National Institutes of Health

139 articles published in JoVE

• Biology

  
  Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Yuki X. Chen*^1,5,6, Kelly Veerasammy*^1,2, Jun Yin*³, Tenzin Choetso^1,4, Tiffany Zhong⁷, Muniyat A. Choudhury^1,4,5, Cory Weng^1,4,5, Ethan Xu⁸, Mayan A. Hein^9,10, Rinat Abzalimov², Ye He^{1 1}Advanced Science Research Center, Neuroscience Initiative, the City University of New York, Graduate Center New York, ²Advanced Science Research Center, Structural Biology Initiative, the City University of New York, Graduate Center, ³National Institute of Neurological Disorders and Stroke, National Institutes of Health, ⁴The City College of New York, CUNY, ⁵Macaulay Honors College, CUNY, ⁶Graduate School of Public Health and Health Policy, The City University of New York, ⁷Princeton University, ⁸Ardrey Kell High School, ⁹The Borough of Manhattan Community College, CUNY, ¹⁰Gallatin School of Individualized Study, New York University The aim of this protocol is to provide detailed guidance on the proper sample preparation for lipid and metabolite analysis in small tissues, such as the Drosophila brain, using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging.

• Biochemistry

  
  Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry Matthew Payea¹, Myriam Gorospe¹, Nathan Basisty^{2 1}Laboratory of Genetics and Genomics, National Institute on Aging Intramural Research Program, National Institutes of Health, ²Translational Gerontology Branch, National Institute on Aging Intramural Research Program, National Institutes of Health This protocol describes the workflow for metabolic labeling of senescent and non-dividing cells with pulsed SILAC, untargeted mass spectrometry analysis, and a streamlined calculation of protein half-lives.

• Neuroscience

  
  Acquisition of Resting-State Functional Magnetic Resonance Imaging Data in the Rat Diana J. Wallin*^1,2, Emily D. K. Sullivan*^1,2, Elise M. Bragg¹, Jibran Y. Khokhar^2,4, Hanbing Lu^2,3, Wilder T. Doucette^{1,2 1}Dartmouth-Hitchcock Medical Center, ²Geisel School of Medicine at Dartmouth, ³National Institute on Drug Abuse, National Institutes of Health, ⁴University of Guelph This protocol describes a method for obtaining stable resting-state functional magnetic resonance imaging (rs-fMRI) data from a rat using low dose isoflurane in combination with low dose dexmedetomidine.

• Neuroscience

  
  Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples Ke Jiang¹, Jacob Nellissery¹, Anand Swaroop^{1 1}Neurobiology, Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

• Biology

  
  Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements Ji Won Park*¹, Samantha M. Thomas*¹, Lee J. Wylie², Andrew M. Jones², Anni Vanhatalo², Alan N. Schechter¹, Barbora Piknova^{1 1}Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, ²Sport and Health Sciences, College of Life and Environmental Sciences, St Luke’s Campus, University of Exeter We present protocols for three different methods for the homogenization of four different muscle groups of rat skeletal muscle tissue to measure and compare the levels of nitrate and nitrite. Furthermore, we compare different sample weights to investigate whether tissue sample size affects the results of homogenization.

• Biochemistry

  
  Polysome Profiling without Gradient Makers or Fractionation Systems Mack Sobhany¹, Robin E. Stanley^{1 1}Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Department of Health and Human Services, National Institutes of Health This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

• Biochemistry

  
  Rapid Determination of Antibody-Antigen Affinity by Mass Photometry Di Wu¹, Grzegorz Piszczek^{1 1}Biophysics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

• Biochemistry

  
  Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays Ananya Tripathi¹, Charles Bond^1,2, James R. Sellers¹, Neil Billington¹, Yasuharu Takagi^{1 1}Laboratory of Molecular Physiology, Cell and Developmental Biology Center, National Heart, Lung and Blood Institute, National Institutes of Health, ²Department of Physiology, Perelman School of Medicine, University of Pennsylvania Presented here is a procedure to express and purify myosin 5a followed by a discussion of its characterization, using both ensemble and single molecule in vitro fluorescence microscopy-based assays, and how these methods can be modified for the characterization of nonmuscle myosin 2b.

• Genetics

  
  Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis Isabelle Louradour¹, Kashinath Ghosh¹, Ehud Inbar¹, David L. Sacks¹, Channa Aluvihare², Robert A. Harrell II^{2 1}Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, ²University of Maryland Insect Transformation Facility, The Institute for Bioscience and Biotechnology Research This protocol details the steps of CRISPR/Cas9 targeted mutagenesis in sand flies: embryo collection, injection, insect rearing, and identification as well as selection of mutations of interest.

• Biochemistry

  
  Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology Sayuri L. Higashi^1,2, Kazuya Yagyu^1,2, Haruna Nagase^1,2, Craig S. Pearson¹, Herbert M. Geller¹, Yasuhiro Katagiri^{1 1}Laboratory of Developmental Neurobiology, Cell and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, ²United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University An ultra-high-speed western blotting technique is developed by improving the kinetics of antigen-antibody binding through cyclic draining and replenishing (CDR) technology in conjunction with an immunoreaction enhancing agent.

• Neuroscience

  
  In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity Matthew J. Fischl¹, Catherine J. C. Weisz^{1 1}Section on Neuronal Circuitry, National Institute on Deafness and Other Communication Disorders, NIH Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

• Chemistry

  
  Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium Kiyoharu J. Miyagishima¹, Congxiao Zhang*², Volha V. Malechka*³, Kapil Bharti², Wei Li^{1 1}Retinal 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.

• Immunology and Infection

  
  Analysis of Human Natural Killer Cell Metabolism Javier Traba^1,2, Thomas A. Waldmann³, Olga M. Anton^{3 1}Cardiovascular Branch, National Heart, Lung and Blood Institute, National Institutes of Health, ²Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), ³Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health In this paper, we describe a method to measure glycolysis and mitochondrial respiration in primary human Natural Killer (NK) cells isolated from peripheral blood, at rest or following IL15-induced activation. The protocol described could be easily extended to primary human NK cells activated by other cytokines or soluble stimuli.

