Northwestern University View Institution's Website 63 articles published in JoVE Neuroscience Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina Shichu Chang1, Wenjin Xu1, Weijia Fan2, John A. McDaniel1, Marta Grannonico1, David A. Miller2, Mingna Liu1, Hao F. Zhang2, Xiaorong Liu1,3,4,5 1Department of Biology, University of Virginia, 2Department of Biomedical Engineering, Northwestern University, 3Department of Ophthalmology, University of Virginia, 4Program in Fundamental Neuroscience, University of Virginia, 5Department of Psychology, University of Virginia The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images. Environment A Complete Method for Evaluating the Performance of Photocatalysts for the Degradation of Antibiotics in Environmental Remediation Bing Wang1, XuXia Zhang1, LeCheng Li1, MengTing Ji1, Zheng Zheng1, ChuanHui Shi1, Zhuo Li1, Hong Hao1 1School of Chemical Engineering, Northwest University Presented here is a protocol to explore a universal set of experimental procedures for comprehensive laboratory evaluation of photocatalysts in the field of environmental purification, using the example of photocatalytic removal of antibiotic organic pollutant molecules from water by phthalocyanine sensitized silver phosphate composites. Biochemistry Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET Chiranjib Banerjee1, Brandon Wey-Hung Liauw1, Reza Vafabakhsh1 1Department of Molecular Biosciences, Northwestern University This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis. Neuroscience Slicing the Embryonic Chicken Auditory Brainstem to Evaluate Tonotopic Gradients and Microcircuits Sandesh Mohan1, Abhijit Roy1, George Ordiway1, Jason Tait Sanchez1,2,3 1Roxelyn and Richard Department of Communication Sciences and Disorders, Northwestern University, 2Knowles Hearing Research Center, Northwestern University, 3Department of Neurobiology, Northwestern University Here we present a protocol for obtaining non-coronal auditory brainstem slices of the chicken embryo for the investigation of tonotopic properties and developmental trajectories within one brainstem slice. These slices include sagittal, horizontal, and horizontal/transverse sections encompassing larger tonotopic regions within an individual slice plane that the traditional coronal sections. Neuroscience Evaluation of Auditory Brainstem Response in Chicken Hatchlings George Ordiway1, Miranda McDonnell1, Sandesh Mohan1, Jason Tait Sanchez1,2,3 1Roxelyn and Richard Pepper Department of Communication Sciences and Disorders, Northwestern University, 2Knowles Hearing Research Center, Northwestern University, 3Department of Neurobiology, Northwestern University We have used standard auditory brainstem response (ABR) techniques and applied them to hatchling chickens, a precocious avian model for auditory function. The protocol outlines animal preparation and ABR acquisition techniques in detail, with steps that could translate to other avian or rodent models. Biology A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes Timothy A. Crombie1, Robyn E. Tanny1, Claire M. Buchanan1, Nicole M. Roberto1, Erik C. Andersen1 1Department of Molecular Biosciences, Northwestern University This protocol can be used to perform large-scale ecological surveys of selfing Caenorhabditis nematodes. The primary advantage of this method is the efficient organization and analysis of ecological and molecular data associated with the nematodes collected from nature. Neuroscience Implantation and Control of Wireless, Battery-free Systems for Peripheral Nerve Interfacing Hongkai Wang1,2, Dom D’Andrea1, Yeon Sik Choi3,4, Yasmine Bouricha1, Grace Wickerson3,4, Hak-Young Ahn3, Hexia Guo3,4, Yonggang Huang3,4,5,6, Milap S. Sandhu7, Sumanas W. Jordan8, John A. Rogers3,4,6,9,10,11,12, Colin K. Franz1,3,13 1Laboratory of Regenerative Rehabilitation, Shirley Ryan AbilityLab, Department of Physical Medicine and Rehabilitation, Northwestern University Feinberg School of Medicine, 2Northwestern University Interdepartmental Neuroscience Program, 3Center for Bio-integrated Electronics, Querrey Simpson Institute for Bioelectronics, Northwestern University, 4Department of Materials Science and Engineering, Northwestern University, 5Department of Civil and Environmental Engineering, Northwestern University, 6Department of Mechanical Engineering, Northwestern University, 7Arms and Hands Lab, Shirley Ryan AbilityLab, Department of Physical Medicine and Rehabilitation, Northwestern University Feinberg School of Medicine, 8Division of Plastic and Reconstructive Surgery, Biologics, Shirley Ryan AbilityLab, Northwestern University, 9Department of Biomedical Engineering, Northwestern University, 10Department of Neurological Surgery, Northwestern University, 11Department of Chemistry, Northwestern University, 12Department of Electrical and Computer Engineering, Northwestern University, 13The Ken and Ruth Davee Department of