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University of Michigan

117 ARTICLES PUBLISHED IN JoVE

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Biology

Monitoring Acupuncture Effects on Human Brain by fMRI
Kathleen K. S. Hui 1, Vitaly Napadow 1, Jing Liu 1, Ming Li 1, Ovidiu Marina 1,2, Erika E. Nixon 1, Joshua D. Claunch 1, Lauren LaCount 1, Tara Sporko 1, Kenneth K. Kwong 1
1Department of Radiology, Massachusetts General Hospital and Harvard Medical School, 2William Beaumont Hospital

FMRI and physiological monitoring is used to study the effects of Acupuncture on the central and peripheral nervous systems. Acupuncture mobilizes a limbic-paralimbic-neocortical network, with great overlap with the default mode network, to modulate neurological activity, possibly related to its autonomic effect in the peripheral nervous system.

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Neuroscience

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats
Esther Y. Maier 1,2, Sean T. Ma 3, Allison Ahrens 2,4, Timothy J. Schallert 2,4,5, Christine L. Duvauchelle 1,2,4
1College of Pharmacy, Division of Pharmacology and Toxicology, University of Texas at Austin, 2The Waggoner Center of Addiction and Alcohol Research, University of Texas at Austin, 3Department of Psychology, University of Michigan, 4Institute for Neuroscience, University of Texas at Austin, 5Department of Psychology, University of Texas at Austin

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

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Neuroscience

Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification
Kristine M. Haines 1, Eva L. Feldman 2, Stephen I. Lentz 3
1Michigan Research Community, Undergraduate Research Opportunity Program, University of Michigan, 2Department of Neurology, University of Michigan, 3Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan

We developed a sensitive technique to label newly synthesized mitochondrial DNA (mtDNA) in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of EdU together with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons.

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Neuroscience

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds
Michelle K. Leach 1, Zhang-Qi Feng 2, Caitlyn C. Gertz 3, Samuel J. Tuck 3, Tara M. Regan 3, Youssef Naim 3, Andrea M. Vincent 3, Joseph M. Corey 1,3,4
1Department of Biomedical Engineering, University of Michigan, 2State Key Laboratory of Bioelectronics, Southeast University, 3Department of Neurology, University of Michigan , 4Geriatric Research, Education and Clinical Center, Veterans Affairs Ann Arbor Health System

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

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Immunology and Infection

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis
Kelly Schwartz 1, Rachel Stephenson 1, Margarita Hernandez 1, Nicolays Jambang 1, Blaise R. Boles 1
1Molecular, Cellular, and Developmental Biology, University of Michigan

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

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Bioengineering

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters
Michelle K. Leach 1, Zhang-Qi Feng 1,2, Samuel J. Tuck 3, Joseph M. Corey 1,3,4
1Department of Biomedical Engineering, University of Michigan, 2State Key Laboratory of Bioelectronics, Southeast University, 3Department of Neurology, University of Michigan, 4Geriatrics Research, Education and Clinical Center, Veterans Affairs Ann Arbor Healthcare Center

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

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Biology

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)
Zhen Wang 1, Christoph Benning 1
1Department of Biochemistry and Molecular Biology, Michigan State University

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

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Biology

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software
Koen J. C. Verbrugghe 1, Raymond C. Chan 1
1Human Genetics, University of Michigan

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

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Biology

Solid Plate-based Dietary Restriction in Caenorhabditis elegans
Tsui-Ting Ching 1, Ao-Lin Hsu 1,2
1Department of Internal Medicine, Division of Geriatric Medicine, University of Michigan, 2Department of Molecular and Integrative Physiology, University of Michigan

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

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Medicine

Electrolytic Inferior Vena Cava Model (EIM) of Venous Thrombosis
Jose A. Diaz 1, Shirley K. Wrobleski 1, Angela E. Hawley 1, Benedict R. Lucchesi 2, Thomas W. Wakefield 1, Daniel D. Myers, Jr. 1
1Conrad Jobst Vascular Research Laboratories, Section of Vascular Surgery, University of Michigan , 2Department of Pharmacology, University of Michigan

The electrolytic induction of endothelial activation to the internal surface of the Inferior Vena Cava results in venous type thrombus formation due to endothelial activation and partial blood stasis, two components of Virchow's triad.

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Neuroscience

Electrode Positioning and Montage in Transcranial Direct Current Stimulation
Alexandre F. DaSilva 1, Magdalena Sarah Volz 2,3, Marom Bikson 4, Felipe Fregni 2
1Headache & Orofacial Pain Effort (H.O.P.E.), Biologic & Material Sciences, School of Dentistry, University of Michigan , 2Laboratory of Neuromodulation, Department of Physical Medicine & Rehabilitation, Spaulding Rehabilitation Hospital and Massachusetts General Hospital, Harvard Medical School, 3Charité, University Medicine Berlin, 4Department of Biomedical Engineering, The City College of New York

Transcranial direct current stimulation (tDCS) is an established technique to modulate cortical excitability1,2. It has been used as an investigative tool in neuroscience due to its effects on cortical plasticity, easy operation, and safe profile. One area that tDCS has been showing encouraging results is pain alleviation 3-5.

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Medicine

Corneal Donor Tissue Preparation for Endothelial Keratoplasty
Maria A. Woodward 1, Michael Titus 2, Kyle Mavin 2, Roni M. Shtein 1
1Department of Ophthalmology, University of Michigan , 2MidWest Eye Banks

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

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Engineering

Multicopter Aerodynamics: Characterizing Thrust on a Hexacopter
Prashin Sharma 1, Ella M. Atkins 1
1Department of Aerospace Engineering, University of Michigan

Multicopter Aerodynamics: Characterizing Thrust on a Hexacopter

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Engineering

Real-time Flight Control: Embedded Sensor Calibration and Data Acquisition
Ella M. Atkins 1
1Department of Aerospace Engineering, University of Michigan

Real-time Flight Control: Embedded Sensor Calibration and Data Acquisition

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Biology

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations
Eric J. Horstick 1, Elizabeth M. Gibbs 1, Xingli Li 1, Ann E. Davidson 1, James J. Dowling 1
1Departments of Pediatrics and Neurology, University of Michigan