• Developmental Biology

  
  Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging Russell H. Knutsen¹, Leah M. Gober¹, Joseph R. Sukinik¹, Danielle R. Donahue², Elise K. Kronquist¹, Mark D. Levin¹, Sean E. McLean³, Beth A. Kozel^{1 1}Translational Vascular Medicine Branch, National Heart Lung and Blood Institute, National Institutes of Health, ²Mouse Imaging Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, ³Division of Pediatric Surgery, Department of Surgery, University of North Carolina at Chapel Hill The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

• Biochemistry

  
  Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates Monica C. Pillon¹, Robin E. Stanley^{1 1}Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Department of Health and Human Services, National Institutes of Health This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

• Cancer Research

  
  Measuring the Motor Aspect of Cancer-Related Fatigue using a Handheld Dynamometer Li Rebekah Feng¹, Jeniece Regan¹, Joseph Shrader², Josephine Liwang¹, Sarah Alshawi¹, Jamell Joseph², Alexander Ross¹, Leorey Saligan^{1 1}National Institute of Nursing Research, National Institutes of Health, ²Clinical Center Rehabilitation Medicine, National Institutes of Health Simple and accessible methods were developed to measure the motor aspect of cancer-related fatigue objectively and quantitatively. We describe, in detail, ways to administer the physical fatigue test using a simple handgrip device as well as methods to calculate fatigue indices.

• Bioengineering

  
  Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells Angela Ballesteros¹, Kenton J. Swartz^{1 1}Molecular Physiology and Biophysics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health Here, we present a method for visualizing the uptake of 3 kDa Texas Red-labeled dextran in auditory hair cells with functional mechanotransduction channels. In addition, dextrans of 3–10 kDa can be used to study endocytosis in hair and supporting cells of the organ of Corti.

• Developmental Biology

  
  Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells Kilsoo Jeon¹, Kyeyoon Park², Anton M. Jetten^{1 1}Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, ²NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

• Immunology and Infection

  
  Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods Xin Hu¹, Jaimee R. Compton², Patricia M. Legler^{2 1}National Center for Advancing Translational Sciences, National Institutes of Health, ²United States Naval Research Laboratory We present a general protocol for identifying short stretches of homologous host-pathogen protein sequences (SSHHPS) embedded in the viral polyprotein. SSHHPS are recognized by viral proteases and direct the targeted destruction of specific host proteins by several Group IV viruses.

• Developmental Biology

  
  A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development Renat N. Khaliullin^1,2,3, Jeffrey M. Hendel^1,2, Adina Gerson-Gurwitz^1,2, Shaohe Wang^1,4,5, Stacy D. Ochoa^1,6, Zhiling Zhao^1,7, Arshad Desai^1,2, Karen Oegema^1,2, Rebecca A. Green^{1,2 1}Ludwig Institute for Cancer Research, San Diego, ²Department of Cellular and Molecular Medicine, University of California, San Diego, ³Recursion Pharmaceuticals, ⁴Biomedical Sciences Graduate Program, University of California, San Diego, ⁵Cell Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, ⁶Department of Biology, San Diego State University, ⁷Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80–100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

• Neuroscience

  
  Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons Chengyu Sheng¹, Uzma Javed¹, Justin Rosenthal¹, Jun Yin¹, Bo Qin¹, Quan Yuan^{1 1}National Institute of Neurological Disorders and Stroke, National Institutes of Health Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

• Biochemistry

  
  A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput Nasir Malik¹, Rohini Manickam¹, Muznabanu Bachani¹, Joseph P. Steiner^{1 1}National 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.

• Biochemistry

  
  A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors Laszlo Radnai^1,2, Rebecca F. Stremel^1,2, James R. Sellers³, Gavin Rumbaugh², Courtney A. Miller^{1,2 1}Department of Molecular Medicine, The Scripps Research Institute, ²Department of Neuroscience, The Scripps Research Institute, ³Laboratory of Molecular Physiology, NHLBI, National Institutes of Health A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 µL per well. The platform should be applicable to virtually any ADP producing enzyme.

• Neuroscience

  
  Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo R. Michelle Saré¹, Anita Torossian¹, Michael Rosenheck¹, Tianjian Huang¹, Carolyn Beebe Smith^{1 1}Section on Neuroadaptation and Protein Metabolism, National Institute of Mental Health, National Institutes of Health Protein synthesis is a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-¹⁴C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

• Developmental Biology

  
  Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution Leighton H. Duncan^1,5, Mark W. Moyle^1,5, Lin Shao^1,5, Titas Sengupta^1,5, Richard Ikegami^1,5, Abhishek Kumar^4,5, Min Guo^4,5, Ryan Christensen^4,5, Anthony Santella^2,5, Zhirong Bao^2,5, Hari Shroff^4,5, William Mohler^3,5, Daniel A. Colón-Ramos^{1,5,6 1}Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine, ²Developmental Biology Program, Sloan Kettering Institute, ³Department of Genetics and Genome Sciences and Center for Cell Analysis and Modeling, University of Connecticut Health Center, ⁴Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, ⁵WormGUIDES.org, ⁶Instituto de Neurobiología, Recinto de Ciencias Médicas, Universidad de Puerto Rico Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

• Bioengineering

  
  Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts Armel N. Femnou^1,2, Abigail Giles¹, Robert S. Balaban^{1 1}Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, ²Department of Biomedical Engineering, The George Washington University Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

• Medicine

  
  Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using ¹⁸F-FDG PET/CT Katherine Kim¹, Shan Huang², Laura A. Fletcher¹, Alana E. O'Mara¹, Ilan Tal³, Robert J. Brychta¹, Aaron M. Cypess¹, Kong Y. Chen¹, Brooks P. Leitner^{1 1}Diabetes, Endocrinology, and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, ²National Cancer Institute, National Institutes of Health, ³Division of Nuclear Medicine and Molecular Imaging, Department of Radiology, Beth Israel Deaconess Medical Center, Harvard Medical School Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using ¹⁸F-FDG PET/CT.

• Medicine

  
  Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation Sirada Srihirun^1,2, Alan N. Schechter², Barbora Piknova^{2 1}Department of Pharmacology, Faculty of Dentistry, Mahidol University, ²Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

• Cancer Research

  
  Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions Ayşegül Doğan¹, Selami Demirci², Hüseyin Apdik¹, Ezgi Avşar Apdik¹, Fikrettin Şahin^{1 1}Genetics 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.

• Immunology and Infection

  
  Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture Andrea Introini^1,2, Christophe Vanpouille², Wendy Fitzgerald², Kristina Broliden¹, Leonid Margolis^{2 1}Department of Medicine Solna, Center for Molecular Medicine, Karolinska University Hospital, Karolinska Institutet, ²Section of Intercellular Interactions, Eunice Shriver National Institute of Child Health and Human Development, National Institutes of Health Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

• Behavior

  
  Chronic Sleep Deprivation in Mouse Pups by Means of Gentle Handling Abigail Lemons¹, R. Michelle Saré¹, Carolyn Beebe Smith^{1 1}Section on Neuroadaptation and Protein Metabolism, National Institute of Mental Health, National Institutes of Health We describe a sleep deprivation technique known as gentle handling where investigators gently prod mice any time sleep behavior is observed. This method is a powerful tool that allows researchers to study the effects that chronic sleep restriction throughout development can have on future brain physiology and behavior.

