National Institutes of Health View Institution's Website 151 articles published in JoVE Medicine Rodent Model of Intestinal Ischemia-Reperfusion Injury via Occlusion of the Superior Mesenteric Artery Lucie Henein1,2, Randy Clevenger1, Karen Keeran1, Lauren Brinster3 1Animal Surgery and Resources Core, National Heart, Lung, and Blood Institute, National Institutes of Health, 2College of Veterinary Medicine, Mississippi State University, 3Division of Veterinary Resources, Office of Research Services, National Institutes of Health We describe how to generate a widely used surgical model of intestinal ischemia-reperfusion injury (IRI) in rodents. The procedure involves occlusion of the superior mesenteric artery followed by the restoration of blood flow. This model is useful for studies investigating occlusive causes of intestinal IRI in both veterinary and human medicine. Cancer Research Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia Gisele O. L. Rodrigues1, WenQing Li1, Sarah D. Cramer1,2,3, Hila Y. Winer1, Tu Chun Hsu1,2,3, Timothy Gower4, Julie A. Hixon1, Scott K. Durum1 1Cytokines and Immunity Section, Cancer Innovation Laboratory, National Cancer Institute, National Institutes of Health, 2Comparative Biomedical Scientist Training Program, NIH, 3Department of Pathobiology and Diagnostic Investigation, Veterinary Diagnostic Laboratory, Michigan State University, 4NCI-Frederick Laboratory Animal Sciences Program This protocol describes the isolation of double-negative thymocytes from the mouse thymus followed by retroviral transduction and co-culture on the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4) for further functional analysis. Biochemistry Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications Yilun Sun1 1Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs. Biology Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining Dillon Strepay1, Rafal Olszewski1, Ian Taukulis1, J. Dixon Johns1, Shoujun Gu1, Michael Hoa1 1Auditory Development and Restoration Program, National Institute on Deafness and Other Communication Disorders, National Institutes of Health The stria vascularis is vital to the generation of endocochlear potential. Here, we present the dissection of the adult mouse stria vascularis for single-nucleus sequencing or immunostaining. Biology Assessment of Chemical Toxicity in Adult Drosophila Melanogaster Jessica M. Holsopple*1,2, Shannon R. Smoot*1, Ellen M. Popodi1,2, John K. Colbourne3, Joseph R. Shaw4, Brian Oliver5, Thomas C. Kaufman1, Jason M. Tennessen1 1Department of Biology, Indiana University, 2Bloomington Drosophila Stock Center, Department of Biology, Indiana University, 3School of Biosciences, University of Birmingham, 4O'Neill School of Public and Environmental Affairs, Indiana University, 5Section of Developmental Genomics, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Kidney and Digestive Diseases, National Institutes of Health This protocol describes an efficient and inexpensive method that uses liquid media to assess the effects of chemical toxicants on the viability of adult Drosophila melanogaster. Medicine Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis Smita Joshi*1, Alexis N. Smith1, Kanakanagavalli Shravani Prakhya1, Hammodah R. Alfar1, Joshua Lykins1, Ming Zhang1, Irina Pokrovskaya2, Maria Aronova3, Richard D. Leapman3, Brian Storrie2, Sidney W. Whiteheart*1 1Department of Molecular and Cellular Biochemistry, University of Kentucky, 2Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences, 3Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health The present protocol describes how to use a FeCl3-mediated injury to induce arterial thrombosis, and how to collect and prepare arterial injury samples at various stages of thrombosis for electron microscopy analysis. Bioengineering Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies Amanda D. East1, James E. T. Gebo1, Anna F. Lau1 1Sterility Testing Service, Department of Laboratory Medicine, Clinical Center, National Institutes of Health The protocol summarizes the best practices to minimize microbial bioburden in a cleanroom environment and includes strategies such as environmental monitoring, process monitoring, and product sterility testing. It is relevant for manufacturing and testing facilities that are required to meet current good tissue practice standards and current good manufacturing practice standards. Immunology and Infection Gene Editing of Primary Rhesus Macaque B Cells Harald Hartweger1, Rajeev Gautam2, Yoshiaki Nishimura2, Fabian Schmidt3,5, Kai-Hui Yao1, Amelia Escolano1,6, Mila Jankovic1, Malcolm A. Martin2, Michel C. Nussenzweig1,4 1Laboratory of Molecular Immunology, The Rockefeller University, 2Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 3Laboratory of Retrovirology, The Rockefeller University, 4Howard Hughes Medical Institute, The Rockefeller University, 5Laboratory of Applied Virology and Precision Medicine, King Abdullah University of Science and Technology (KAUST), 6Vaccine and Immunotherapy Center, Wistar Institute We present a method for culturing and gene editing primary rhesus macaque B cells using CRISPR/Cas9 and recombinant adeno-associated virus serotype 6 for the study of B cell therapies. Environment Visualizing Field Data Collection Procedures of Exposure and Biomarker Assessments for