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University of Notre Dame

59 ARTICLES PUBLISHED IN JoVE

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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

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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Basic Biology

Rodent Identification II
Kay Stewart 1, Valerie A. Schroeder 1
1University of Notre Dame

Rodent Identification II

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Basic Biology

Compound Administration I
Kay Stewart 1, Valerie A. Schroeder 1
1University of Notre Dame

Compound Administration I

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Basic Biology

Compound Administration IV
Kay Stewart 1, Valerie A. Schroeder 1
1University of Notre Dame

Compound Administration IV

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Basic Biology

Compound Administration III
Kay Stewart 1, Valerie A. Schroeder 1
1University of Notre Dame

Compound Administration III

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Basic Biology

Rodent Handling and Restraint Techniques
Kay Stewart 1, Valerie A. Schroeder 1
1University of Notre Dame

Rodent Handling and Restraint Techniques

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Basic Biology

Blood Withdrawal II
Kay Stewart 1, Valerie A. Schroeder 1
1University of Notre Dame

Blood Withdrawal II

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Basic Biology

Anesthesia Induction and Maintenance
Kay Stewart 1, Valerie A. Schroeder Schroeder 1
1University of Notre Dame

Anesthesia Induction and Maintenance

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Basic Biology

Considerations for Rodent Surgery
Kay Stewart 1, Valerie A. Schroeder 1
1University of Notre Dame

Considerations for Rodent Surgery

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Basic Biology

Basic Care Procedures
Kay Stewart 1, Valerie A. Schroeder 1
1University of Notre Dame

Basic Care Procedures

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Basic Biology

Fundamentals of Breeding and Weaning
Kay Stewart 1, Valerie A. Schroeder 1
1University of Notre Dame

Fundamentals of Breeding and Weaning

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Basic Biology

Sterile Tissue Harvest
Kay Stewart 1, Valerie A. Schroeder 1
1University of Notre Dame

Sterile Tissue Harvest

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Basic Biology

Compound Administration II
Kay Stewart 1, Valerie A. Schroeder 1
1University of Notre Dame

Compound Administration II

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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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Engineering

Measurement of Coherence Decay in GaMnAs Using Femtosecond Four-wave Mixing
Daniel Webber 1, Tristan de Boer 1, Murat Yildirim 1, Sam March 1, Reuble Mathew 1, Angela Gamouras 1, Xinyu Liu 2, Margaret Dobrowolska 2, Jacek Furdyna 2, Kimberley Hall 1
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.

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JoVE Journal

A Microfluidic Technique to Probe Cell Deformability
David J. Hoelzle 1,2, Bino A. Varghese 1,3, Clara K. Chan 1, Amy C. Rowat 1
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.

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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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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. Bennion 1, Katherine R. Mickley Steinmetz 2, Elizabeth A. Kensinger 1, Jessica D. Payne 3
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.

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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

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation
Emmanuel Bikorimana 1,2, Danica Lapid 3, Hyewon Choi 3, Richard Dahl 1,2,3
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.

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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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Bioengineering

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models
Dorothy R. Ahlf Wheatcraft 1,2, Xin Liu 1,2, Amanda B. Hummon 1,2
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.

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Biology

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays
Nydia Morales-Soto 1,2, Morgen E. Anyan 1, Anne E. Mattingly 1, Chinedu S. Madukoma 1, Cameron W. Harvey 3, Mark Alber 3, Eric Déziel 4, Daniel B. Kearns 5, Joshua D. Shrout 1,2,6
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.

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Biology

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae
Xin Zhang *1, Keshava Mysore *2,3, Ellen Flannery 3,4, Kristin Michel 1, David W. Severson 3,4, Kun Yan Zhu 5, Molly Duman-Scheel 2,3,4
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.

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Engineering

Preparation and Reactivity of Gasless Nanostructured Energetic Materials
Khachatur V. Manukyan 1, Christopher E. Shuck 2, Alexander S. Rogachev 3, Alexander S. Mukasyan 2
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.

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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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Chemistry

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation
Michelle A. Pillers 1, Rebecca Shute 1, Adam Farchone 2, Keenan P. Linder 3, Rose Doerfler 2, Corey Gavin 4, Valerie Goss 3, Marya Lieberman 1
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.

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Medicine

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis
Kyle A. Lewellen *1, Matthew N. Metzinger *1, Yueying Liu 1, M. Sharon Stack 1
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.

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Engineering

The Measurement of Unsteady Surface Pressure Using a Remote Microphone Probe
Yaoyi Guan 1, Carl R. Berntsen 1, Michael J. Bilka 1, Scott C. Morris 1
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.

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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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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. Flaherty 1, Shaun W. Lee 1
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.

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Developmental Biology

Rapid Acquisition of 3D Images Using High-resolution Episcopic Microscopy
Haochuan Zhang *1,2,3, JunGang Huang *2,3,4, Xin Liu 2,3, Ping Zhu 4, Zhongrong Li 1, Xue Li 2,3
1Department of Pediatric Surgery, The Second Affiliated Hospital & Yuying Children's Hospital, Wenzhou Medical University, 2Departments of Urology, Boston Children's Hospital, 3Department of Surgery, Harvard Medical School, 4Department of Cardiovascular Surgery, Guangdong Cardiovascular Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences

We describe a detailed protocol using high-resolution episcopic microscopy to acquire three-dimensional (3D) images of mouse embryos. This improved protocol utilizes a modified tissue preparation method to enhance penetration of the fluorescent dye, thereby permitting morphometric analysis of both small and large-sized specimens.

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Behavior

The Deese-Roediger-McDermott (DRM) Task: A Simple Cognitive Paradigm to Investigate False Memories in the Laboratory
Enmanuelle Pardilla-Delgado 1, Jessica D. Payne 1
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.