• Developmental Biology

  
  Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome Yang Jo Chung¹, Ghanwa Khawaja¹, Karen M. Wolcott², Peter D. Aplan^{1 1}Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, ²LGI Flow Cytometry Core Facility, Center for Cancer Research, National Cancer Institute, National Institutes of Health We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

• Immunology and Infection

  
  Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4⁺ T Cells Isolated from Rhesus macaques Andrey Tokarev¹, Matthew Creegan¹, Michael A. Eller¹, Mario Roederer², Diane L. Bolton^{1 1}US Military HIV Research Program, Henry M. Jackson Foundation, Walter Reed Army Institute of Research, ²Vaccine Research Center, NIAID, NIH Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4⁺ T cells isolated from rhesus macaques.

• Genetics

  
  Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents Naemeh Pourshafie¹, Philip R. Lee², Ke-lian Chen¹, George G. Harmison¹, Laura C. Bott^1,3, Kenneth H. Fischbeck¹, Carlo Rinaldi^{1,4 1}Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, ²Section on Nervous System Development and Plasticity, The Eunice Kennedy Shriver National Institute of Child and Human Development, National Institutes of Health, ³Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University, ⁴Department of Physiology, Anatomy and Genetics, University of Oxford Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

• Neuroscience

  
  Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains Giulia Quattrocolo¹, Maria Isaac², Yajun Zhang², Timothy J. Petros^{2 1}Kavli Institute for Systems Neuroscience and Centre for Neural Computation, Norwegian University of Science and Technology, ²Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

• Cancer Research

  
  Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue Li Rebekah Feng¹, Quang Nguyen¹, Alexander Ross¹, Leorey N. Saligan^{1 1}National Institute of Nursing Research, National Institutes of Health Our goal was to develop a practical protocol to evaluate mitochondrial dysfunction associated with fatigue in cancer patients. This innovative protocol is optimized for clinical use involving only standard phlebotomy and basic laboratory procedures.

• Developmental Biology

  
  Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies Xuesong Feng*¹, Faiza Naz*², Aster H. Juan¹, Stefania Dell'Orso², Vittorio Sartorelli^{1 1}Laboratory 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.

• Behavior

  
  Noninvasive, High-throughput Determination of Sleep Duration in Rodents R. Michelle Saré¹, Abigail Lemons¹, Anita Torossian¹, Carolyn Beebe Smith^{1 1}Section on Neuroadaptation and Protein Metabolism, National Institute of Mental Health, National Institutes of Health We describe a high-throughput method of measuring sleep by means of activity-based home-cage monitoring. This method offers advantages over traditional EEG-based methods. It is well validated for the determination of total sleep duration and can be a powerful tool to monitor sleep in rodent models of human disease.

• Developmental Biology

  
  Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution Tomoko Yamazaki^1,2, Wenling Li¹, Yoh-Suke Mukouyama^{1 1}Laboratory of Stem Cell and Neuro-Vascular Biology, Genetics and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, ²Earle A. Chiles Research Institute, Robert W. Franz Cancer Center, Providence Portland Medical Center Here, we describe a high resolution whole-mount imaging method in the entire adult mouse ear skin, which enables us to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution.

• Cancer Research

  
  Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay Michael M. Lizardo^1,2, Poul H. Sorensen^{2,3 1}Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, ²BC Cancer Agency, Provincial Health Services Authority, ³Department of Pathology and Laboratory Medicine, University of British Columbia The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

• Medicine

  
  Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse Kevin Isgrig¹, Wade W. Chien^{1,2 1}National Institute on Deafness and Other Communication Disorders, National Institutes of Health, ²Department of Otolaryngology-Head & Neck Surgery, Johns Hopkins School of Medicine In this study, we describe the posterior semicircular canal approach as a reliable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear.

• Genetics

  
  Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome Vid Leko^1,2, Smitha Sripathy¹, Robin L. Adrianse¹, Taylor Loe¹, Angela Park¹, Uyen Lao¹, Eric J. Foss¹, Marisa S. Bartolomei³, Antonio Bedalov^{1,4 1}Clinical Research Division, Fred Hutchinson Cancer Research Center, ²Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, ³Epigenetics Program, Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, ⁴Departments of Medicine and Biochemistry, University of Washington We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

• Immunology and Infection

  
  Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence Riti Sharan*¹, Hee-Jeong Yang*², Preeti Sule¹, Jeffrey D. Cirillo^{1 1}Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, ²Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

• Developmental Biology

  
  Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations Yuho Kim^1,2,3, Jonathan M. Memme^1,2, David A. Hood^{1,2 1}Muscle Health Research Centre, York University, ²School of Kinesiology and Health Science, York University, ³National Heart, Lung, and Blood Institute, National Institutes of Health This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

• Immunology and Infection

  
  Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry Nermi L. Parrow*¹, Pierre-Christian Violet*², Hongbin Tu², James Nichols³, Corinne A. Pittman⁴, Courtney Fitzhugh⁴, Robert E. Fleming^1,5, Narla Mohandas⁶, John F. Tisdale³, Mark Levine^{2 1}Department of Pediatrics, Saint Louis University School of Medicine, ²Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, ³Molecular and Clinical Hematology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, ⁴Sickle Cell Branch, National Heart, Lung and Blood Institute, National Institutes of Health, ⁵Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, ⁶Red Cell Physiology Laboratory, New York Blood Center Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

• Biochemistry

  
  Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae Yu-Hua Lo¹, Monica C. Pillon¹, Robin E. Stanley^{1 1}Signal Transduction Laboratory, National Institutes of Environmental Health Sciences, Department of Health and Human Services, National Institutes of Health This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

• Cancer Research

  
  Using the Dot Assay to Analyze Migration of Cell Sheets Christina H. Stuelten^{1 1}Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

