University of Notre Dame View Institution's Website 43 articles published in JoVE Cancer Research Shear Assay Protocol for the Determination of Single-Cell Material Properties Luke J. Holen*1, Killian Onwudiwe*1, Julian Najera1, Maksym Zarodniuk1, John D. Obayemi2, Winston O. Soboyejo2, Meenal Datta1 1Department of Aerospace and Mechanical Engineering, University of Notre Dame, 2Departments of Mechanical and Biomedical Engineering, Worcester Polytechnic Institute This protocol outlines the quantification of the mechanical properties of cancerous and non-cancerous cell lines in vitro. Conserved differences in the mechanics of cancerous and normal cells can act as a biomarker that may have implications in prognosis and diagnosis. Neuroscience Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish James Hentig1,2,3, Kaylee Cloghessy1,2,3, David R. Hyde1,2,3 1Department of Biological Sciences, University of Notre Dame, 2Center for Zebrafish Research, University of Notre Dame, 3Center for Stem Cells and Regenerative Medicine, University of Notre Dame Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition. Neuroscience A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish James Hentig1,2,3, Kaylee Cloghessy1,2,3, Chloe Dunseath2,3, David R. Hyde1,2,3 1Department of Biological Sciences, University of Notre Dame, 2Center for Zebrafish Research, University of Notre Dame, 3Center for Stem Cells and Regenerative Medicine, University of Notre Dame We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI. Bioengineering Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications Megan Levis*1,2, Fernando Ontiveros*3, Jonathan Juan1, Anthony Kavanagh1, Jeremiah J. Zartman1,2 1Department of Chemical and Biomolecular Engineering, University of Notre Dame, 2Bioengineering Graduate Program, University of Notre Dame, 3Biology Department, St. John Fisher College Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational settings. Biochemistry Nitropeptide Profiling and Identification Illustrated by Angiotensin II Shan Feng1,2, Xiaofei Wen3, Xin Lu2,4,5,6 1Mass Spectrometry Core Facility, School of Life Sciences, Westlake University, 2Department of Biological Sciences, University of Notre Dame, 3Department of Urology, Shanghai East Hospital, Tongji University School of Medicine, 4Boler-Parseghian Center for Rare and Neglected Diseases, University of Notre Dame, 5Harper Cancer Research Institute, University of Notre Dame, 6Tumor Microenvironment and Metastasis Program, Indiana University Melvin and Bren Simon Cancer Center Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems. Biochemistry Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum Gustavo Diaz1, Chandler Bridges1, Megan Lucas1, Yong Cheng2, Jeff S. Schorey2, Karen M. Dobos1, Nicole A. Kruh-Garcia1 1Department of Microbiology, Immunology and Pathology, Colorado State University, 2Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization. Developmental Biology Single-cell Photoconversion in Living Intact Zebrafish Lauren Green1,2, Cody J. Smith1,2 1Department of Biological Sciences, University of Notre Dame, 2Center for Stem Cells and Regenerative Medicine, University of Notre Dame Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals. Immunology and Infection Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion James J. Faust1,2, Wayne Christenson3,4,5, Kyle Doudrick6, John Heddleston7, Teng-Leong Chew7, Marko Lampe8, Arnat Balabiyev1,2, Robert Ros3,4,5, Tatiana P. Ugarova1,2 1Center for Metabolic and Vascular Biology, Mayo Clinic, 2Molecular and Cellular Biosciences, School of Life Sciences, Arizona State University, 3Department of Physics, Arizona State University, 4Center for Biological Physics, Arizona State University, 5Biodesign Institute, Arizona State University, 6Department of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame, 7Advanced Imaging Center, HHMI Janelia Research Campus, 8Advanced Light Microscopy Facility, European Molecular Biology Laboratory This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens. Chemistry Total Internal Reflection Absorption Spectroscopy (TIRAS) for the Detection of Solvated Electrons at a Plasma-liquid Interface Hernan E. Delgado1, Paul Rumbach2, David M. Bartels3, David B. Go1,2 1Department of Chemical and Biomolecular Engineering, University of Notre Dame, 2Department of Aerospace and Mechanical Engineering, University of Notre Dame, 3Department of Chemistry and Biochemistry, Notre Dame Radiation Laboratory, University of Notre Dame This article presents a total internal reflection absorption spectroscopy (TIRAS) method for measuring short-lived free radicals at a plasma-liquid interface. In particular, TIRAS is used to identify solvated electrons based on their optical absorbance of red light near 700 nm. Developmental Biology Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence Amanda N. Marra1, Marisa Ulrich1, Audra White1, Meghan Springer1, Rebecca A. Wingert1 1Department of Biological Sciences, University of Notre Dame Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish. Developmental Biology Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo Veronica Akle1, Nathalie Agudelo-Dueñas*1,2, Maria A. Molina-Rodriguez*1, Laurel Brianne