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

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Medicine

Technique and Considerations in the Use of 4x1 Ring High-definition Transcranial Direct Current Stimulation (HD-tDCS)
Mauricio F. Villamar 1,2, Magdalena Sarah Volz 1,3, Marom Bikson 4, Abhishek Datta 1,4, Alexandre F. DaSilva *5, Felipe Fregni *1
1Laboratory of Neuromodulation, Department of Physical Medicine & Rehabilitation, Spaulding Rehabilitation Hospital and Massachusetts General Hospital, Harvard Medical School, 2School of Medicine, Pontifical Catholic University of Ecuador, 3Charité University Medicine Berlin, 4The City College of The City University of New York, 5Headache & Orofacial Pain Effort (H.O.P.E.), Biologic & Materials Sciences, School of Dentistry, University of Michigan

High-definition transcranial direct current stimulation (HD-tDCS), with its 4x1-ring montage, is a noninvasive brain stimulation technique that combines both the neuromodulatory effects of conventional tDCS with increased focality. This article provides a systematic demonstration of the use of 4x1 HD-tDCS, and the considerations needed for safe and effective stimulation.

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Medicine

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies
Jacqueline R. Dauch 1, Chelsea N. Lindblad 1, John M. Hayes 1, Stephen I. Lentz 2, Hsinlin T. Cheng 1
1Department of Neurology, University of Michigan , 2Department of Internal Medicine, University of Michigan

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

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Education

High-speed Particle Image Velocimetry Near Surfaces
Louise Lu 1, Volker Sick 1
1Department of Mechanical Engineering, University of Michigan

A procedure for studying transient flows near boundaries using high-resolution, high-speed particle image velocimetry (PIV) is described here. PIV is a non-intrusive measurement technique applicable to any optically accessible flow by optimizing several parameter constraints such as the image and recording properties, the laser sheet properties, and analysis algorithms.

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Immunology and Infection

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells
Qiao Li 1, Lin Lu 1, Huimin Tao 1, Carolyn Xue 1, Seagal Teitz-Tennenbaum 2, John H. Owen 3, Jeffrey S Moyer 3, Mark E.P. Prince 3, Alfred E. Chang 1, Max S. Wicha 2
1Department of Surgery, University of Michigan, 2Department of Internal Medicine, University of Michigan, 3Department of Otolaryngology, University of Michigan

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

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Medicine

3D-Neuronavigation In Vivo Through a Patient's Brain During a Spontaneous Migraine Headache
Alexandre F. DaSilva *1,2,3, Thiago D. Nascimento *1, Tiffany Love *3, Marcos F. DosSantos 1, Ilkka K. Martikainen 1,3, Chelsea M. Cummiford 3, Misty DeBoer 1, Sarah R. Lucas 1, MaryCatherine A. Bender 1, Robert A. Koeppe 4, Theodore Hall 5, Sean Petty 5, Eric Maslowski 5, Yolanda R. Smith 6, Jon-Kar Zubieta 3
1Headache & Orofacial Pain Effort (H.O.P.E.), Biological & Materials Sciences Department, University of Michigan School of Dentistry, 2Michigan Center for Oral Health Research (MCOHR), University of Michigan School of Dentistry, 3Translational Neuroimaging Laboratory, Molecular & Behavioral Neuroscience Institute, University of Michigan, 4PET Physics Section, Division of Nuclear Medicine, Radiology Department, University of Michigan, 53DLab, University of Michigan, 6Department of Obstetrics and Gynecology, University of Michigan

In this study, the authors report for the first time a novel 3D-Immersive & Interactive Neuronavigation (3D-IIN) through the impact of a spontaneous migraine headache attack in the μ-opioid system of a patient's brain in vivo.

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Bioengineering

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae
Bibhudatta Mishra 1, Mostafa Ghannad-Rezaie 2, Jiaxing Li 1, Xin Wang  1, Yan Hao 1, Bing Ye 3,4, Nikos Chronis 2,5, Catherine A. Collins 1
1Department of Molecular, Cellular and Developmental Biology, University of Michigan, 2Department of Biomedical Engineering, University of Michigan, 3Life Sciences Institute, University of Michigan, 4Department of Cell and Developmental Biology, University of Michigan, 5Department of Mechanical Engineering, University of Michigan

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the 'larva chip' for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

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Chemistry

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation
Karthik Pillai 1,2,3, Fernando Navarro Arzate 2,4, Wei Zhang 2,5, Scott Renneckar 1,2,5,6
1Institute for Critical Technology and Applied Science, Virginia Tech, 2Macromolecules and Interfaces Institute, Virginia Tech, 3Institute for Food Safety and Health, Illinois Institute of Technology- Moffett Campus, 4Wood, Cellulose, and Paper Research Department, University of Guadalajara, 5Department of Sustainable Biomaterials, Virginia Tech, 6Sustainable Nanotechnology Interdisciplinary Graduate Education Program, Virginia Tech

The objective of this research was to form synthetic plant cell wall tissue using layer-by-layer assembly of nanocellulose fibrils and isolated lignin assembled from dilute aqueous suspensions.  Surface measurement techniques of quartz crystal microbalance and atomic force microscopy were used to monitor the formation of the polymer-polymer nanocomposite material.

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JoVE Journal

Fabrication and Testing of Microfluidic Optomechanical Oscillators
Kewen Han 1, Kyu Hyun Kim 2, Junhwan Kim 1, Wonsuk Lee 2,3, Jing Liu 3, Xudong Fan 3, Tal Carmon 2, Gaurav Bahl 1
1Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, 2Electrical Engineering and Computer Science, University of Michigan, 3Biomedical Engineering, University of Michigan

Parametric optomechanical excitations have recently been experimentally demonstrated in microfluidic optomechanical resonators by means of optical radiation pressure and stimulated Brillouin scattering. This paper describes the fabrication of these microfluidic resonators along with methodologies for generating and verifying optomechanical oscillations.