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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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Bioengineering

Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1
Merrel T. Holley *1, Neerajha Nagarajan *2, Christian Danielson 1, 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.

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Bioengineering

Cardiac Muscle Cell-based Actuator and Self-stabilizing Biorobot - Part 2
Neerajha Nagarajan *1, Merrel T. Holley *2, Christian Danielson 2, 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.

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JoVE Journal

Synthesis of High Purity Nonsymmetric Dialkylphosphinic Acid Extractants
Junlian Wang 1, Meiying Xie 1, Xinyu Liu 1, Shengming Xu 2
1School of Civil and Resource Engineering, University of Science and Technology Beijing, 2Institute of Nuclear and New Energy Technology, Tsinghua University

A protocol for the synthesis of high purity nonsymmetric dialkylphosphinic acid extractants is presented, taking (2,3-dimethylbutyl)(2,4,4'-trimethylpentyl)phosphinic acid as an example.

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Developmental Biology

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo
Veronica Akle 1, Nathalie Agudelo-Dueñas *1,2, Maria A. Molina-Rodriguez *1, Laurel Brianne Kartchner 1,3,4,6, Annette Marie Ruth 1,3,5,6, John M. González 3, Manu Forero-Shelton 2
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.

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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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Chemistry

Total Internal Reflection Absorption Spectroscopy (TIRAS) for the Detection of Solvated Electrons at a Plasma-liquid Interface
Hernan E. Delgado 1, Paul Rumbach 2, David M. Bartels 3, David B. Go 1,2
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.

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Immunology and Infection

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion
James J. Faust 1,2, Wayne Christenson 3,4,5, Kyle Doudrick 6, John Heddleston 7, Teng-Leong Chew 7, Marko Lampe 8, Arnat Balabiyev 1,2, Robert Ros 3,4,5, Tatiana P. Ugarova 1,2
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.

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Developmental Biology

Single-cell Photoconversion in Living Intact Zebrafish
Lauren Green 1,2, Cody J. Smith 1,2
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.

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Biochemistry

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum
Gustavo Diaz 1, Chandler Bridges 1, Megan Lucas 1, Yong Cheng 2, Jeff S. Schorey 2, Karen M. Dobos 1, Nicole A. Kruh-Garcia 1
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.

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Medicine

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of β-aminopropionitrile-induced Aortic Aneurysm and Dissection
Brittany O. Aicher 1, Subhradip Mukhopadhyay 1, Xin Lu 2, Selen C. Muratoglu 1, Dudley K. Strickland 1, Areck A. Ucuzian 1,3
1Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, 2Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, 3Division of Vascular Surgery, University of Maryland School of Medicine

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

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Biochemistry

Nitropeptide Profiling and Identification Illustrated by Angiotensin II
Shan Feng 1,2, Xiaofei Wen 3, Xin Lu 2,4,5,6
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.

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Immunology and Infection

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)
Xue Bai *1,2, Lin Zhang *3, Jin Hu 2, Xiuxiu Zhao 2, Jinheng Pan 2, Haiteng Deng 3, Shan Feng 1,2
1Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, 2Mass Spectrometry Core Facility, School of Life Sciences, Westlake University, 3MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University

Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) is novel for the elimination of harmful antibodies. Here, we describe a detailed protocol for the synthesis of DCAF1 molecule, which can selectively block 4G2 antibody to eliminate antibody dependent enhancement effect during Dengue virus infection.

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Bioengineering

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications
Megan Levis *1,2, Fernando Ontiveros *3, Jonathan Juan 1, Anthony Kavanagh 1, Jeremiah J. Zartman 1,2
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.

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Biology

Detecting Establishment of Shared Blood Supply in Parabiotic Mice by Caudal Vein Glucose Injection
Xin Liu 1, Xue Bai 1, Mingqi Li 1, Huimin Li 1, Yong Zhang 1,2, Baofeng Yang 1,3
1Department of Pharmacology, The State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education, College of Pharmacy, Harbin Medical University, 2Institute of Metabolic Disease, Heilongjiang Academy of Medical Science, 3Department of Pharmacology and Therapeutics, Melbourne School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences University of Melbourne

Here we describe a new method of detecting successful establishment of shared blood circulation of two parabionts through a caudal vein injection of glucose, which causes minimal damage and is not fatal to the parabionts.

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Neuroscience

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish
James Hentig 1,2,3, Kaylee Cloghessy 1,2,3, Chloe Dunseath 2,3, David R. Hyde 1,2,3
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.

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Neuroscience

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish
James Hentig 1,2,3, Kaylee Cloghessy 1,2,3, David R. Hyde 1,2,3
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.

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Cancer Research

Shear Assay Protocol for the Determination of Single-Cell Material Properties
Luke J. Holen *1, Killian Onwudiwe *1, Julian Najera 1, Maksym Zarodniuk 1, John D. Obayemi 2, Winston O. Soboyejo 2, Meenal Datta 1
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.

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Biology

Low-cost Polyethylene Terephthalate Lamination Microfluidics Designs for Multiplexed Zebrafish Imaging
Shelly Tan 1, Xiaoguang Zhu 2, Jeremiah J. Zartman 3,4, Qing Deng 1
1Department of Biological Sciences, Purdue University, 2Bindley Bioscience Center, Purdue University, 3Department of Chemical and Biomolecular Engineering, University of Notre Dame, 4Department of Biological Sciences, University of Notre Dame

An innovative method for fabricating microfluidic devices using polyethylene terephthalate (PET) lamination significantly reduces the cost and complexity of entrapping and imaging multiple live zebrafish embryos.

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