the Household Air Pollution Intervention Network Trial in India Karthikeyan D. Rajamani1, Sankar Sambandam1, Krishnendu Mukhopadhyay1, Naveen Puttaswamy1, Gurusamy Thangavel1, Durairaj Natesan1, Rengaraj Ramasamy1, Saritha Sendhil1, Amudha Natarajan1, Vigneswari Aravindalochan1, Ajay Pillarisetti2, Michael Johnson3, Joshua Rosenthal*4, Kyle Steenland5, Ricardo Piedhrahita3, Jennifer Peel6, Maggie L. Clark6, Dana Boyd Barr5, Sarah Rajkumar6, Bonnie Young6, Shirin Jabbarzadeh7, Ghislaine Rosa8, Miles Kirby9, Lindsay J. Underhill10, Anaite Diaz-Artiga11, Amy Lovvorn5, William Checkley12, Thomas Clasen5, Kalpana Balakrishnan1 1Department of Environmental Health Engineering, ICMR Center for Advanced Research on Air Quality, Climate and Health, Faculty of Public Health, Sri Ramachandra Institute of Higher Education and Research (Deemed University), 2Division of Environmental Health Sciences, University of California, Berkeley, 3Berkeley Air Monitoring Group, 4Division of International Epidemiology and Population Studies, National Institutes of Health, 5Gangarosa Department of Environmental Health, Rollins School of Public Health, Emory University, 6Department of Environmental and Radiological Health Sciences, Colorado State University, 7Department of Biostatistics and Informatics, Rollins School of Public Health, Emory University, 8Department of Disease Control, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, 9Department of Global Health & Population, Harvard, T.H. Chan School of Public Health, 10Cardiovascular Division, Washington University School of Medicine, Washington University, 11Centro de Estudios en Salud, Universidad del Valle de Guatemala, 12Division of Pulmonary and Critical Care, School of Medicine, Johns Hopkins University We detail the consistent, high-quality procedures used throughout air and biological sampling processes at Indian field sites during a large randomized controlled trial. Insights gathered from the oversight of applications of innovative technologies, adapted for exposure assessment in rural regions, enable better field data collection practices with more reliable outcomes. 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 Choetso1,4, Tiffany Zhong7, Muniyat A. Choudhury1,4,5, Cory Weng1,4,5, Ethan Xu8, Mayan A. Hein9,10, Rinat Abzalimov2, Ye He1 1Advanced 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 Basisty2 1Laboratory 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. Genetics Reusable Single Cell for Iterative Epigenomic Analyses Hidetaka Ohnuki1, David J. Venzon2, Alexei Lobanov3,4, Giovanna Tosato1 1Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2Biostatistics and Data Management Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 3CCR Collaborative Bioinformatics Resource (CCBR), Center for Cancer Research, National Cancer Institute, National Institutes of Health, 4Advanced Biomedical Computational Science, Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute The present protocol describes a single-cell method for iterative epigenomic analyses using a reusable single cell. The reusable single cell allows analyses of multiple epigenetic marks in the same single cell and statistical validation of the results. Bioengineering High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues Ravi Lokwani1, Kaitlyn Sadtler1 1Section on Immuno-Engineering, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis. 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. Khokhar2,4, Hanbing Lu2,3, Wilder T. Doucette1,2 1Dartmouth-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 Swaroop1 1Neurobiology, 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 Piknova1 1Molecular 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. Biology Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion Jing Zhang*1, Jing Huang*2, Ishani Majumdar1, Ryan C. James3, Julia Gichimu1, Manikandan Paramasivam4, Durga Pokharel5, Himabindu Gali6, Marina A. Bellani1, Michael M Seidman1 1Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, 2Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, 3Department of Molecular Biology and Genetics, Cornell University, 4Department of Cellular and Molecular Medicine, University of Copenhagen, 5Horizon Discovery, 6Boston University School of Medicine While replication fork collisions with DNA adducts can induce double strand breaks, less is known about the interaction between replisomes and blocking lesions. We have employed the proximity ligation assay to visualize these encounters and to characterize the consequences for replisome composition. Biochemistry Polysome Profiling without Gradient Makers or Fractionation Systems Mack Sobhany1, Robin E. Stanley1 1Signal 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 Piszczek1 1Biophysics 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 Bond1,2, James R. Sellers1, Neil Billington1, Yasuharu Takagi1 1Laboratory 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 II2 1Laboratory 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. Higashi1,2, Kazuya Yagyu1,2, Haruna Nagase1,2, Craig S. Pearson1, Herbert M. Geller1, Yasuhiro Katagiri1 1Laboratory 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. Weisz1 1Section 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 Li1 1Retinal Neurophysiology Section, National Eye Institute, National