• Medicine

  
  In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice Evandro F. Fang^1,6, Konstantinos Palikaras², Nuo Sun³, Elayne M. Fivenson¹, Ryan D. Spangler⁴, Jesse S. Kerr¹, Stephanie A. Cordonnier¹, Yujun Hou¹, Eszter Dombi⁵, Henok Kassahun⁶, Nektarios Tavernarakis^2,7, Joanna Poulton⁵, Hilde Nilsen⁶, Vilhelm A. Bohr^{1,8 1}Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, ²Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology - Hellas, ³Center for Molecular Medicine, National Heart Lung and Blood Institute, National Institutes of Health, ⁴Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, ⁵Nuffield Department of Obstetrics and Gynaecology, University of Oxford, ⁶Department of Clinical Molecular Biology, University of Oslo and Akershus University Hospital, ⁷Department of Basic Sciences, Faculty of Medicine, University of Crete, ⁸Danish Center for Healthy Aging, University of Copenhagen Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

• Bioengineering

  
  Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System Anjali Nandal^1,2, Barbara Mallon³, Bhanu P. Telugu^{1,2,4 1}Department of Animal and Avian Sciences, University of Maryland, ²Animal Bioscience and Biotechnology Laboratory, ARS, USDA, ³NIH Stem Cell Unit, Bethesda, National Institutes of Health, ⁴RenOVAte Biosciences Inc This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

• Biology

  
  Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection Kangsun Yun^{1 1}Cancer and Developmental Biology Laboratory, National Cancer Institute, National Institutes of Health Methylene blue dye injection into the renal pelvis facilitates the assessment of urinary tract junction obstruction defects during mouse embryonic urinary tract development. Here, a protocol for methylene blue dye injection into the renal pelvis is described.

• Medicine

  
  A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor Karen J. Keeran*¹, Kenneth R. Jeffries¹, Arthur D. Zetts¹, Joni Taylor¹, Shawn Kozlov¹, Timothy J. Hunt*^{1 1}Animal Surgery and Resources Core, National Heart, Lung, and Blood Institute, National Institutes of Health The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

• Behavior

  
  Reducing State Anxiety Using Working Memory Maintenance Nicholas L. Balderston¹, Abigail Hsiung¹, Jeffrey Liu¹, Monique Ernst¹, Christian Grillon^{1 1}Section 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.

• Developmental Biology

  
  Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method Jason P. Stanko¹, Suzanne E. Fenton^{1 1}National Toxicology Program Laboratory, Division of the National Toxicology Program, National Institute of Environmental Health Sciences Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

• Neuroscience

  
  New Methods to Study Gustatory Coding Alejandra Boronat-García*¹, Sam Reiter*^1,2, Kui Sun¹, Mark Stopfer^{1 1}National 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.

• Immunology and Infection

  
  Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM Jason Yi¹, Asit Manna¹, Valarie A. Barr¹, Jennifer Hong², Keir C. Neuman², Lawrence E. Samelson^{1 1}Laboratory of Cellular & Molecular Biology, National Cancer Institute, National Institutes of Health, ²Laboratory of Single Molecule Biophysics, National Heart, Lung, and Blood, Institute, National Institutes of Health We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

• Genetics

  
  Single Molecule Analysis of Laser Localized Psoralen Adducts Jing Huang¹, Himabindu Gali¹, Julia Gichimu¹, Marina A. Bellani¹, Durga Pokharel¹, Manikandan Paramasivam¹, Michael M. Seidman^{1 1}Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

• Neuroscience

  
  Analyzing Dendritic Morphology in Columns and Layers Chun-Yuan Ting¹, Philip G. McQueen², Nishith Pandya³, Evan S. McCreedy³, Matthew McAuliffe³, Chi-Hon Lee^{1 1}Section 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.

• Cancer Research

  
  A Mouse Model of Fatigue Induced by Peripheral Irradiation Brian S. Wolff¹, Michael A. Renner¹, Danielle A. Springer², Leorey N. Saligan^{1 1}Symptom Biology Unit, National Institute of Nursing Research, National Institutes of Health, ²Murine Phenotyping Core, National Heart, Lung, and Blood Institute, National Institutes of Health We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

• Engineering

  
  3D Printing of Biomolecular Models for Research and Pedagogy Eduardo Da Veiga Beltrame¹, James Tyrwhitt-Drake², Ian Roy³, Raed Shalaby⁴, Jakob Suckale⁴, Daniel Pomeranz Krummel^{5 1}Department of Physics, Brandeis University, ²Bioinformatics and Computational Biosciences Branch (BCBB), NIH/NIAID/OD/OSMO/OCICB, ³Library/LTS/MakerLab, Brandeis University, ⁴Interfaculty Institute of Biochemistry (IFIB), University of Tübingen, ⁵Winship Cancer Institute, Emory University School of Medicine Physical models of biomolecules can facilitate an understanding of their structure-function for the researcher, aid in communication between researchers, and serve as an educational tool in pedagogical endeavors. Here, we provide detailed guidance for the 3D printing of accurate models of biomolecules using fused filament fabrication desktop 3D printers.

• Behavior

  
  Feeding Experimentation Device (FED): Construction and Validation of an Open-source Device for Measuring Food Intake in Rodents Katrina P. Nguyen¹, Mohamed A. Ali¹, Timothy J. O'Neal¹, Ilona Szczot¹, Julia A. Licholai^1,2, Alexxai V. Kravitz^{1,3 1}National Insttitute of Diabetes and Digestive and Kidney Diseases, ²National Center for Complementary and Integrative Health, ³National Institute on Drug Abuse, National Institutes of Health Feeding Experimentation Device (FED) is an open-source device for measuring food intake in mice. FED can also synchronize food intake measurements with other techniques via a real-time digital output. Here, we provide a step-by-step tutorial for the construction, validation, and usage of FED.

• Behavior

  
  A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training Eric Augier¹, Russell S. Dulman², Erick Singley², Markus Heilig^{1 1}Center for Social and Affective Neuroscience, IKE, Linköping University, ²Laboratory of Clinical and Translational Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health This protocol describes a novel and efficient method to quickly initiate operant responding for ethanol in rats that, contrary to standard methods, does not require water deprivation or saccharin/sucrose fading to initiate responding.