Neurology, Northwestern University Feinberg School of Medicine This is a protocol for the surgical implantation and operation of a wirelessly powered interface for peripheral nerves. We demonstrate the utility of this approach with examples from nerve stimulators placed on either the rat sciatic or phrenic nerve. Genetics Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations Amanda O. Shaver1, Goncalo J. Gouveia2, Pamela S. Kirby3, Erik C. Andersen4, Arthur S. Edison1,2,3 1Department of Genetics, University of Georgia, 2Department of Biochemistry and Molecular Biology, University of Georgia, 3Complex Carbohydrate Research Center, University of Georgia, 4Department of Molecular Biosciences, Northwestern University To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented. Medicine Obtaining Quality Extended Field-of-View Ultrasound Images of Skeletal Muscle to Measure Muscle Fascicle Length Amy N. Adkins1,4,5, Wendy M. Murray1,2,3,4,5 1Department of Biomedical Engineering, Northwestern University, 2Department of Physical Medicine & Rehabilitation, Northwestern University Feinberg School of Medicine, 3Department of Physical Therapy and Human Movement Sciences, Northwestern University Feinberg School of Medicine, 4Shirley Ryan AbilityLab, 5Edward Hines, Jr. VA Hospital This study describes how to obtain high quality musculoskeletal images using the extended field-of-view ultrasound (EFOV-US) method for the purpose of making muscle fascicle length measures. We apply this method to muscles with fascicles that extend past the field-of-view of common traditional ultrasound (T-US) probes. Developmental Biology Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids Maxwell E Edmonds1, Micah D. Forshee1, Teresa K. Woodruff1 1Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro. Neuroscience Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve Nicholas P. Boyer1, Maite Azcorra1,2, Peter Jung3,4, Anthony Brown1 1Department of Neuroscience, The Ohio State University, 2Present address: Interdepartmental Neuroscience Graduate Program and Department of Neurobiology, Northwestern University, 3Quantitative Biology Institute, Ohio University, 4Department of Physics and Astronomy, Ohio University We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein. Medicine Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat Feibo Zheng1,3, Andy Tully1,2, Kyle M Koss1, Xiaomin Zhang1, Longhui Qiu1, Jiao-Jing Wang1, Bilal A Naved1,4, David Z Ivancic1, James M Mathew1, Jason A Wertheim1, Zheng Jenny Zhang1 1Comprehensive Transplant Center and Department of Surgery, Feinberg School of Medicine, Northwestern University, 2Department of Surgery, University of Illinois at Chicago, 3Department of Surgery, Tianjin Occupational Diseases Precaution and Therapeutic Hospital, 4Department of Biomedical Engineering, McCormick School of Engineering, Northwestern University This protocol presents a robust, reproducible model of vascularized composite allotransplant (VCA) geared toward simultaneous study of immunology and functional recovery. The time invested in meticulous technique in a right mid-thigh hind limb orthotopic transplant with hand sewn vascular anastomoses and neural coaptation yields the ability to study functional recovery. Immunology and Infection Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes Steven Droho1, Carla M. Cuda*2, Jeremy A. Lavine*1 1Department of Ophthalmology, Feinberg School of Medicine, Northwestern University, 2Department of Medicine, Division of Rheumatology, Feinberg School of Medicine, Northwestern University This protocol provides a method to digest whole eyes into a single cell suspension for the purpose of multi-parameter flow cytometric analysis in order to identify specific ocular mononuclear phagocytic populations, including monocytes, microglia, macrophages, and dendritic cells. Immunology and Infection A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections Dinesh Jaishankar1, Cormac Cosgrove1, Ryan J. Deaton2, I. Caroline Le Poole1,3 1Robert Lurie Comprehensive Cancer Center and Department of Dermatology, Feinberg School of Medicine, Northwestern University, 2Department of Pathology, University of Illinois at Chicago, 3Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University We describe a rapid staining method to perform multispectral imaging on frozen tissues. Chemistry Sample Preparation in Quartz Crystal Microbalance Measurements of Protein Adsorption and Polymer Mechanics Gwen E. dePolo*1, Emily Schafer*2, Kazi Sadman1, Jonathan Rivnay2, Kenneth R. Shull1 1Department of Materials Science and Engineering, Northwestern University, 2Department of Biomedical Engineering, Northwestern University The quartz crystal microbalance can provide accurate mass and viscoelastic properties for films in the micron or submicron range, which is relevant for investigations in biomedical and environmental sensing, coatings, and polymer science. The