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Bioengineering

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting
Christopher W. Coyne 1, Karan Patel 1, Johanna Heureaux 1, Jeanne Stachowiak 3, Daniel A. Fletcher 4,5, Allen P. Liu 1,2
1Department of Mechanical Engineering, University of Michigan, 2Department of Biomedical Engineering, University of Michigan, 3Department of Biomedical Engineering, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 4Department of Bioengineering, University of California, Berkeley, 5Physical Biosciences Division, Lawrence Berkeley National Laboratory

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

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Biology

SIVQ-LCM Protocol for the ArcturusXT Instrument
Jason D. Hipp *1, Jerome Cheng *2, Jeffrey C. Hanson *1, Avi Z. Rosenberg 1, Michael R. Emmert-Buck 1, Michael A. Tangrea 1, Ulysses J. Balis 2
1Laboratory of Pathology, National Cancer Institute, National Institutes of Health, 2Department of Pathology, University of Michigan

SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings.

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Biology

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development
Katherine D. Walton 1, Åsa Kolterud 2
1Cell and Developmental Biology, University of Michigan, 2Department of Biosciences and Nutrition, Karolinska Instituet Novum

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

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Immunology and Infection

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins
Natalie Maricic 1, Suzanne Dawid 1,2
1Microbiology and Immunology, University of Michigan, 2Pediatrics and Communicable Diseases, University of Michigan

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.  Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

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Medicine

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty
Hassan N. Tausif 1, Lauren Johnson 2, Michael Titus 2, Kyle Mavin 2, Navasuja Chandrasekaran 1, Maria A. Woodward 1, Roni M. Shtein 1, Shahzad I. Mian 1
1Department of Ophthalmology, University of Michigan, 2MidWest Eye Banks

Endothelial keratoplasty is a surgical technique continuously evolving as advancements result in transplantation of thinner grafts with each succession1. Here we present a technique for controlled manual tissue dissection to allow safe and repeatable separation of endothelium and Descemet's membranes from donor corneoscleral buttons for transplantation2.

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Medicine

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery
Wei Zhang 1, Mohamed Trebak 1
1Nanobioscience, State University of New York College of Nanoscale Science and Engineering (SUNY CNSE)

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

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Developmental Biology

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells
Ieng Lam Lei 1, Lei Bu 2, Zhong Wang 1
1Cardiac Surgery, University of Michigan, 2Leon H Charney Division of Cardiology, New York University School of Medicine

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

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Medicine

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys
Liwei Huang 1, An Xiao 1, Andrea Wecker 1, Daniel A. McBride 1, Soo Young Choi 2, Weibin Zhou 3, Joshua H. Lipschutz 2
1Department of Medicine, Eastern Virginia Medical School, 2Department of Medicine, Medical University of South Carolina, 3Department of Pediatrics, University of Michigan

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

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Biology

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution
Francine E. Garrett-Bakelman *1, Caroline K. Sheridan *1, Thadeous J. Kacmarczyk 1, Jennifer Ishii 1, Doron Betel 1,2, Alicia Alonso 1, Christopher E. Mason 3, Maria E. Figueroa 4, Ari M. Melnick 1
1Department of Medicine, Weill Cornell Medical College, 2Institute for Computational Biomedicine, Weill Cornell Medical College, 3Department of Physiology and Biophysics, Weill Cornell Medical College, 4Department of Pathology, University of Michigan

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

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Medicine

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma
Anda-Alexandra Calinescu 1, Felipe Javier Núñez 1, Carl Koschmann 2, Bradley L. Kolb 1, Pedro R. Lowenstein 1,3, Maria G. Castro 1,3
1Department of Neurosurgery, University of Michigan School of Medicine, 2Department of Pediatrics, Division of Hematology-Oncology, University of Michigan School of Medicine, 3Department of Cell and Developmental Biology, University of Michigan

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

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Bioengineering

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity
Xiaobao Li 1, Jisun L. Song 2, Alessandro Culotti 1, Wei Zhang 1, David L. Chopp 3, Nanxi Lu 1, Aaron I. Packman 1
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.

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Immunology and Infection

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination
Lukasz J. Ochyl 1,2, James J Moon 1,2,3
1Department of Pharmaceutical Sciences, University of Michigan, 2Biointerfaces Institute, University of Michigan, 3Department of Biomedical Engineering, University of Michigan

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

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Neuroscience

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain
Alzahra J. Al Omran 1, Hannah C. Saternos 1, Tongyu Liu 2, Surya M. Nauli 3, Wissam A. AbouAlaiwi 1
1Department of Pharmacology and Experimental Therapeutics, University of Toledo, College of Pharmacy and Pharmaceutical Sciences, 2Life Sciences Institute, University of Michigan, 3Department of Biomedical & Pharmaceutical Sciences, Chapman University, School of Pharmacy, Rinker Health Science campus

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

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Immunology and Infection

Isolation of Leukocytes from the Human Maternal-fetal Interface
Yi Xu 1, Olesya Plazyo 1, Roberto Romero 1,2,3,4, Sonia S. Hassan 1,5, Nardhy Gomez-Lopez 1,5,6
1Perinatology Research Branch, NICHD/NIH/DHHS, 2Department of Obstetrics and Gynecology, University of Michigan, 3Department of Epidemiology and Biostatistics, Michigan State University, 4Department of Molecular Obstetrics and Genetics, Wayne State University, 5Department of Obstetrics and Gynecology, Wayne State University School of Medicine, 6Department of Immunology and Microbiology, Wayne State University School of Medicine

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

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Medicine

Direct Mouse Trauma/Burn Model of Heterotopic Ossification
Jonathan R. Peterson 1, Shailesh Agarwal 1, R. Cameron Brownley 1, Shawn J. Loder 1, Kavitha Ranganathan 1, Paul S. Cederna 1, Yuji Mishina 2, Stewart C. Wang 1, Benjamin Levi 1
1Department of Surgery, University of Michigan Medical School, 2Department of Biologic and Materials Sciences, University of Michigan School of Dentistry

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

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Bioengineering

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5
Eric Ehrke-Schulz 1, Wenli Zhang 1, Maren Schiwon 1, Thorsten Bergmann 1, Manish Solanki 1, Jing Liu 1, Philip Boehme 1, Theo Leitner 1, Anja Ehrhardt 1
1Institute of Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, University Witten/Herdecke