• Immunology and Infection

  
  Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles Anush Arakelyan¹, Wendy Fitzgerald¹, Sonia Zicari¹, Murad Vagida², Jean-Charles Grivel³, Leonid Margolis^{1 1}Section on Intercellular Interactions, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, ²Laboratory of Atherothrombosis, Cardiology Department, Moscow State University of Medicine and Dentistry, ³Sidra Medical and Research Center Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

• Genetics

  
  Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform Nicolaas H. Fourie¹, Ralph M. Peace^1,2, Sarah K. Abey¹, LeeAnne B. Sherwin¹, John W. Wiley³, Wendy A. Henderson^{1 1}Digestive Disorders Unit, National Institute of Nursing Research, National Institutes of Health, DHHS, ²National Institutes of Health Research Scholar, Howard Hughes Medical Institute, ³Internal Medicine, Medical School, University of Michigan We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

• Immunology and Infection

  
  An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes Javier Traba*¹, Pietro Miozzo*², Billur Akkaya³, Susan K. Pierce², Munir Akkaya^{2 1}Laboratory of Mitochondrial Biology and Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, ²Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, ³Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

• Genetics

  
  In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining Zhe Chen¹, Hong Xu^{1 1}Lab of Molecular Genetics, National Heart, Lung and Blood Institute, National Institutes of Health Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2´-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

• Immunology and Infection

  
  Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices Steven Mazur¹, Michael R. Holbrook¹, Tracey Burdette¹, Nicole Josleyn¹, Jason Barr¹, Daniela Pusl¹, Laura Bollinger¹, Linda Coe¹, Peter B. Jahrling¹, Matthew G. Lackemeyer¹, Jiro Wada¹, Jens H. Kuhn¹, Krisztina Janosko^{1 1}Integrated 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.

• 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 Janosko¹, Michael R. Holbrook¹, Ricky Adams¹, Jason Barr¹, Laura Bollinger¹, Je T'aime Newton², Corrie Ntiforo², Linda Coe¹, Jiro Wada¹, Daniela Pusl¹, Peter B. Jahrling¹, Jens H. Kuhn¹, Matthew G. Lackemeyer^{1 1}Integrated 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.

• Immunology and Infection

  
  Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 4. Medical Imaging Procedures Russell Byrum¹, Lauren Keith¹, Christopher Bartos¹, Marisa St. Claire¹, Matthew G. Lackemeyer¹, Michael R. Holbrook¹, Krisztina Janosko¹, Jason Barr¹, Daniela Pusl¹, Laura Bollinger¹, Jiro Wada¹, Linda Coe¹, Lisa E. Hensley¹, Peter B. Jahrling¹, Jens H. Kuhn¹, Margaret R. Lentz^{1 1}Integrated 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.

• Immunology and Infection

  
  Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology J. Kyle Bohannon¹, Krisztina Janosko¹, Michael R. Holbrook¹, Jason Barr¹, Daniela Pusl¹, Laura Bollinger¹, Linda Coe¹, Lisa E. Hensley¹, Peter B. Jahrling¹, Jiro Wada¹, Jens H. Kuhn¹, Matthew G. Lackemeyer^{1 1}Integrated 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.

• Immunology and Infection

  
  A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice Jennifer H. Madenspacher¹, Michael B. Fessler^{1 1}Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

• Biology

  
  Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells Xi Wen¹, Tian Jin¹, Xuehua Xu^{1 1}Chemotaxis Signal Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

• Chemistry

  
  From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins Nicholas Noinaj¹, Stephen Mayclin², Ann M. Stanley^2,3, Christine C. Jao^2,3, Susan K. Buchanan^{2 1}Department of Biological Sciences, Markey Center for Structural Biology, Purdue University, ²National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, ³National Institute of General Medical Sciences (NIGMS), National Institutes of Health β-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of β-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

• Immunology and Infection

  
  Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages Xueting Jin¹, Howard S. Kruth^{1 1}Experimental Atherosclerosis Section, National Heart, Lung, and Blood Institute, National Institutes of Health A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

• Medicine

  
  Cutaneous Surgical Denervation: A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease Shelby C. Peterson¹, Isaac Brownell*², Sunny Y. Wong*^{1 1}Dermatology, Cell and Developmental Biology, University of Michigan, ²Dermatology Branch, National Cancer Institute, National Institutes of Health This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount β-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

• Biology

  
  Maintenance of a Drosophila melanogaster Population Cage Juan Manuel Caravaca¹, Elissa P. Lei^{1 1}Laboratory of Cellular and Developmental Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

• Neuroscience

  
  High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses Jun Zhang¹, Ronald S. Petralia², Ya-Xian Wang², Jeffrey S. Diamond^{1 1}Synaptic Physiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, ²Advanced Imaging Core, National Institute on Deafness and Other Communication Disorders, National Institutes of Health The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

• Medicine

  
  Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration Jianhua Xiong¹, Jian Hou^{2 1}Center for Molecular Medicine, National Heart, Lung, and Blood Institute, National Institutes of Health, ²State Key Laboratory for Agrobiotechnology, College of Biological Science, China Agricultural University A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

• Medicine

  
  A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine Mitsuhiro Yoneda¹, Alfredo A. Molinolo², Jerrold M. Ward³, Shioko Kimura¹, Robert A. Goodlad^{4 1}National Cancer Institute, Laboratory of Metabolism, National Institutes of Health, ²National Institute of Dental and Craniofacial Research, Oral and Pharyngeal Cancer Branch, National Institutes of Health, ³Global VetPathology, ⁴Centre for Pathology, Hammersmith Hospital, Imperial College The use of a simple device to cut and ‘roll’ mouse intestines to rapidly prepare whole mount preparations is described.

• Biology

  
  Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP Mark J. Henderson*¹, Emily S. Wires*¹, Kathleen A. Trychta¹, Xiaokang Yan¹, Brandon K. Harvey^{1 1}National Institute on Drug Abuse, National Institutes of Health Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

• Biology

  
  Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification Nathan P. Manes¹, Jessica M. Mann¹, Aleksandra Nita-Lazar^{1 1}Cellular Networks Proteomics Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

• Biology

  
  Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80 Kentaro Ohkuni¹, Yoshimitsu Takahashi¹, Munira A. Basrai^{1 1}Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institute of Health This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

• Medicine

  
  Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay Christopher J. Johnson¹, Christina M. Carlson², Aaron R. Morawski³, Alyson Manthei⁴, Neil R. Cashman^{5 1}USGS National Wildlife Health Center, ²Department of Soil Science, University of Wisconsin–Madison, ³Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, ⁴Merial Veterinary Scholars Program, School of Veterinary Medicine, University of Wisconsin–Madison, ⁵Department of Neurology, University of British Columbia Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

• Developmental Biology

  
  Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor Trevor Cerbini¹, Yongquan Luo¹, Mahendra S. Rao², Jizhong Zou^{1 1}National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, ²Q Therapeutics TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

• Medicine

  
  Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model Jing Feng¹, Yvonne Fitz¹, Yan Li¹, Melinda Fernandez¹, Irene Cortes Puch¹, Dong Wang¹, Stephanie Pazniokas², Brandon Bucher³, Xizhong Cui¹, Steven B. Solomon^{1 1}Critical Care Medicine Department, Clinical Center, National Institutes of Health, ²Harvard Apparatus, ³ADInstruments Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