Institutes of Health, 2Ocular and Stem Cell Translational Research Section, National Eye Institute, National Institutes of Health, 3Human Visual Function Core, National Eye Institute, National Institutes of Health Here, we present a method for recording light-evoked electrical responses of the retinal pigment epithelium (RPE) in mice using a technique known as DC-ERGs first described by Marmorstein, Peachey, and colleagues in the early 2000s. Immunology and Infection Analysis of Human Natural Killer Cell Metabolism Javier Traba1,2, Thomas A. Waldmann3, Olga M. Anton3 1Cardiovascular 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. Kozel1 1Translational 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. Stanley1 1Signal 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 Saligan1 1National 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. Swartz1 1Molecular 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. Jetten1 1Immunity, 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. Legler2 1National 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. Khaliullin1,2,3, Jeffrey M. Hendel1,2, Adina Gerson-Gurwitz1,2, Shaohe Wang1,4,5, Stacy D. Ochoa1,6, Zhiling Zhao1,7, Arshad Desai1,2, Karen Oegema1,2, Rebecca A. Green1,2 1Ludwig 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 Yuan1 1National 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. Steiner1 1National Institute of Neurological Disorders and Stroke (NINDS), Neurotherapeutic Development Unit (NTDU), National Institutes of Health (NIH) It is often necessary to assess the potential cytotoxicity of a set of compounds on cultured cells. Here, we describe a strategy to reliably screen for toxic compounds in a 96-well format. Biochemistry A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors Laszlo Radnai1,2, Rebecca F. Stremel1,2, James R. Sellers3, Gavin Rumbaugh2, Courtney A. Miller1,2 1Department 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 Smith1 1Section 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. Duncan1,5, Mark W. Moyle1,5, Lin Shao1,5, Titas Sengupta1,5, Richard Ikegami1,5, Abhishek Kumar4,5, Min Guo4,5, Ryan Christensen4,5, Anthony Santella2,5, Zhirong Bao2,5, Hari Shroff4,5, William Mohler3,5, Daniel A. Colón-Ramos1,5,6 1Department 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. Femnou1,2, Abigail Giles1, Robert S. Balaban1 1Laboratory 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. Leitner1 1Diabetes, 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 Srihirun1,2, Alan N. Schechter2, Barbora Piknova2 1Department 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 Şahin1 1Genetics and Bioengineering, Yeditepe University, 2National Heart, Lung, and Blood Institute (NHLBI), Sickle Cell Branch, National Institutes of Health (NIH) We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction. Immunology and Infection Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture Andrea Introini1,2, Christophe Vanpouille2, Wendy Fitzgerald2, Kristina Broliden1, Leonid Margolis2 1Department 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 Smith1 1Section 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. Aplan1 1Genetics 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. Bolton1 1US 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. Bott1,3, Kenneth H. Fischbeck1, Carlo Rinaldi1,4 1Neurogenetics 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. Petros2 1Kavli 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. Saligan1 1National 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 Sartorelli1 1Laboratory of Muscle Stem Cells and Gene Regulation, National Institutes of Health, 2Office of Science and Technology, National Institute of Arthritis, Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining. Behavior Noninvasive, High-throughput Determination of Sleep Duration in Rodents R. Michelle Saré1, Abigail Lemons1, Anita Torossian1, Carolyn Beebe Smith1 1Section 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 Yamazaki1,2, Wenling Li1, Yoh-Suke Mukouyama1 1Laboratory 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. Lizardo1,2, Poul H. Sorensen2,3 1Pediatric 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. Chien1,2 1National 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 Leko1,2, Smitha Sripathy1, Robin L. Adrianse1, Taylor Loe1, Angela Park1, Uyen Lao1, Eric J. Foss1, Marisa S. Bartolomei3, Antonio Bedalov1,4 1Clinical 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. Cirillo1 1Department 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 Kim1,2,3, Jonathan M. Memme1,2, David A. Hood1,2 1Muscle 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. Fleming1,5, Narla Mohandas6, John F. Tisdale3, Mark Levine2 1Department 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. Stanley1 1Signal 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. Stuelten1 1Laboratory 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. Fang1,6, Konstantinos Palikaras2, Nuo Sun3, Elayne M. Fivenson1, Ryan D. Spangler4, Jesse S. Kerr1, Stephanie A. Cordonnier1, Yujun Hou1, Eszter Dombi5, Henok Kassahun6, Nektarios Tavernarakis2,7, Joanna Poulton5, Hilde Nilsen6, Vilhelm A. Bohr1,8 1Laboratory 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 Nandal1,2, Barbara Mallon3, Bhanu P. Telugu1,2,4 1Department 