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

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Biology

Cryosectioning Method for Microdissection of Murine Colonic Mucosa
Attila E. Farkas 1, Christian Gerner-Smidt 1, Loukia Lili 1, Asma Nusrat 1, Christopher T. Capaldo 1
1Epithelial Pathobiology and Mucosal Inflammation Research Unit, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

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Developmental Biology

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye
Sarah J. Smith *1,2, Eric J. Horstick *3,4, Ann E. Davidson 1,2, James Dowling 1,2,4
1Program in Genetics & Genome Biology, The Hospital for Sick Children, 2Department of Molecular Genetics, The University of Toronto, 3Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, 4Departments of Pediatrics and Neurology, University of Michigan

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

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Neuroscience

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers
Hussein S. Hamid 1, John M. Hayes 2, Eva L. Feldman 2, Stephen I. Lentz 3
1University of Michigan Medical School, 2Department of Neurology, University of Michigan, 3Department of Internal Medicine, University of Michigan

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

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Behavior

Pavlovian Conditioned Approach Training in Rats
Christopher J. Fitzpatrick 1, Jonathan D. Morrow 2
1Neuroscience Graduate Program, University of Michigan, 2Department of Psychiatry, University of Michigan

Here, we present a protocol to elicit Pavlovian conditioned approach behavior in rats. This procedure can be used to measure individual differences in the tendency to approach and attribute incentive salience to reward-related cues and investigate addiction vulnerability.

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Immunology and Infection

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells
Gregory J. Baker 1,2, Maria G. Castro 1,2, Pedro R. Lowenstein 1,2
1Department of Neurosurgery, University of Michigan, 2Department of Cell and Developmental Biology, University of Michigan

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

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Chemistry

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability
Hui Gao 1, Eshita Khera 2, Jung-Kul Lee 1, Fei Wen 2
1Department of Chemical Engineering, Konkuk University, 2Department of Chemical Engineering, University of Michigan

Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads.

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Medicine

Cutaneous Surgical Denervation: A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease
Shelby C. Peterson 1, Isaac Brownell *2, Sunny Y. Wong *1
1Dermatology, Cell and Developmental Biology, University of Michigan, 2Dermatology Branch, National Cancer Institute, National Institutes of Health

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount β-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

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Biology

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture
Qianfan Bai *1, Zhiqiang Bai *1, Shaohai Xu 3, Lei Sun 1,2
1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School, 2Institute of Molecular and Cell Biology, Singapore, 3Division of Bioengineering, School of Chemical & Biomedical Engineering, Nanyang Technological University

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

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Immunology and Infection

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes
Balaji Olety 1, Sarah L. Veatch 2, Akira Ono 1
1Department of Microbiology and Immunology, University of Michigan Medical School, 2Department of Biophysics, University of Michigan

We illustrate here an in vitro membrane binding assay in which interactions between HIV-1 Gag and lipid membranes are visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique.

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Biology

Measuring In Vitro ATPase Activity for Enzymatic Characterization
Chelsea S. Rule 1, Marcella Patrick 1, Maria Sandkvist 1
1Department of Microbiology and Immunology, University of Michigan

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

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Developmental Biology

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes
Hidehito Saito 1,2, Kumiko Iwabuchi 3, Noemi Fusaki 4,5, Fumito Ito 3,6
1Department of Surgery, University of Michigan, 2Department of Biochemistry II, Kanazawa Medical University, 3Center for Immunotherapy, Roswell Park Cancer Institute, 4DNAVEC Corporation, 5Department of Ophthalmology, Keio University School of Medicine, 6Department of Surgical Oncology, Roswell Park Cancer Institute

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

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Biochemistry

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo
Jan-Ulrik Dahl 1, Philipp Koldewey 1,2, James C. A. Bardwell 1,2, Ursula Jakob 1
1Department of Molecular, Cellular, and Developmental Biology, University of Michigan, 2Howard Hughes Medical Institute, University of Michigan

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

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Biochemistry

Sheathless Capillary Electrophoresis–Mass Spectrometry for Metabolic Profiling of Biological Samples
Wei Zhang 1, M. Can Gulersonmez 1, Thomas Hankemeier 1, Rawi Ramautar 1
1Division of Analytical Biosciences, Leiden Academic Center for Drug Research, Leiden University

A protocol for metabolic profiling of biological samples by capillary electrophoresis–mass spectrometry using a sheathless porous tip interface design is presented.

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Genetics

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform
Nicolaas H. Fourie 1, Ralph M. Peace 1,2, Sarah K. Abey 1, LeeAnne B. Sherwin 1, John W. Wiley 3, Wendy A. Henderson 1
1Digestive Disorders Unit, National Institute of Nursing Research, National Institutes of Health, DHHS, 2National Institutes of Health Research Scholar, Howard Hughes Medical Institute, 3Internal Medicine, Medical School, University of Michigan

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

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Biology

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay
Ansley Semack 1, Rabia U. Malik 2, Sivaraj Sivaramakrishnan 1
1Genetics, Cell Biology, and Development, University of Minnesota, 2Department of Cell and Developmental Biology, University of Michigan

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, Fӧrster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

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Developmental Biology

Dissection of Larval Zebrafish Gonadal Tissue
Xinjian Wang *1, Sijie Chen *1, Wei Zhang 1, Yiyuan Ren 1, Quan Zhang 1, Gang Peng 1
1Institutes of Brain Science, State Key Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science, Fudan University

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

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Biology

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy
Jingwen Yang 1, Yunjun Yan 1, Xiaowei Xiang 1, Yuchao Xu 1, Naiming Zhou 2, Tianming Wang 1
1National Engineering Research Center of Marine Facilities Aquaculture, Marine Science College, Zhejiang Ocean University, 2Institute of Biochemistry, College of Life Sciences, Zhejiang University

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

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JoVE Core

Scalable Solution-processed Fabrication Strategy for High-performance, Flexible, Transparent Electrodes with Embedded Metal Mesh
Arshad Khan 1, Sangeon Lee 2, Taehee Jang 3, Ze Xiong 4, Cuiping Zhang 1,5, Jinyao Tang 4, L. Jay Guo 2,3, Wen-Di Li 1,5
1Department of Mechanical Engineering, University of Hong Kong, 2Department of Mechanical Engineering, University of Michigan, 3Department of Electrical Engineering and Computer Science, University of Michigan, 4Department of Chemistry, University of Hong Kong, 5HKU-Shenzhen Institute of Research and Innovation

This protocol describes a solution-based fabrication strategy for high-performance, flexible, transparent electrodes with fully-embedded, thick metal mesh. Flexible transparent electrodes fabricated by this process demonstrate among the highest reported performances, including ultra-low sheet resistance, high optical transmittance, mechanical stability under bending, strong substrate adhesion, surface smoothness, and environmental stability.

