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University of Notre Dame

18 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging
Joel R. Meyerson 1,2, Tommi A. White 1, Donald Bliss 3, Amy Moran 3, Alberto Bartesaghi 1, Mario J. Borgnia 1, M. Jason V. de la Cruz 1, David Schauder 1, Lisa M. Hartnell 1, Rachna Nandwani 1,4, Moez Dawood 5, Brianna Kim 6, Jun Hong Kim 7, John Sununu 8, Lisa Yang 9, Siddhant Bhatia 10, Carolyn Subramaniam 1, Darrell E. Hurt 11, Laurent Gaudreault 12, Sriram Subramaniam 1
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.

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Biology

Dissection of the Adult Zebrafish Kidney
Gary F. Gerlach *1, Lauran N. Schrader *1, Rebecca A. Wingert 1
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.

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Biology

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration
Corbin S. Johnson *1, Nicholas F. Holzemer *1, Rebecca A. Wingert 1
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.

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Biology

Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups
Miriam M. Gillett-Kunnath 1, Slavi C. Sevov 1
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.

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Biology

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina
Ryan Thummel 1, Travis J. Bailey 2,3, David R. Hyde 2,3
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.

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Biology

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin
David R. Hyde 1, Alan R. Godwin 2, Ryan Thummel 3
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.

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Medicine

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography
Todd A. Sasser 1, Sarah E. Chapman 2, Shengting Li 1, Caroline Hudson 2, Sean P. Orton 1, Justin M. Diener 3, Seth T. Gammon 1, Carlos Correcher 4, W. Matthew Leevy 2
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.

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Biology

3D Printing of Preclinical X-ray Computed Tomographic Data Sets
Evan Doney 1, Lauren A. Krumdick 1, Justin M. Diener 2, Connor A. Wathen 3, Sarah E. Chapman 4, Brian Stamile 5, Jeremiah E. Scott 3, Matthew J. Ravosa 6, Tony Van Avermaete 4, W. Matthew Leevy 1,4,7
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.

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Medicine

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG
Rashna D. Balsara 1,2, Sarah E. Chapman 3, Ian M. Sander 4, Deborah L. Donahue 1, Lucas Liepert 4, Francis J. Castellino 1,2, W. Matthew Leevy 3,4,5
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.

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Biology

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization
Christina N. Cheng 1, Yue Li 1, Amanda N. Marra 1, Valerie Verdun 1, Rebecca A. Wingert 1
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.

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Biology

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Kristen K. McCampbell 1, Kristin N. Springer 1, Rebecca A. Wingert 1
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.

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Biology

Production of Haploid Zebrafish Embryos by In Vitro Fertilization
Paul T. Kroeger Jr. 1, Shahram Jevin Poureetezadi 1, Robert McKee 1, Jonathan Jou 1, Rachel Miceli 1, Rebecca A. Wingert 1
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.

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Biology

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos
Shahram Jevin Poureetezadi 1, Eric K. Donahue 1, Rebecca A. Wingert 1
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.

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Developmental Biology

Microbead Implantation in the Zebrafish Embryo
Gary F. Gerlach 1, Elvin E. Morales 1, Rebecca A. Wingert 1
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.

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Biology

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury
Robert A. McKee 1,2, Rebecca A. Wingert 1,2
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.

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Developmental Biology

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy
Manuela Lahne 1, Ryne A. Gorsuch 1, Craig M. Nelson 1,2, David R. Hyde 1
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.

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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. Dethlefs 1, Justin Piotrowicz 2, Tony Van Avermaete 1, Jeff Maki 2, Steve Gerstler 3, W. M. Leevy 1,4,5
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.

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Developmental Biology

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence
Amanda N. Marra 1, Marisa Ulrich 1, Audra White 1, Meghan Springer 1, Rebecca A. Wingert 1
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.

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