• Developmental Biology

  
  Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells Tongguang Wang¹, Elliot Choi¹, Maria Chiara G. Monaco², Eugene O. Major², Marie Medynets¹, Avindra Nath^{1 1}Translational Neuroscience Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, ²Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, National Institutes of Health A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

• Biology

  
  Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies Matthias Garten¹, Sophie Aimon², Patricia Bassereau¹, Gilman E. S. Toombes^{3 1}Institut Curie, Centre de Recherche, CNRS, UMR 168, PhysicoChimie Curie, Université Pierre et Marie Curie, ²Kavli Institute for Brain and Mind, University of California, San Diego, ³Molecular Physiology and Biophysics Section, National Institute for Neurological Disorders and Stroke, National Institute of Health The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods — electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

• Medicine

  
  Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling Jerome D. Robin¹, Woody E. Wright¹, Yaqun Zou², Stacy A. Cossette³, Michael W. Lawlor³, Emanuela Gussoni^{4 1}Department of Cell Biology, UT Southwestern Medical Center, ²National Institute of Neurological Disorders and Stroke, National Institute of Health, ³Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, ⁴ This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

• Developmental Biology

  
  Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation Khujista D. Haque¹, Atul K. Pandey¹, Matthew W. Kelley², Chandrakala Puligilla^{1 1}Department of Pathology and Laboratory Medicine, Medical University of South Carolina, College of Medicine, ²Laboratory of Cochlear Development, NIDCD, NIH We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

• Behavior

  
  Functional Near Infrared Spectroscopy of the Sensory and Motor Brain Regions with Simultaneous Kinematic and EMG Monitoring During Motor Tasks Theresa Sukal-Moulton¹, Ana Carolina de Campos¹, Christopher J. Stanley¹, Diane L. Damiano^{1 1}Functional and Applied Biomechanics Section, Rehabilitation Medicine Department, Clinical Center, National Institutes of Health Monitoring brain activity during upright motor tasks is of great value when investigating the neural source of movement disorders. Here, we demonstrate a protocol that combines functional near infrared spectroscopy with continuous monitoring of muscle and kinematic activity during 4 types of motor tasks.

• Immunology and Infection

  
  Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes Thomas Hoenen¹, Ari Watt¹, Anita Mora², Heinz Feldmann^{1 1}Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, ²Research Technology Branch, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

• Biology

  
  Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis Kevin G. Chen¹, Rebecca S. Hamilton¹, Pamela G. Robey², Barbara S. Mallon^{1 1}NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, ²Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

• Biology

  
  SIVQ-LCM Protocol for the ArcturusXT Instrument Jason D. Hipp*¹, Jerome Cheng*², Jeffrey C. Hanson*¹, Avi Z. Rosenberg¹, Michael R. Emmert-Buck¹, Michael A. Tangrea¹, Ulysses J. Balis^{2 1}Laboratory of Pathology, National Cancer Institute, National Institutes of Health, ²Department of Pathology, University of Michigan SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings.

• Neuroscience

  
  Live Imaging of Drosophila Larval Neuroblasts Dorothy A. Lerit¹, Karen M. Plevock¹, Nasser M. Rusan^{1 1}National Heart, Lung, and Blood Institute, National Institutes of Health This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

• Immunology and Infection

  
  Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking Olga Pavlova¹, Raffaele Ieva¹, Harris D Bernstein^{1 1}Genetics and Biochemistry Branch of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution “snapshots” of an ordered assembly pathway in a living cell.

• Medicine

  
  Dual-phase Cone-beam Computed Tomography to See, Reach, and Treat Hepatocellular Carcinoma during Drug-eluting Beads Transarterial Chemo-embolization Vania Tacher¹, MingDe Lin², Nikhil Bhagat¹, Nadine Abi Jaoudeh³, Alessandro Radaelli⁴, Niels Noordhoek⁴, Bart Carelsen⁴, Bradford J. Wood³, Jean-François Geschwind^{1 1}Russell H. Morgan Department of Radiology and Radiological Science, Division of Vascular and Interventional Radiology, The Johns Hopkins Hospital, ²Clinical Informatics, Interventional, and Translational Solutions (CIITS), Philips Research North America, ³Center for Interventional Oncology, Interventional Radiology Section, National Institutes of Health, ⁴Interventional X-ray, Philips Healthcare Dual-phase cone-beam computed tomography (DP-CBCT) is a useful intraprocedural imaging technique for transarterial chemo-embolization treatment with drug-eluting beads of hepatocellular carcinoma. DP-CBCT has been used to perform three major steps in oncologic interventional radiology: tumor localization (see), navigation and intraprocedural catheter guidance (reach), and intraprocedural evaluation of treatment success (treat).

• Neuroscience

  
  Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis Natalia B. Pivovarova¹, S. Brian Andrews^{1 1}Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health This paper describes the application of cryoanalytical electron microscopy to the quantitative measurement of total calcium content and distribution at subcellular resolution in physiologically defined biological specimens.

• Medicine

  
  Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung Yan Cai¹, Shioko Kimura^{1 1}Laboratory of Metabolism, National Cancer Institute, National Institutes of Health The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

• Biology

  
  Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins Andrius Masedunskas^1,2, Natalie Porat-Shliom¹, Muhibullah Tora¹, Oleg Milberg^1,3, Roberto Weigert^{1 1}Intracellular Membrane Trafficking Unit, Oral and Pharyngeal Cancer Branch National Institute of Dental and Craniofacial Research, National Institutes of Health, ²Department of Biology, University of North Carolina at Chapel Hill, ³Department of Chemical & Biochemical Engineering and Department of Biomedical Engineering, Rutgers University Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

• Chemistry

  
  Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity Akira Sassa¹, William A. Beard¹, David D. Shock¹, Samuel H. Wilson^{1 1}Laboratory of Structural Biology, NIEHS, National Institutes of Health Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

• Neuroscience

  
  Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents Annalisa Scimemi¹, Jeffrey S. Diamond^{1 1}Synaptic Physiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

• Behavior

  
  Simultaneous Scalp Electroencephalography (EEG), Electromyography (EMG), and Whole-body Segmental Inertial Recording for Multi-modal Neural Decoding Thomas C. Bulea^1,2, Atilla Kilicarslan², Recep Ozdemir^2,3,4, William H. Paloski^3,4, Jose L. Contreras-Vidal^{2,4,5 1}Functional and Applied Biomechanics Group, National Institutes of Health, ²Laboratory for Non-invasive Brain-Machine Interface Systems, Department of Electrical and Computer Engineering, University of Houston, ³Department of Health and Human Performance, University of Houston, ⁴Center for Neuromotor & Biomechanics Research, University of Houston, ⁵Department of Biomedical Engineering, University of Houston Development of an effective brain-machine-interface (BMI) system for restoration and rehabilitation of bipedal locomotion requires accurate decoding of user's intent. Here we present a novel experimental protocol and data collection technique for simultaneous non-invasive acquisition of neural activity, muscle activity, and whole-body kinematics during various locomotion tasks and conditions.