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Developmental Biology

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos
Antionette L. Williams 1, Brenda L. Bohnsack 1
1Department of Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

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Genetics

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing
Ahmed Elaswad *1,2, Karim Khalil *1,3, David Cline 1, Patrick Page-McCaw 4, Wenbiao Chen 4, Maximilian Michel 5, Roger Cone 5, Rex Dunham 1
1School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, 2Department of Animal Wealth Development, Faculty of Veterinary Medicine, Suez Canal University, 3Anatomy and Embryology Department, Faculty of Veterinary Medicine, Cairo University, 4Department of Molecular Physiology and Biophysics, Vanderbilt University, 5Life Science Institute, University of Michigan

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

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Medicine

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate (HoLEP).
Huan Xu *1, Xiang Wan *1, Meng Gu 1, Yanbo Chen 1, Qiling Shi 1, Qi Chen 1, Zhong Wang 1
1Department of Urology, Shanghai 9th People's Hospital, Shanghai Jiaotong University School of Medicine

Here we present a safe and efficient protocol, holmium laser enucleation of the prostate, to treat benign prostatic hyperplasia.

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Developmental Biology

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids
David R. Hill 1, Sha Huang 1, Yu-Hwai Tsai 1, Jason R. Spence 1,2, Vincent B. Young 1,3
1Department of Internal Medicine, Division of Gastroenterology, University of Michigan, 2Department of Cell and Developmental Biology, University of Michigan, 3Department of Internal Medicine, Division of Infectious Disease, University of Michigan

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

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Cancer Research

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres
Flor M. Mendez 1, Felipe J. Núñez 1,2, Rocío I. Zorrilla-Veloz 3,4, Pedro R. Lowenstein 1,2, Maria G. Castro 1,2
1Department of Cell and Developmental Biology, University of Michigan Medical School, 2Department of Neurosurgery, University of Michigan Medical School, 3Cancer Research Summer Internship Program (CARSIP), Cancer Biology Program, University of Michigan Medical School, 4Department of Biology, University of Puerto Rico-Río Piedras Campus

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

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Engineering

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes
Chao Tian 1, Wei Zhang 2,3, Van Phuc Nguyen 1, Xueding Wang 2,4, Yannis M. Paulus 1,2
1Kellogg Eye Center, Department of Ophthalmology and Visual Sciences, University of Michigan, 2Department of Biomedical Engineering, University of Michigan, 3Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, 4Department of Radiology, University of Michigan

This manuscript describes the novel setup and operating procedure of a photoacoustic microscopy and optical coherence tomography dual-modality system for noninvasive, label-free chorioretinal imaging of larger animals, such as rabbits.

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Behavior

Methods of Pairing and Pair Maintenance of New Zealand White Rabbits (Oryctolagus Cuniculus) Via Behavioral Ethogram, Monitoring, and Interventions
Sarah Thurston 1,2, Lisa Burlingame 1,2, Patrick A. Lester 1,2, Jennifer Lofgren 1,2
1Unit for Laboratory Animal Medicine, University of Michigan, 2Refinement and Enrichment Advancements Laboratory, University of Michigan

Though European rabbits are a social species, socially housing them can be challenging. Therefore, there must be a thorough understanding of behaviors and social structures of pair-housed laboratory rabbits. Here we present a protocol to identify pairing methods, species-typical hierarchy establishment behaviors and behaviors that warrant appropriate intervention.

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Education

Collecting Hair Samples for Hair Cortisol Analysis in African Americans
Kathy D. Wright 1, Jodi L. Ford 1, Joseph Perazzo 2, Lenette M. Jones 3, Sherrilynn Mahari 4, Brent A. Sullenbarger 5,6,7, Mark L. Laudenslager 8
1College of Nursing, The Ohio State University, 2College of Nursing, University of Cincinnati, 3School of Nursing, University of Michigan, 4Licensed Cosmetologist Consultant, 5Clinical Research Manager, The Ohio State University, 6Biomedical Core Research Coordinator, The Ohio State University, 7College of Nursing Biomedical Core Research Laboratory, The Ohio State University, 8Department of Psychiatry, Behavioral Immunology & Endocrinology Laboratory, University of Colorado Denver Anschutz Medical Campus

Hair cortisol concentration analysis provides an alternative to traditional measures of cortisol; however, to collect hair samples from African Americans, scientists need to be culturally informed and competent. The purpose of this protocol is to demonstrate a culturally informed technique to collect hair samples for cortisol analysis from African Americans.

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Medicine

Photoselective Vaporesection of the Prostate via an End-firing Lithium Triborate Crystal Laser
Xin Wang  1, Yao-Guang Zhang 1, Sheng-Cai Zhu 1, Ben Wan 1, Ming Liu 1, Jian-Ye Wang 1
1Department of Urology, Beijing Hospital, National Center of Gerontology

Here we present a protocol to perform Photoselective Vaporesection of the Prostate (PVRP) for benign prostatic hyperplasia (BPH) treatment.