Kartchner1,3,4,6, Annette Marie Ruth1,3,5,6, John M. González3, Manu Forero-Shelton2 1Laboratory of Neurosciences and Circadian Rhythms, School of Medicine, Universidad de los Andes, 2Biophysics Group, Department of Physics, Universidad de los Andes, 3Laboratory of Basic Medical Sciences, School of Medicine, Universidad de los Andes, 4Department of Microbiology and Immunology, University of North Carolina, 5Notre Dame Initiative for Global Development, University of Notre Dame, 6USAID Research and Innovation Fellowship program In this protocol, fluorescently labeled T. cruzi were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy. Bioengineering Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1 Merrel T. Holley*1, Neerajha Nagarajan*2, Christian Danielson1, Pinar Zorlutuna*2, Kidong Park*1 1Division of Electrical and Computer Engineering, Louisiana State University, 2Department of Aerospace and Mechanical Engineering, Bioengineering Graduate Program, University of Notre Dame In this two-part study, a biological actuator was developed using highly flexible polydimethylsiloxane (PDMS) cantilevers and living muscle cells (cardiomyocytes), and characterized. The biological actuator was incorporated with a base made of modified PDMS materials to build a self-stabilizing, swimming biorobot. Bioengineering Cardiac Muscle Cell-based Actuator and Self-stabilizing Biorobot - Part 2 Neerajha Nagarajan*1, Merrel T. Holley*2, Christian Danielson2, Kidong Park*2, Pinar Zorlutuna*1 1Department of Aerospace and Mechanical Engineering, Bioengineering Graduate Program, University of Notre Dame, 2Division of Electrical and Computer Engineering, Louisiana State University In this study, a biological actuator and a self-stabilizing, swimming biorobot with functionalized elastomeric cantilever arms are seeded with cardiomyocytes, cultured, and characterized for their biochemical and biomechanical properties over time. Bioengineering Scaled Anatomical Model Creation of Biomedical Tomographic Imaging Data and Associated Labels for Subsequent Sub-surface Laser Engraving (SSLE) of Glass Crystals Aislinn M. Betts*1, Matthew T. McGoldrick*1, Christopher R. Dethlefs1, Justin Piotrowicz2, Tony Van Avermaete1, Jeff Maki2, Steve Gerstler3, W. M. Leevy1,4,5 1Department of Biological Sciences, University of Notre Dame, 2Models Plus Incorporated, 3Saint Joseph Regional Medical Center, 4Harper Cancer Research Institute, University of Notre Dame, 5Notre Dame Integrated Imaging Facility, University of Notre Dame A methodology is described herein for representing anatomical imaging data within crystals. We create scaled three-dimensional models of biomedical imaging data for use in Sub-Surface Laser Engraving (SSLE) of crystal glass. This tool offers a useful complement to computational display or three-dimensionally printed models used within clinical or educational settings. Developmental Biology Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy Manuela Lahne1, Ryne A. Gorsuch1, Craig M. Nelson1,2, David R. Hyde1 1Department of Biological Sciences, University of Notre Dame, 2Department of Neurosurgery, Mayo Clinic Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy. Behavior The Deese-Roediger-McDermott (DRM) Task: A Simple Cognitive Paradigm to Investigate False Memories in the Laboratory Enmanuelle Pardilla-Delgado1, Jessica D. Payne1 1Psychology, University of Notre Dame Here we present the Deese, Roediger and McDermott (DRM) task, a tool to study false memories in the laboratory. Subjects study lists of semantically related words (e.g., nurse, sick, etc.), and later falsely remember an unstudied word (doctor) that represents the gist, or theme, of the word list. Engineering The Measurement of Unsteady Surface Pressure Using a Remote Microphone Probe Yaoyi Guan1, Carl R. Berntsen1, Michael J. Bilka1, Scott C. Morris1 1Department of Aerospace and Mechanical Engineering, University of Notre Dame Here, we present a protocol to measure, with high spatial resolution, the unsteady surface pressure in turbulent flows. This method demonstrates the construction of a remote microphone probe (RMP) and the determination of its frequency-dependent, complex transfer function. An analytical determination of the dynamic response is presented and validated. Immunology and Infection Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells Rebecca A. Flaherty1, Shaun W. Lee1 1Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection. Medicine Quantitation of Intra-peritoneal Ovarian Cancer Metastasis Kyle A. Lewellen*1, Matthew N. Metzinger*1, Yueying Liu1, M. Sharon Stack1 1Department of Chemistry & Biochemistry, Harper Cancer Research Institute, University of Notre Dame Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging. Biology Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury Robert A. McKee1,2, Rebecca A. Wingert1,2 1Center for Zebrafish Research, Department of Biological Sciences, University of Notre Dame, 2Center for Stem Cells and Regenerative Medicine, Department of Biological Sciences, University of Notre Dame Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures. Developmental Biology Microbead Implantation in the Zebrafish Embryo Gary F. Gerlach1, Elvin E. Morales1, Rebecca A. Wingert1 1Department