection

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils
Shuichiro Nakabo 1, Mariana J. Kaplan 1, Sarthak Gupta 1
1Systemic Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health (NIH)

We present an automated high-throughput method to quantify neutrophil extracellular traps (NETs) utilizing the live cell analysis system, coupled with a membrane permeability-dependent dual-dye approach.

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