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Neuroscience

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method
Weiyue Zhang *1, Wei Zhang *1, Ning Dai *1, Chenxia Han 2, Fengzhi Wu 1, Xu Wang 1, Libo Tan 1, Jie Li 1, Feng Li 1, Qingjia Ren 3
1School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, 2Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, 3Institute of Tibetan Medicine, Tibetan Traditional Medical College

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

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Immunology and Infection

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration
Emma Teal 1, Nina G. Steele 2, Jayati Chakrabarti 1, Loryn Holokai 3, Yana Zavros 1
1Department of Pharmacology and Systems Physiology, University of Cincinnati, 2Department of Cell & Developmental Biology, University of Michigan, 3Department of Molecular Genetics, Biochemistry & Microbiology, University of Cincinnati

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

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Genetics

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins
Harriet Prior *1, Lauren MacConnachie *1, Jose L. Martinez 1, Georgina C.B. Nicholl 1, Asim A. Beg 1
1Department of Pharmacology, University of Michigan

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

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Cancer Research

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity
Yanlan Wang 1,2, Jiayu Liu 1,2, Haitao Pan 1,2, Jieyu Xing 1,2, Xiaoqiong Wu 1,2, Qing Li 1,2, Zhong Wang 1,2
1School of Pharmaceutical Sciences, Sun Yat-Sen University, 2Center for Cellular & Structural Biology, Sun Yat-Sen University

Here, we present a protocol to produce a bispecific antibody GPC3-S-Fab in Escherichia coli. The purified GPC3-S-Fab has potent cytotoxicity against GPC3 positive liver cancer cells.

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Biology

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse
Zhen Wang 1,4, Ramon Ocadiz-Ruiz 1, Sinju Sundaresan 1, Lin Ding 1, Michael Hayes 1, Nirakar Sahoo 3, Haoxing Xu 1,2, Juanita L. Merchant 1,2,5
1Department of Internal Medicine-Gastroenterology, University of Michigan, 2Department of Molecular and Integrative Physiology, University of Michigan, 3Department of Molecular, Cellular and Developmental Biology, University of Michigan, 4Department of Gastrointestinal Surgery, The First Affiliated Hospital of Guangxi Medical University, 5Division of Gastroenterology, University of Arizona College of Medicine

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

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JoVE Core

Bulk and Thin Film Synthesis of Compositionally Variant Entropy-stabilized Oxides
Sai Sivakumar *1, Elizabeth Zwier *1, Peter Benjamin Meisenheimer *1, John T. Heron 1
1Department of Materials Science and Engineering, University of Michigan

The synthesis of high quality bulk and thin film (Mg0.25(1-x)CoxNi0.25(1-x)Cu0.25(1-x)Zn0.25(1-x))O and (Mg0.25(1-x)Co0.25(1-x)Ni0.25(1-x)CuxZn0.25(1-x))O entropy-stabilized oxides is presented.

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Bioengineering

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex
Monika Goss-Varley 1,2, Andrew J. Shoffstall 1,2, Keith R. Dona 1,2, Justin A. McMahon 1,2, Sydney C. Lindner 1,2, Evon S. Ereifej 1,2, Jeffrey R. Capadona 1,2
1Advanced Platform Technology Center, Rehabilitation Research and Development, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, 2Department of Biomedical Engineering, Case Western Reserve University

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.

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Biochemistry

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity
Antonio E. Chambers *1, Adam E. Richardson *1, David F. Read 2, Thomas J. Waller 3, Douglas A. Bernstein 1, Philip J. Smaldino 1
1Department of Biology, Ball State University, 2Department of Genome Sciences, University of Washington, 3Department of Molecular, Cellular, and Developmental Biology, University of Michigan

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

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Neuroscience

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion
Qiao Li 1, Cheng Qian 1, Feng-Quan Zhou 1,2
1Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, 2The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

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Engineering

Design and Fabrication of an Optical Fiber Made of Water
Mark L. Douvidzon 1, Shai Maayani 2, Leopoldo L. Martin 3, Tal Carmon 4
1Department of Nanoscience and Nanotechnology, Russell Berrie Nanotechnology Institute (RBNI), Technion - Israel Institute of Technology, 2Department of Material Sciences and Engineering, MIT, 3Centro de Tecnologia Nanofotónica, Universitat Politècnica de València, 4Department of Mechanical Engineering, Technion - Israel Institute of Technology

This protocol describes the design and manufacture of a water bridge and its activation as a water fiber. The experiment demonstrates that capillary resonances of the water fiber modulate its optical transmission.

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Medicine

Complete and Partial Aortic Occlusion for the Treatment of Hemorrhagic Shock in Swine
Aaron M. Williams *1, Umar F. Bhatti *1, Isabel S. Dennahy 1, Kiril Chtraklin 1, Panpan Chang 1, Nathan J. Graham 1, Basil M. Baccouche 1, Shalini Roy 1, Mohammed Harajli 1, Jing Zhou 1, Vahagn C. Nikolian 1, Qiufang Deng 1, Yuzi Tian 1, Baoling Liu 1, Yongqing Li 1, Gregory L. Hays 1,2, Julia L. Hays 1,2, Hasan B. Alam 1
1Department of Surgery, University of Michigan, 2Hays Innovations

Here, we present a protocol demonstrating a hemorrhagic shock model in swine that uses aortic occlusion as a bridge to definitive care in trauma. This model has application in testing a wide range of surgical and pharmacological therapeutic strategies.

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Medicine

Introduction of Intracapsular Rotary-cut Procedures (IRCP): A Modified Hysteromyomectomy Procedures Facilitating Fertility Preservation
Liping Zeng 1,2, Weixia Wei 1,2, Huiru Tang 1,2, Fang He 1,2, Shilin Zhong 1,2, Wei Zhang 1,2, Yuting Xiang 1, Xinfeng Qu 3, Ruifang Wu 1,2
1Department of Obstetrics and Gynecology, Peking University Shenzhen Hospital, China, 2Shenzhen Key Laboratory of Gynecological Diagnostic Technology Research, China, 3Specialist of SZSM Project, Peking University Shenzhen Hospital, China

Here, we present a protocol for performing an intracapsular rotary-cut procedure (IRCP), a modified laparoscopic intracapsular myomectomy that promotes fertility preservation.