sample thickness influences which information can be obtained from the material in contact with the sensor. Developmental Biology Generation of Multicellular Human Primary Endometrial Organoids Alina R. Murphy*1, Teerawat Wiwatpanit*2, Zhenxiao Lu1, Batzaya Davaadelger1, J. Julie Kim1 1Department of Obstetrics and Gynecology, Northwestern University, 2National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA) A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids. Neuroscience Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury Steven J Schwulst1, Mecca B.A.R. Islam1 1Department of Surgery, Northwestern University Here we describe a protocol for the induction of murine traumatic brain injury via an open-head controlled cortical impact. Neuroscience An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures Andrew M. Chiu1, Levi Barse1, Pavla Hubalkova1,2, Antonio Sanz-Clemente1 1Department of Pharmacology, Feinberg School of Medicine, Northwestern University, 2Department of Cellular Neurophysiology, Institute of Physiology CAS This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes. Medicine A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity Jiao-Jing Wang*1, Longhui Qiu*1, Ramiro Fernandez2, Xin Yi Yeap1, Charlie Xiaoying Lin3, Zheng Jenny Zhang1,2 1Comprehensive Transplant Center, Northwestern University, 2Department of surgery, Feinberg School of Medicine, Northwestern University, 3Weinberg art and science college, Northwestern University This protocol details the surgical steps of a mouse model of vascularized heterotopic spleen transplantation, a technically challenging model that can serve as a powerful tool in studying the fate and longevity of spleen cells, the mechanisms of distinct spleen cell populations in disease progression, and transplant immunity. Immunology and Infection A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis Guillermo J. Ares1, Christie Buonpane1, Carrie Yuan1, Douglas Wood1, Catherine J. Hunter1,2 1Department of Pediatrics, Northwestern University, 2 Enteroids are emerging as a novel model in the study of human disease. The protocol describes how to simulate an enteroid model of human necrotizing enterocolitis using lipopolysaccharide (LPS) treatment of enteroids generated from neonatal tissue. Collected enteroids demonstrate inflammatory changes akin to those seen in human necrotizing enterocolitis. Chemistry Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology Max Z. Levine*1,2, Nicole E. Gregorio*2,3, Michael C. Jewett4,5,6,7, Katharine R. Watts2,3, Javin P. Oza2,3 1Department of Biological Sciences, California Polytechnic State University, San Luis Obispo, 2Center for Applications in Biotechnology, California Polytechnic State University, San Luis Obispo, 3Department of Chemistry and Biochemistry, California Polytechnic State University, 4Department of Chemical and Biological Engineering, Northwestern University, 5Chemistry of Life Processes Institute, Northwestern University, 6Center for Synthetic Biology, Northwestern University, 7Interdisciplinary Biological Sciences Program, Northwestern University This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized. Behavior Defining the Role Of Language in Infants' Object Categorization with Eye-tracking Paradigms Alexander LaTourrette1, Sandra R. Waxman1,2 1Department of Psychology, Northwestern University, 2Institute for Policy Research, Northwestern University Here we present a protocol for familiarization-test paradigms which provide a direct test of infant categorization and help to define the role of language in early category learning. Environment Production and Measurement of Organic Particulate Matter in a Flow Tube Reactor Yue Zhang1,2, Pengfei Liu1, Zhaoheng Gong1, Franz M. Geiger3, Scot T. Martin1,4 1School of Engineering and Applied Sciences, Harvard University, 2Department of Environmental Science and Engineering, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, 3Department of Chemistry, Northwestern University, 4Department of Earth and Planetary Sciences, Harvard University This paper describes the operation procedure for the flow tube reactor and related data collection. It shows the protocols for setting the experiments, recording data and generating the number-diameter distribution as well as the particle mass information, which gives useful information about chemical and physical properties of the organic aerosols. Environment Production and Measurement of Organic Particulate Matter in the Harvard Environmental Chamber Yue Zhang1,2, Zhaoheng Gong1, Suzane de Sa1, Adam P. Bateman1, Yingjun Liu1, Yongjie Li1, Franz M. Geiger3, Scot T. Martin1,4 1School of Engineering and Applied Sciences, Harvard University, 2Department of Environmental Science and Engineering, Gillings School of Global Public Health, University of North Carolina, 3Department of Chemistry, Northwestern University, 4Department of Earth and Planetary Sciences, Harvard University This paper describes operation procedures for the Harvard Environmental Chamber (HEC) and related instrumentation for measuring gaseous and particle species. The environmental chamber is used to produce and study secondary organic species produced from the organic precursors, especially related to atmospheric organic particulate matter. Bioengineering Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation Sean Allen1, Michael Vincent1, Evan Scott1,2 1Interdisciplinary Biological Sciences, Northwestern University, 2Department of Biomedical Engineering, Northwestern University Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation. 