of Biological Sciences, University of Notre Dame The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo. Chemistry Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation Michelle A. Pillers1, Rebecca Shute1, Adam Farchone2, Keenan P. Linder3, Rose Doerfler2, Corey Gavin4, Valerie Goss3, Marya Lieberman1 1Department of Chemistry and Biochemistry, University of Notre Dame, 2Department of Chemical and Biomolecular Engineering, University of Notre Dame, 3Department of Chemistry, Physics, and Engineering Studies, Chicago State University, 4Department of Technology, Ivy Tech Community College, South Bend, Indiana Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated. Biology Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays Nydia Morales-Soto1,2, Morgen E. Anyan1, Anne E. Mattingly1, Chinedu S. Madukoma1, Cameron W. Harvey3, Mark Alber3, Eric Déziel4, Daniel B. Kearns5, Joshua D. Shrout1,2,6 1Department of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame, 2Eck Institute for Global Health, University of Notre Dame, 3Department of Applied and Computational Mathematics and Statistics, University of Notre Dame, 4INRS-Institut Armand-Frappier, 5Department of Biology, Indiana University, 6Department of Biological Sciences, University of Notre Dame Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques. Engineering Preparation and Reactivity of Gasless Nanostructured Energetic Materials Khachatur V. Manukyan1, Christopher E. Shuck2, Alexander S. Rogachev3, Alexander S. Mukasyan2 1Department of Physics, University of Notre Dame, 2Department of Chemical and Biomolecular Engineering, University of Notre Dame, 3Center of Functional Nano-Ceramics, National University of Science and Technology, "MISIS" This protocol describes the preparation of gasless nanostructured energetic materials (Ni+Al, Ta+C, Ti+C) using the short-term high-energy ball milling (HEBM) technique. It also describes a high-speed thermal imaging method to study the reactivity of mechanically fabricated nanocomposites. These protocols can be extended to other reactive nanostructured energetic materials. Biology Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae Xin Zhang*1, Keshava Mysore*2,3, Ellen Flannery3,4, Kristin Michel1, David W. Severson3,4, Kun Yan Zhu5, Molly Duman-Scheel2,3,4 1Division of Biology, Kansas State University, 2Department of Medical and Molecular Genetics, Indiana University School of Medicine, 3Eck Institute for Global Health, University of Notre Dame, 4Department of Biological Sciences, University of Notre Dame, 5Department of Entomology, Kansas State University Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae. Medicine Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG Rashna D. Balsara1,2, Sarah E. Chapman3, Ian M. Sander4, Deborah L. Donahue1, Lucas Liepert4, Francis J. Castellino1,2, W. Matthew Leevy3,4,5 1W. M. Keck Center for Transgene Research, University of Notre Dame, 2Department of Chemistry and Biochemistry, University of Notre Dame, 3Notre Dame Integrated Imaging Facility, University of Notre Dame, 4Department of Biological Sciences, University of Notre Dame, 5Harper Cancer Research Institute, University of Notre Dame Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies. Bioengineering Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models Dorothy R. Ahlf Wheatcraft1,2, Xin Liu1,2, Amanda B. Hummon1,2 1Department of Chemistry and Biochemistry, University of Notre Dame, 2Harper Cancer Research Institute, University of Notre Dame Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis. Biology A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos Shahram Jevin Poureetezadi1, Eric K. Donahue1, Rebecca A. Wingert1 1Department of Biological Sciences, University of Notre Dame The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out. Biology Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation Emmanuel Bikorimana1,2, Danica Lapid3, Hyewon Choi3, Richard Dahl1,2,3 1Harper Cancer Research Institute, 2Microbiology and Immunology, Indiana University School of Medicine, 3Department of Biological Sciences, University of Notre Dame Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation. Bioengineering A Microfluidic Technique to Probe Cell Deformability David J. Hoelzle1,2, Bino A. Varghese1,3, Clara K. Chan1, Amy C. Rowat1 1Department of Integrative Biology and Physiology, University of California, Los Angeles, 2Department of Aerospace and Mechanical Engineering, University of Notre Dame, 3Molecular Imaging Center, University of Southern California We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions. Biology Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney Kristen K. McCampbell1, Kristin N. Springer1, Rebecca A. Wingert1 1Department of Biological Sciences, University of Notre Dame The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption. Biology Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization Christina N. Cheng1, Yue Li1, Amanda N. Marra1, Valerie Verdun1, Rebecca A. Wingert1 1Department of Biological Sciences, University of Notre Dame The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens. Biology Production of Haploid Zebrafish Embryos by In Vitro Fertilization Paul T. Kroeger Jr.1, Shahram Jevin