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Chemistry

Methionine Functionalized Biocompatible Block Copolymers for Targeted Plasmid DNA Delivery
Yang Wu *1, Wei Zhang *2, Jian Zhang 2, Zhi-Xiang Mao 3, Li Ding 2, Hao Li 3, Rong Ma 1, Jin-Hai Tang 2
1Research Center of Clinical Oncology, Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research & Nanjing Medical University Affiliated Cancer Hospital, 2Department of General Surgery, The First Affiliated Hospital with Nanjing Medical University, 3School of Clinical Medicine, Xuzhou Medical University

This work presents the preparation of methionine functionalized biocompatible block copolymers (mBG) via the reversible addition-fragmentation chain transfer (RAFT) method. The plasmid DNA complexing ability of the obtained mBG and their transfection efficiency were also investigated. The RAFT method is very beneficial for polymerizing monomers containing special functional groups.

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Chemistry

A New Straightforward Method for Lipophilicity (logP) Measurement using 19F NMR Spectroscopy
Zhong Wang 1, Benjamin F. Jeffries 1, Hannah R. Felstead 1, Neil J. Wells 1, Elisabetta Chiarparin 2, Bruno Linclau 1
1Chemistry, University of Southampton, 2Medicinal Chemistry, Oncology, IMED Biotech Unit, AstraZeneca

A novel and straightforward variation of the shake-flask method was developed for accurate lipophilicity measurement of fluorinated compounds by 19F NMR spectroscopy.

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Cancer Research

Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics
Xin Wang  *1, Jiamin Zhou *2, Chen Qi *1, Gelin Wang 1,3
1School of Pharmaceutical Sciences, Tsinghua University, 2Department of Radiology, University of California, San Francisco, 3Beijing Advanced Innovation Center for Structural Biology

Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

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Bioengineering

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles
Hisato Konoeda 1, Hong Yang 2, Chengliang Yang 1, Annette Gower 1, Chun Xu 1, Wei Zhang 1, Mingyao Liu 1,3
1Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, 2Respiratory Medicine Research Laboratory, Institute of Translation Medicine, Shanghai General Hospital, Shanghai Jiaotong University, 3Institute of Medical Science, Faculty of Medicine, University of Toronto

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

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Cancer Research

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures
Felipe J. Núñez 1,2, Flor M. Mendez 2, Maria B. Garcia-Fabiani 1,2, Joaquín Pardo 1,3, Marta Edwards 1,2, Pedro R. Lowenstein 1,2, Maria G. Castro 1,2
1Department of Neurosurgery, University of Michigan Medical School, 2Department of Cell & Developmental Biology, University of Michigan, 3INIBIOLP, Histology B-Pathology B, School of Medicine, UNLP

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

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Medicine

Protocol and Guidelines for Point-of-Care Lung Ultrasound in Diagnosing Neonatal Pulmonary Diseases Based on International Expert Consensus
Jing Liu 1,2, Roberto Copetti 3, Erich Sorantin 4, Jovan Lovrenski 5, Javier Rodriguez-Fanjul 6, Dalibor Kurepa 7, Xing Feng 8, Luigi Cattaross 9, Huayan Zhang 10,11, Misun Hwang 12, Tsu F. Yeh 13,14, Yisrael Lipener 7, Abhay Lodha 15, Jia-Qin Wang 16, Hai-Ying Cao 2,17, Cai-Bao Hu 2,18, Guo-Rong Lyu 19, Xin-Ru Qiu 1,2, Li-Qun Jia 20, Xiao-Man Wang 20, Xiao-Ling Ren 1,2, Jiu-Ye Guo 1,2, Yue-Qiao Gao 1,2, Jian-Jun Li 1,2, Ying Liu 1,2, Wei Fu 1,2, Yan Wang 21, Zu-Lin Lu 1,2, Hua-Wei Wang 8, Li-Li Shang 22
1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The Neonatal Lung Ultrasound Training Base, Chinese College of Critical Ultrasound, 3Emergency Department, Cattinara University Hospital, 4Division of Pediatric Radiology, Department of Radiology, Medical University Graz, 5Faculty of Medicine, University of Novi Sad, Radiology Department, Institute for Children and Adolescents Health Care of Vojvodina, 6Pediatric Intensive Care Unit, Pediatric Service Hospital Joan XXIII Tarragona, University Rovira i Virgil, 7Division of Neonatal-Perinatal Medicine, Cohen Children's Medical Center, 8Department of Neonatology, Children's Hospital of Soochow University, 9Department of Neonatology, Udine University Hospital, 10Center for Newborn Care, Guangzhou Women and Children's Medical Center, 11Division of Neonatology, Children's Hospital of Philadelphia, 12Department of Radiology, Children's Hospital of Philadelphia, 13Division of Neonatology and NICU, Cook County Children's Hospital, University of Illinois, 14Division of Neonatology, Department of Pediatrics, Taipei Medical University, 15Department of Pediatrics and Community Health Sciences, University of Calgary, 16Department of Pediatrics, The Third Affiliated Hospital of Xinxiang Medical University, 17Department of Ultrasound, GE Healthcare, 18Intensive Care Unit, Zhejiang Hospital, 19Collaborative Innovation Center for Maternal and Infant Health Service Application Technology, Quanzhou Medical College, 20Department of Ultrasound, Beijing Children's Hospital Affiliated with Capital Medical University, 21Department of Neonatology and NICU, Tai'an City Central Hospital of Shandong Province, 22Department of Intensive Care Unit, The Second Affiliated Hospital of Heilongjiang University of Chinese Medicine

Lung ultrasound is a noninvasive and valuable tool for bedside evaluation of neonatal lung diseases. However, a relative lack of reference standards, protocols and guidelines may limit its application. Here, we aim to develop a standardized neonatal lung ultrasound diagnostic protocol to be used in clinical decision-making.

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JoVE Journal

Experimental Paradigm for Measuring the Effects of Self-distancing in Young Children
Amanda Grenell 1, Rachel E. White 2, Emily O. Prager 1, Catherine Schaefer 1, Ethan Kross 3, Angela L. Duckworth 4, Stephanie M. Carlson 1
1Insitute of Child Development, University of Minnesota-Twin Cities, 2Department of Psychology, Hamilton College, 3Department of Psychology, University of Michigan, 4Department of Psychology, University of Pennsylvania

An experimental paradigm was created to measure the effects of self-distancing in young children (4-6-year-olds). Self-distancing is a process through which individuals adopt a less egocentric perspective. This paradigm has been used to examine the effects of self-distancing on young children's self-regulation.