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. Immunology and Infection Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies Ariel Finkielsztein1, Lorraine Mascarenhas1, Veronika Butin-Israeli1, Ronen Sumagin1 1Department of Pathology, Feinberg School of Medicine, Northwestern University New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function. Bioengineering Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro Corey M. Rountree1, John B. Troy2, Laxman Saggere1 1Department of Mechanical and Industrial Engineering, University of Illinois at Chicago, 2Department of Biomedical Engineering, Northwestern University This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases. Biology Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis Mohammed A. Amin1, Dileep Varma1 1Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions. Biochemistry Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation Pan Liu1, Tomokazu Souma1, Andrew Zu-Sern Wei1, Xueying Xie3, Xunrong Luo2, Jing Jin1 1Division of Nephrology and Hypertension, and the Center for Kidney Research and Therapeutics at the Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, 2Surgery-Organ Transplantation, Northwestern University Feinberg School of Medicine, 3School of Biological Sciences and Medical Engineering, Southeast University Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients. Developmental Biology In Ovo Electroporation in the Chicken Auditory Brainstem Ting Lu1, Ariel Loren Cohen1, Jason Tait Sanchez1,2,3 1Roxelyn and Richard Pepper Department of Communication Sciences and Disorders, Northwestern University, 2Knowles Hearing Research Center, Northwestern University, 3Department of Neurobiology, Northwestern University Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development. Medicine Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model Dou Yu1, Gina Li1, Maciej S. Lesniak1, Irina V. Balyasnikova1 1Department of Neurological Surgery, Feinberg School of Medicine, Northwestern University Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma. Neuroscience Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells Lauren A. Laboissonniere1, Takuma Sonoda2, Seul Ki Lee2, Jeffrey M. Trimarchi1, Tiffany M. Schmidt2 1Department of Genetics, Development, and Cell Biology, Iowa State University, 2Department of Neurobiology, Northwestern University Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity. Behavior Experience is Instrumental in Tuning a Link Between Language and Cognition: Evidence from 6- to 7- Month-Old Infants' Object Categorization Danielle R. Perszyk1, Sandra R. Waxman1 1Psychology Department, Northwestern University At 3-4 months, listening to human and nonhuman primate vocalizations boosts infants' cognition; by 6 months, only human vocalizations exert this cognitive advantage. We describe an exposure manipulation that reveals the powerful shaping role of experience as infants specify which sounds to link to cognition and which to tune out. Biology Activating Autophagy by Aerobic Exercise in Mice Altea Rocchi1, Congcong He1 1Department of Cell and Molecular Biology, Northwestern University Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice. Biochemistry Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b Ye Han*1, Kyle A. Lyman*1, Matt Clutter2, Gary E. Schiltz3, Quratul-Ain Ismail1, Xiangying Cheng1, Chi-Hao Luan4, Dane M. Chetkovich1,5 1Davee Department of Neurology and Clinical Neurosciences, Feinberg School of Medicine, Northwestern University, 2Center for Molecular Innovation and Drug Discovery, Northwestern University, 3Department of Pharmacology, Feinberg School of Medicine, Northwestern University, 4High Throughput Analysis Laboratory, Department of Molecular Biosciences, Northwestern University, 5Department of Physiology, Feinberg School of Medicine, Northwestern University The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented. Biology Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method Sally M. McFall1, Mário F. Neto1, Jennifer L. Reed1, Robin L. Wagner2 1Center for Innovation in Global Health Technologies (CIGHT), Department of Biomedical Engineering, Northwestern University, 2Pritzker School of Medicine, The University of Chicago