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 Yun1 1Cancer 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 1Animal 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 Grillon1 1Section on Neurobiology of Fear and Anxiety, National Institute of Mental Health, National Institutes of Health (NIH) This protocol demonstrates how to measure anxiety-potentiated startle during the Sternberg Working Memory paradigm. Developmental Biology Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method Jason P. Stanko1, Suzanne E. Fenton1 1National 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 Stopfer1 1National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), 2Max Planck Institute for Brain Research We present three new methods to study gustatory coding. Using a simple animal, the moth Manduca sexta (Manduca), we describe a dissection protocol, the use of extracellular tetrodes to record the activity of multiple gustatory receptor neurons, and a system for delivering and monitoring precisely timed pulses of tastants. 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. Samelson1 1Laboratory 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. Seidman1 1Laboratory 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 Lee1 1Section on Neuronal Connectivity, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (NIH), 2Mathematical and Statistical Computing Laboratory, Center for Information Technology, National Institutes of Health (NIH), 3Biomedical Imaging Research Services Section, Center for Information Technology, National Institutes of Health (NIH) Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space. Cancer Research A Mouse Model of Fatigue Induced by Peripheral Irradiation Brian S. Wolff1, Michael A. Renner1, Danielle A. Springer2, Leorey N. Saligan1 1Symptom 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 Krummel5 1Department 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. Licholai1,2, Alexxai V. Kravitz1,3 1National 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 Heilig1 1Center 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 Margolis1 1Section 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. Peace1,2, Sarah K. Abey1, LeeAnne B. Sherwin1, John W. Wiley3, Wendy A. Henderson1 1Digestive 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 Akkaya2 1Laboratory 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 Xu1 1Lab 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 Janosko1 1Integrated Research Facility at Frederick, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal. 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. Lackemeyer1 1Integrated Research Facility at Frederick, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), 2Environmental Health and Safety, Biological and Chemical Safety Program, University of Texas Medical Branch Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures. 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. Lentz1 1Integrated Research Facility at Frederick, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) Here, we present an overview of the preparation and animal handling procedures required to safely perform medical imaging in an animal biosafety level 4 laboratory. Computed tomography of a mock-infected guinea pig illustrates these procedures that may be used to evaluate the disease caused by a high consequence pathogen. 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. Lackemeyer1 1Integrated Research Facility at Frederick, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment. 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. Fessler1 1Immunity, 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 Xu1 1Chemotaxis 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. Stanley2,3, Christine C. Jao2,3, Susan K. Buchanan2 1Department 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. Kruth1 1Experimental 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 1Dermatology, 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. Lei1 1Laboratory 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. Diamond1 1Synaptic 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 Hou2 1Center 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. Goodlad4 1National 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. Harvey1 1National 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-Lazar1 1Cellular 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. Basrai1 1Genetics 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. Cashman5 1USGS 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 Zou1 1National 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. Solomon1 1Critical 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 Nath1 1Translational 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. Toombes3 1Institut 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 Gussoni4 1Department 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 Puligilla1 1Department 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. Damiano1 1Functional 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 Feldmann1 1Laboratory 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. Mallon1 1NIH 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. Balis2 1Laboratory 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. Rusan1 