Poureetezadi1, Robert McKee1, Jonathan Jou1, Rachel Miceli1, Rebecca A. Wingert1 1Department of Biological Sciences, University of Notre Dame The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis. Behavior Eye Tracking, Cortisol, and a Sleep vs. Wake Consolidation Delay: Combining Methods to Uncover an Interactive Effect of Sleep and Cortisol on Memory Kelly A. Bennion1, Katherine R. Mickley Steinmetz2, Elizabeth A. Kensinger1, Jessica D. Payne3 1Department of Psychology, Boston College, 2Department of Psychology, Wofford College, 3Department of Psychology, University of Notre Dame We present a protocol used to discover an interactive effect between sleep and cortisol on memory consolidation, particularly for negative arousing images. Specifically, the experimental design utilizes eye tracking, salivary cortisol analysis, and behavioral memory testing – methods that can be used with both healthy and clinical participants. Engineering Measurement of Coherence Decay in GaMnAs Using Femtosecond Four-wave Mixing Daniel Webber1, Tristan de Boer1, Murat Yildirim1, Sam March1, Reuble Mathew1, Angela Gamouras1, Xinyu Liu2, Margaret Dobrowolska2, Jacek Furdyna2, Kimberley Hall1 1Department of Physics and Atmospheric Science, Dalhousie University, 2Department of Physics, University of Notre Dame The technique of femtosecond four-wave mixing is described, including spectrally-resolved and time-resolved configurations. We illustrate the utility of this technique for the investigation of crucial physical properties in the III-V diluted magnetic semiconductors, afforded by its nonlinearity and high temporal resolution. Biology 3D Printing of Preclinical X-ray Computed Tomographic Data Sets Evan Doney1, Lauren A. Krumdick1, Justin M. Diener2, Connor A. Wathen3, Sarah E. Chapman4, Brian Stamile5, Jeremiah E. Scott3, Matthew J. Ravosa6, Tony Van Avermaete4, W. Matthew Leevy1,4,7 1Department of Chemistry and Biochemistry, University of Notre Dame, 2Freimann Life Science Center, University of Notre Dame, 3Department of Biological Sciences, University of Notre Dame, 4Notre Dame Integrated Imaging Facility, University of Notre Dame, 5MakerBot Industries LLC, 6Departments of Biological Sciences, Aerospace and Mechanical Engineering, and Anthropology, University of Notre Dame, 7Harper Cancer Research Institute, University of Notre Dame Using modern plastic extrusion and printing technologies, it is now possible to quickly and inexpensively produce physical models of X-ray CT data taken in a laboratory. The three -dimensional printing of tomographic data is a powerful visualization, research, and educational tool that may now be accessed by the preclinical imaging community. Medicine Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography Todd A. Sasser1, Sarah E. Chapman2, Shengting Li1, Caroline Hudson2, Sean P. Orton1, Justin M. Diener3, Seth T. Gammon1, Carlos Correcher4, W. Matthew Leevy2 1Carestream Molecular Imaging, 2Department of Chemistry and Biochemistry, University of Notre Dame, 3Freimann Life Science Center, University of Notre Dame, 4Research and Development, Oncovision, GEM-Imaging S.A. Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization. Biology In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin David R. Hyde1, Alan R. Godwin2, Ryan Thummel3 1Department of Biological Sciences, Center for Zebrafish Research, University of Notre Dame, 2Department of Microbiology, Immunology, and Pathology, Colorado State University, 3Departments of Anatomy and Cell Biology and Ophthalmology, Wayne State University School of Medicine We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein’s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth. Biology Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups Miriam M. Gillett-Kunnath1, Slavi C. Sevov1 1Department of Chemistry and Biochemistry, University of Notre Dame We present the high-temperature synthesis of intermetallic precursors K4Ge9, their dissolution in ethylenediamine to form Ge94- deltahedral Zintl ions, and the reaction of the clusters with alkynes to form organo-Zintl ions. The latter are characterized by electrospray mass spectrometry in solutions and by single-crystal X-ray diffraction in the solid state. Biology In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina Ryan Thummel1, Travis J. Bailey2,3, David R. Hyde2,3 1Departments of Anatomy and Cell Biology and Ophthalmology, Wayne State University School of Medicine, 2Department of Biological Sciences, University of Notre Dame, 3Center for Zebrafish Research, University of Notre Dame A method to conditionally knockdown a target protein’s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein’s role in the regenerating or intact retina. 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 Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration Corbin S. Johnson*1, Nicholas F. Holzemer*1, Rebecca A. Wingert1 1Department of Biological Sciences, University of Notre Dame Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron. Biology Dissection of the Adult Zebrafish Kidney Gary F. Gerlach*1, Lauran N. Schrader*1, Rebecca A. Wingert1 1Department of Biological Sciences, University of Notre Dame The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.