We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods. Behavior Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice Carmen Lin1, John Disterhoft1, Craig Weiss1 1Department of Physiology, Northwestern University The preparation presented here for whisker-signaled eyeblink conditioning in head-fixed mice precisely stimulates specific whiskers while allowing mice to ambulate on a cylindrical treadmill. A whisker stimulation conditioned stimulus (CS) paired with a periorbital shock unconditioned stimulus (US) results in reliable associative learning on this apparatus. Chemistry CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis Rafael D. Melani*1,2, Henrique S. Seckler*1, Owen S. Skinner1, Luis H. F. Do Vale1,3, Adam D. Catherman1, Pierre C. Havugimana1, Marcelo Valle de Sousa3, Gilberto B. Domont2, Neil L. Kelleher1, Philip D. Compton1 1Departments of Chemistry and Molecular Biosciences, Chemistry of Life Processes Institute, Proteomics Center of Excellence, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 2Institute of Chemistry, Proteomics Unit, Federal University of Rio de Janeiro, 3Department of Cell Biology, Brazilian Center for Protein Research, Laboratory of Biochemistry and Protein Chemistry, University of Brasilia This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques. Biology DNA Electroporation, Isolation and Imaging of Myofibers Alexis R. Demonbreun1, Elizabeth M. McNally1 1Center for Genetic Medicine, Northwestern University This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure. Engineering Visualizing Hyporheic Flow Through Bedforms Using Dye Experiments and Simulation Susa H. Stonedahl1, Kevin R. Roche2, Forrest Stonedahl3, Aaron I. Packman2 1Engineering and Physical Science, St. Ambrose University, 2Civil and Environmental Engineering, Northwestern University, 3Mathematics and Computer Science, Augustana College This manuscript describes how to create regular bedforms in a flume, visualize flow through the bedforms, and use computer simulations to simulate the hyporheic flow. The computer simulations compare well with the experimental observations. This coupled simulation and experiment is well-suited for both research and educational purposes. Bioengineering Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development Joseph S. Uzarski1,2, Jimmy Su1,2,3,4, Yan Xie1,2, Zheng J. Zhang1,2, Heather H. Ward5, Angela Wandinger-Ness6, William M. Miller7,8, Jason A. Wertheim1,2,3,4,8,9 1Comprehensive Transplant Center, Feinberg School of Medicine, Northwestern University, 2Department of Surgery, Feinberg School of Medicine, Northwestern University, 3Department of Biomedical Engineering, Northwestern University, 4Simpson Querrey Institute for BioNanotechnology in Medicine, Northwestern University, 5Department of Internal Medicine, University of New Mexico HSC, 6Department of Pathology, University of New Mexico HSC, 7Department of Chemical and Biological Engineering, Northwestern University, 8Chemistry of Life Processes Institute, Northwestern University, 9Department of Surgery, Jesse Brown VA Medical Center This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors. Developmental Biology Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart Lisa D. Wilsbacher1,2, Shaun R. Coughlin2 1Feinberg Cardiovascular Research Institute, Northwestern University, 2Cardiovascular Research Institute, University of California, San Francisco Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart. Chemistry Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation Lydia A. Finney1, Qiaoling Jin2 1X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, 2Department of Physics and Astronomy, Northwestern University Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays. Bioengineering Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity Xiaobao Li1, Jisun L. Song2, Alessandro Culotti1, Wei Zhang1, David L. Chopp3, Nanxi Lu1, Aaron I. Packman1 1Department of Civil and Environmental Engineering, Northwestern University, 2Department of Chemical and Biological Engineeering, Northwestern University, 3Department of Applied Mathematics and Engineering Sciences, Northwestern University Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies. Biology Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans Carmen I. Nussbaum-Krammer1, Mário F. Neto1, Renée M. Brielmann1, Jesper S. Pedersen1, Richard I. Morimoto1 1Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena. Immunology and Infection Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis Baobing Zhao1, Yang Mei1, Jing Yang1, Peng Ji1 1Department of Pathology, Northwestern University We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages. Chemistry Synthesis and Characterization of Functionalized Metal-organic Frameworks Olga Karagiaridi1, Wojciech Bury1,2, Amy A. Sarjeant1, Joseph T. Hupp1, Omar K. Farha1,3 1Department of Chemistry, Northwestern University, 2Department of Chemistry, Warsaw University of