• Neuroscience

  
  In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons Vania Y. Cao^1,2, Yizhou Ye¹, Surjeet S. Mastwal¹, David M. Lovinger³, Rui M. Costa⁴, Kuan H. Wang^{1 1}Unit on Neural Circuits and Adaptive Behaviors, Genes Cognition and Psychosis Program, National Institute of Mental Health, ²Department of Neuroscience, Brown University - National Institutes of Health Graduate Partnership Program, ³Section on Synaptic Pharmacology, Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, ⁴Champalimaud Neuroscience Programme, Champalimaud Center for the Unknown Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

• Neuroscience

  
  Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain Maria Chiara G. Monaco¹, Dragan Maric², Alexandra Bandeian¹, Emily Leibovitch¹, Wan Yang¹, Eugene O. Major^{1 1}Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, National Institutes of Health, ²Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

• Neuroscience

  
  Ex vivo Culturing of Whole, Developing Drosophila Brains Ranjini Prithviraj^1,2, Svetlana Trunova^1,2, Edward Giniger^{1,2 1}National Institute of Neurological Disorders and Stroke, ²National Human Genome Research Institute, National Institutes of Health, Bethesda, MD This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

• Neuroscience

  
  Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation Emilie Pacary¹, Matilda A. Haas¹, Hendrik Wildner¹, Roberta Azzarelli¹, Donald M. Bell², Djoher Nora Abrous³, François Guillemot^{1 1}Division of Molecular Neurobiology, MRC National Institute for Medical Research, ²Confocal and Image Analysis Laboratory, National Institute for Medical Research, ³Physiopathologie de la plasticité neuronale, Neurocentre Magendie, Université de Bordeaux This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

• Biology

  
  Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal Xiaofeng Zheng¹, Guang Hu^{1 1}Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

• Neuroscience

  
  Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes Dvir Blivis¹, Michael J. O'Donovan^{1 1}Developmental Neurobiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

• Neuroscience

  
  Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection Carlene S. Brandon¹, Christina Voelkel-Johnson², Lindsey A. May³, Lisa L. Cunningham^{3 1}Department of Pathology and Laboratory Medicine, Medical University of South Carolina, ²Department of Microbiology & Immunology, Medical University of South Carolina, ³National Institute on Deafness and Other Communication Disorders, National Institutes of Health Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles.

• Immunology and Infection

  
  A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins Travis J. Crites¹, Lirong Chen¹, Rajat Varma^{1 1}Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

• Immunology and Infection

  
  Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers Kathleen C. Prins*^1,2, Gaia Vasiliver-Shamis*^2,3, Michael Cammer^1,2, David Depoil^1,2, Michael L. Dustin², Catarina E. Hioe^{1,4 1}Department of Pathology, New York University Langone School of Medicine, ²Program in Molecular Pathogenesis, Marty and Helen Kimmel Center for Biology and Medicine and Skirball Institute for Biomolecular Medicine, ³Laboratory of Molecular Immunogenetics, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, ⁴Veteran Affairs New York Harbor Healthcare System This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

• Medicine

  
  Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model Tamalee R. Kramp¹, Kevin Camphausen^{1 1}Radiation Oncology Branch, National Cancer Institute The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

• 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. White¹, Donald Bliss³, Amy Moran³, Alberto Bartesaghi¹, Mario J. Borgnia¹, M. Jason V. de la Cruz¹, David Schauder¹, Lisa M. Hartnell¹, Rachna Nandwani^1,4, Moez Dawood⁵, Brianna Kim⁶, Jun Hong Kim⁷, John Sununu⁸, Lisa Yang⁹, Siddhant Bhatia¹⁰, Carolyn Subramaniam¹, Darrell E. Hurt¹¹, Laurent Gaudreault¹², Sriram Subramaniam^{1 1}Laboratory 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.

• Biology

  
  Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry Jaime M. Ross^{1,2 1}Department of Neuroscience, Karolinska Institutet, ²National Institute on Drug Abuse (NIDA) The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

• Biology

  
  Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum Xuehua Xu¹, Tian Jin^{1 1}Chemotaxis Signal Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

• Biology

  
  Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans Lynn Boyd¹, Connie Hajjar¹, Kevin O'Connell^{2 1}Department of Biological Sciences, University of Alabama in Huntsville, ²NIDDK-National Institutes of Health This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

• Medicine

  
  A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study Zhongshu Tang¹, Fan Zhang¹, Yang Li¹, Pachiappan Arjunan¹, Anil Kumar¹, Chunsik Lee¹, Xuri Li^{1 1}National Eye Institute The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

• Biology

  
  Competitive Genomic Screens of Barcoded Yeast Libraries Andrew M. Smith*^1,2, Tanja Durbic*^2,3, Julia Oh*⁴, Malene Urbanus^1,2, Michael Proctor⁵, Lawrence E. Heisler^2,3, Guri Giaever^2,6, Corey Nislow^{1,2,3 1}Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, ²Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, ³Donnelly Sequencing Centre, University of Toronto, ⁴Genetics and Molecular Biology Branch, National Human Genome Research Institute, NIH, ⁵Stanford Genome Technology Center, Stanford School of Medicine, Stanford University, ⁶Department of Pharmaceutical Sciences, University of Toronto We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

• Medicine

  
  An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth Dalit Barkan¹, Jeffrey E. Green^{2 1}Department of Biology, University of Haifa, ²Transgenic Oncogenesis and Genomics Section, Laboratory of Cancer Biology and Genetics, National Cancer Institute A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

• Neuroscience

  
  Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures Dietmar Plenz¹, Craig V. Stewart¹, Woodrow Shew¹, Hongdian Yang¹, Andreas Klaus¹, Tim Bellay^{1 1}Section on Critical Brain Dynamics, National Institute of Mental Health A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

• Neuroscience

  
  Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals Sang Beom Jun^1,2, Verginia Cuzon Carlson¹, Stephen Ikeda³, David Lovinger^{1 1}Section on Synaptic Pharmacology/Laboratory for Integrative Neuroscience, National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism, ²Department of Electronics Engineering, Ewha Womans University, ³Section on Transmitter Signaling/Laboratory of Molecular Physiology, National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

• Biology

  
  Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo Wenling Li¹, Yoh-suke Mukouyama^{1 1}Laboratory of Stem Cell and Neuro-Vascular Biology, Genetics and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

• Medicine

  
  Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct Yusuke Kuriki¹, Younan Liu¹, Dengsheng Xia¹, Eva M. Gjerde¹, Saeed Khalili¹, Brennan Mui¹, Changyu Zheng², Simon D. Tran^{1 1}Faculty of Dentistry, McGill University, ²National Institutes of Health, Bethesda, MD, USA A protocol for the cannulation of the mouse submandibular salivary gland via the Wharton's duct is described. For this experiment, the trypan blue solution is used as a dyer to demonstrate how this technique effectively delivers infusions into the targeted gland, and to suggest the reliability of this new approach as a potential clinical drug/cell therapy for the regeneration of salivary glands.