1National 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 Bernstein1 1Genetics 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 Geschwind1 1Russell 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 Andrews1 1Laboratory 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 Kimura1 1Laboratory 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 Masedunskas1,2, Natalie Porat-Shliom1, Muhibullah Tora1, Oleg Milberg1,3, Roberto Weigert1 1Intracellular 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. Wilson1 1Laboratory 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. Diamond1 1Synaptic 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. Bulea1,2, Atilla Kilicarslan2, Recep Ozdemir2,3,4, William H. Paloski3,4, Jose L. Contreras-Vidal2,4,5 1Functional 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. Cao1,2, Yizhou Ye1, Surjeet S. Mastwal1, David M. Lovinger3, Rui M. Costa4, Kuan H. Wang1 1Unit 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. Major1 1Laboratory 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 Prithviraj1,2, Svetlana Trunova1,2, Edward Giniger1,2 1National 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 Guillemot1 1Division 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 Hu1 1Laboratory 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'Donovan1 1Developmental 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. Cunningham3 1Department 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 Varma1 1Laboratory 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 Cammer1,2, David Depoil1,2, Michael L. Dustin2, Catarina E. Hioe1,4 1Department 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 Camphausen1 1Radiation 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. Meyerson1,2, Tommi A. White1, Donald Bliss3, Amy Moran3, Alberto Bartesaghi1, Mario J. Borgnia1, M. Jason V. de la Cruz1, David Schauder1, Lisa M. Hartnell1, Rachna Nandwani1,4, Moez Dawood5, Brianna Kim6, Jun Hong Kim7, John Sununu8, Lisa Yang9, Siddhant Bhatia10, Carolyn Subramaniam1, Darrell E. Hurt11, Laurent Gaudreault12, Sriram Subramaniam1 1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2The Medical Research Council Mitochondrial Biology Unit, University of Cambridge, 3National Library of Medicine, National Institutes of Health, 4Massachusetts Institute of Technology, 5William Fremd High School, 6University of Virginia, 7Duke University, 8Yale University, 9University of Notre Dame, 10Washington University in St. Louis, 11Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12Thomas Jefferson High School for Science and Technology The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis. Biology Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry Jaime M. Ross1,2 1Department 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 Jin1 1Chemotaxis 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'Connell2 1Department 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 Li1 1National 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 Urbanus1,2, Michael Proctor5, Lawrence E. Heisler2,3, Guri Giaever2,6, Corey Nislow1,2,3 1Banting 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. Green2 1Department 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 Bellay1 1Section 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 Jun1,2, Verginia Cuzon Carlson1, Stephen Ikeda3, David Lovinger1 1Section 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 Mukouyama1 1Laboratory 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. Tran1 1Faculty 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 Zhang1,2, Chunsik Lee1, Anil Kumar1, Pachiappan Arjunan1, Yang Li1, Fan Zhang1, Xuri Li1 1National 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 Shimizu1 1Department 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 Kachar1 1National 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 Hara1 1Department 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 Gadina1 1Translational 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 Hanifudin1 1Department 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 Li1,2, Xin-zhuan Su1 1National 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. Zinselmeyer1,2, Jeremiah R. McDole1, Peggy A. Gieselman1, Mark J. Miller1 1Department 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. Miller1 1National Eye Institute, National Institutes of Health We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases. 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. Alvarez1 1Section 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 1Laboratory 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. Ikeda1 1Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism (NIAAA), National Institutes of Health (NIH) Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays. Biology Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell Andrea Balbo1, Huaying Zhao1, Patrick H. Brown1, Peter Schuck1 1National 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. Iadarola1 1Neurobiology 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. Saravanamuthu1 1Laboratory 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 Liang1,2, Shawn M. Burgess1 1Genome 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 Wu1 1National 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.