Technology, 3Department of Chemistry, Faculty of Science, King Abdulaziz University Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges. Biology Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice Keng Jin Lee1, Lyubov Czech1, Gregory B. Waypa1, Kathryn N. Farrow1 1Department of Pediatrics, Northwestern University Feinberg School of Medicine We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation. Medicine An Orthotopic Murine Model of Human Prostate Cancer Metastasis Janet Pavese1, Irene M. Ogden1, Raymond C. Bergan1,2,3 1Department of Medicine, Northwestern University, 2Robert H. Lurie Cancer Center, Northwestern University, 3Center for Molecular Innovation and Drug Discovery, Northwestern University This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans. Medicine A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects Liang Feng1,2, Hui Chen1, Genn Suyeoka1, Xiaorong Liu1,2 1Department of Ophthalmology, Northwestern University, 2Department of Neurobiology, Northwestern University Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test. Neuroscience Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model Jonathan C. Chou1, Stuart D. Rollins1, Amani A. Fawzi1 1Department of Ophthalmology, Northwestern University Feinberg School of Medicine Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels. Immunology and Infection Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis Marsha L Quick1, Joseph D Done1, Praveen Thumbikat1 1Department of Urology, Northwestern University Feinberg School of Medicine Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time. Bioengineering Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography Wei Song*1,2, Qing Wei*1, Shuliang Jiao3, Hao F. Zhang1,4 1Department of Biomedical Engineering, Northwestern University, 2Department of Physics, Harbin Institute of Technology, 3Department of Ophthalmology, University of Southern California, 4Department of Ophthalmology, Northwestern University Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats. Neuroscience Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo Marin Manuel1, C.J. Heckman1 1Department of Physiology, Northwestern University Feinberg School of Medicine This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system. Neuroscience In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development Ben Yang*1, Lauren B. Geary*1, Yong-Chao Ma1,2 1Northwestern University Feinberg School of Medicine, Children's Hospital of Chicago Research Center, 2Departments of Pediatrics, Neurology and Physiology, Northwestern University Feinberg School of Medicine To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation. Medicine Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure Evyn Loucks1, Sara Ahlgren1,2 1Program in Developmental Biology, Children's Memorial Research Center, 2Department of Pediatrics, Northwestern University In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model. Immunology and Infection Colonization of Euprymna scolopes Squid by Vibrio fischeri Lynn M. Naughton1, Mark J. Mandel1 1Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described. Neuroscience Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling Norman Atkins1, Charlie M. Miller1, Joseph R. Owens1, Fred W. Turek1 1Center for Sleep and Circadian Biology, Northwestern University RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method. Neuroscience Analysis of Dendritic Spine Morphology in Cultured CNS Neurons Deepak P. Srivastava1, Kevin M. Woolfrey1, Peter Penzes1,2 1Department of Physiology, Northwestern University Feinberg School of Medicine, 2Department of Psychiatry and Behavioral Sciences, Northwestern University Feinberg School of Medicine Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility. Biology Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells Rita Nokes Cook1, Su Fen Ang*1, Richard Seung-on Kang*1, Heike Fölsch1 1Department of Cell and Molecular Biology, Northwestern University This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique. Bioengineering A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System Ariella Shikanov1, Min Xu2, Teresa K. Woodruff2,3,4, Lonnie D. Shea1,4,5 1Institute for BioNanotechnology in Advanced Medicine, Northwestern University, 2Department of Obstetrics and Gynecology, Northwestern University, Feinberg School of Medicine, 3Center for Reproductive Research, Northwestern University, 4The Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 5Department of Chemical and Biological Engineering, Northwestern University A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion. Biology Methods for the Study of the Zebrafish Maxillary Barbel Elizabeth E. LeClair1, Jacek Topczewski2 1Department of Biological Sciences, DePaul University, 2Children’s Memorial Research Center, Department of Pediatrics, Northwestern University Feinberg School of Medicine The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.