• Neuroscience

  
  An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival Zhongshu Tang¹, Shuihua Zhang^1,2, Chunsik Lee¹, Anil Kumar¹, Pachiappan Arjunan¹, Yang Li¹, Fan Zhang¹, Xuri Li^{1 1}National Eye Institute, NIH, ²Ophthalmology Department, The Second Hospital of Harbin Medical University This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

• Immunology and Infection

  
  Particle Agglutination Method for Poliovirus Identification Minetaro Arita¹, Souji Masujima², Takaji Wakita¹, Hiroyuki Shimizu^{1 1}Department of Virology II, National Institute of Infectious Diseases, ²New Product Design Department, Fujirebio Inc. A recently developed novel particle agglutination (PA) assay utilizing virus receptor molecule allowed a rapid and easy identification of poliovirus (PV). In this article, we will show the procedure for the PA assay for PV identification.

• Neuroscience

  
  Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons Chan-Ying Zheng¹, Ronald S. Petralia¹, Ya-Xian Wang¹, Bechara Kachar^{1 1}National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

• Biology

  
  Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture Abraham Kim¹, German Kilimnik¹, Charles Guo¹, Joshua Sung¹, Junghyo Jo², Vipul Periwal², Piotr Witkowski³, Philip Dilorio⁴, Manami Hara^{1 1}Department of Medicine, University of Chicago, ²Laboratory of Biological Modeling, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, ³Department of Surgery, University of Chicago, ⁴Diabetes Division, University of Massachusetts Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

• Medicine

  
  Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1β using a Whole Blood Stimulation Assay Barbara Yang*¹, Tuyet-Hang Pham*², Raphaela Goldbach-Mansky², Massimo Gadina^{1 1}Translational Immunology Section, Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, ²Translational Autoinflammatory Disease Section, Office of the Clinical Director, National Institute of Arthritis and Musculoskeletal and Skin Diseases We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

• Medicine

  
  A Novel Technique of Rescuing Capsulorhexis Radial Tear-out using a Cystotome Shah M. R. Karim*¹, Chin T. Ong*², Mizanur R. Miah³, Tamsin Sleep⁴, Abdul Hanifudin^{1 1}Department of Ophthalmology, Hairmyres Hospital, NHS Lanarkshire, ²Department of Ophthalmology, Royal Devon and Exeter NHS Foundation Trust, ³National Institute of Ophthalmology, ⁴Department of Ophthalmology, South Devon Healthcare NHS Trust Capsulorhexis is an important step in phacoemulsification surgery. A surgeon creates a continous curvilinear tear on the anterior lens capsule by controlling the tearing vector forces. A peripherally extended tear is a serious complication. This video demonstrates a novel technique of rescuing capsular radial tear out using a cystotome.

• Immunology and Infection

  
  Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites Sittiporn Pattaradilokrat¹, Jian Li^1,2, Xin-zhuan Su^{1 1}National Institute of Allergy and Infectious Diseases, National Institutes of Health, ²School of Life Science, Xiamen University Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

• Immunology and Infection

  
  Non-invasive Imaging of Leukocyte Homing and Migration in vivo Baomei Wang¹, Bernd H. Zinselmeyer^1,2, Jeremiah R. McDole¹, Peggy A. Gieselman¹, Mark J. Miller^{1 1}Department of Pathology and Immunology, Washington University in St. Louis, ²National Institute of Neurological Disorders and Stroke, NINDS, NIH - National Institute of Health Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

• Neuroscience

  
  Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology Arvydas Maminishkis¹, Sheldon S. Miller^{1 1}National 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.

• Neuroscience

  
  DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices Gail K. Seabold¹, James B. Daunais², Andrew Rau³, Kathleen A. Grant³, Veronica A. Alvarez^{1 1}Section on Neuronal Structure, Laboratory for Integrative Neuroscience, NIAAA, NIH, ²Department Physiology and Pharmacology, Wake Forest University Health Sciences, ³Oregon National Primate Research Center, Division of Neuroscience, Oregon Health and Science University We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

• Biology

  
  Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System Monika Aggarwal¹, Robert M. Brosh Jr.^{1 1}Laboratory of Molecular Gerontology, National Institute on Aging, NIH Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

• Biology

  
  Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons Van B. Lu¹, Damian J. Williams¹, Yu-Jin Won¹, Stephen R. Ikeda^{1 1}Laboratory 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.

• Biology

  
  Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell Andrea Balbo¹, Huaying Zhao¹, Patrick H. Brown¹, Peter Schuck^{1 1}National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Dynamics of Macromolecular Assembly, Laboratory of Bioengineering and Physical Science The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

• Biology

  
  Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS) Peter D. Burbelo¹, Kathryn H. Ching¹, Caitlin M. Klimavicz¹, Michael J. Iadarola^{1 1}Neurobiology and Pain Therapeutics Section, National Institute of Dental and Craniofacial Research, National Institutes of Health The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

• Biology

  
  Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation Peggy S. Zelenka¹, Chun Y. Gao¹, Senthil S. Saravanamuthu^{1 1}Laboratory of Molecular and Developmental Biology, National Eye Institute (NEI), National Institutes of Health (NIH) Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

• Biology

  
  Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio) Jin Liang^1,2, Shawn M. Burgess^{1 1}Genome Technology Branch, National Human Genome Research Institute, ²Neuroscience and Cognitive Science Program, University of Maryland The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

• Biology

  
  Methods for Patch Clamp Capacitance Recordings from the Calyx Kenneth Paradiso¹, Wei Wu¹, Ling-Gang Wu^{1 1}National Institute of Neurological Disorders and Stroke, National Institute of Health We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.