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Developmental Biology

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone
Jingwen Yang *1,2, Haichun Pan *2, Yuji Mishina 2
1The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory for Oral Biomedicine of Ministry of Education, School and Hospital of Stomatology, Wuhan University, 2Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

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Biology

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly
Chang Li *1,2, Zhu Hui Liu *1,2, Jia Wei Chen 1,2, Xin Yi Shu 1,2, Ying Shen 1, Feng Hua Ding 1, Rui Yan Zhang 1, Wei Feng Shen 1, Lin Lu 1, Xiao Qun Wang 1
1Department of Cardiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 2Institute of Cardiovascular Diseases, Shanghai Jiao Tong University

Here, we present a protocol for immunofluorescence staining to observe the endothelial cells of the mouse aorta directly. This technique is useful when studying the cellular and molecular phenotype of endothelial cells in different flow patterns and in the development of atherosclerosis.

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Cancer Research

Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction
Mohammed E. Sayed 1, Aaron L. Slusher 1, Andrew T. Ludlow 1
1School of Kinesiology, University of Michigan

We successfully converted the standard telomere repeat amplification protocol (TRAP) assay to be employed in droplet digital polymerase chain reactions. This new assay, called ddTRAP, is more sensitive and quantitative, allowing for better detection and statistical analysis of telomerase activity within various human cells.

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Cancer Research

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells
Michael E. Bregenzer *1, Ciara Davis *1, Eric N. Horst *1, Pooja Mehta *2, Caymen M. Novak *1, Shreya Raghavan *2, Catherine S. Snyder *2, Geeta Mehta 1,2,3,4
1Department of Biomedical Engineering, University of Michigan, 2Department of Materials Science and Engineering, University of Michigan, 3Rogel Cancer Center, School of Medicine, University of Michigan, 4Macromolecular Science and Engineering, University of Michigan

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates’ potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

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Cancer Research

Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry
Jenny Lazarus 1, Yagiz Akiska 1, Mirna Perusina Lanfranca 1, Lawrence Delrosario 1, Lei Sun 1, Daniel Long 2, Jiaqi Shi 3, Howard Crawford 2, Marina P. Di Magliano 1, Weiping Zou 1, Timothy Frankel 1
1Department of Surgery, University of Michigan, 2Department of Molecular and Cellular Physiology, University of Michigan, 3Department of Pathology, University of Michigan

Multiplex fluorescent immunohistochemistry is an emerging technology that enables the visualization of multiple cell types within intact formalin-fixed, paraffin embedded (FFPE) tissue. Presented are guidelines for ensuring a successful 7-color multiplex with instructions for optimizing antibodies and reagents, preparing slides, design and tips for avoiding common problems.

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Behavior

Automated Rat Single-Pellet Reaching with 3-Dimensional Reconstruction of Paw and Digit Trajectories
Alexandra Bova 1, Krista Kernodle 1, Kaitlyn Mulligan 2, Daniel Leventhal 2,3,4
1Neuroscience Graduate Program, University of Michigan, 2Department of Neurology, University of Michigan, 3Department of Biomedical Engineering, University of Michigan, 4Department of Neurology, VA Ann Arbor Health System

Rodent skilled reaching is commonly used to study dexterous skills, but requires significant time and effort to implement the task and analyze the behavior. We describe an automated version of skilled reaching with motion tracking and three-dimensional reconstruction of reach trajectories.

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Bioengineering

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes
Shreya Mahajan 1, Jonah A. Sharkins 2,4, Allen H. Hunter 5, Amir Avishai 6, Evon S. Ereifej 2,3,4
1Department of Electrical Engineering and Computer Science, University of Michigan, 2Veteran Affairs Ann Arbor Healthcare System, 3Department of Neurology, School of Medicine, University of Michigan, 4Department of Biomedical Engineering, University of Michigan, 5Michigan Center for Materials Characterization, University of Michigan, 6Carl Zeiss SMT, Inc.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

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Medicine

A Training and Testing System for Performing Vascular Reconstruction In Vitro
Yue Wang 1,2, Lijun Mu 3, Wei Zhang 1,2, Huan Chen 1,2, Qingshan Li 1,2, Aihua Shi 1, Bo Tang 2, Xiaogang Zhang 1,2, Dinghui Dong 1,2, Yi Lv 1,2
1National Local Joint Engineering Research Center for Precision Surgery and Regenerative Medicine, First Affiliated Hospital of Xi'an Jiaotong University, 2Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi'an Jiaotong University, 3Department of General Surgery, First Affiliated Hospital of Xi'an Jiaotong University

Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

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JoVE Journal

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly
Samuel C. Leguizamon 1, Abdulla F. Alqubati 1, Timothy F. Scott 2,3
1Department of Chemical Engineering, University of Michigan, 2Department of Chemical Engineering, Monash University, 3Department of Materials Science and Engineering, Monash University

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

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Bioengineering

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk
Carmen G. Flesher 1, Nicki A. Baker 1, Clarissa Strieder-Barboza 1,2, Dominic Polsinelli 5, Phillip J. Webster 1,2, Oliver A. Varban 1, Carey N. Lumeng 2,3,4, Robert W. O'Rourke 1,6
1Department of Surgery, University of Michigan Medical School, 2Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, 3Graduate Program in Immunology, University of Michigan Medical School, 4Graduate Program in Cellular and Molecular Biology, University of Michigan Medical School, 5Undergraduate Research Opportunity Program, University of Michigan, 6Department of Surgery, Ann Arbor Veterans Affairs Healthcare System

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

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Biology

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells
Tianming Wang 1, Xu Chen 1, Ke Xu 1, Bing Zhang 1, Dexiang Huang 1, Jingwen Yang 1
1National Engineering Research Center of Marine Facilities Aquaculture, Marine Science College, Zhejiang Ocean University

This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

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Neuroscience

Measuring and Manipulating Functionally Specific Neural Pathways in the Human Motor System with Transcranial Magnetic Stimulation
Elana R. Goldenkoff 1, Amir Mashni 1, Katherine J. Michon 1, Hannah Lavis 1, Michael Vesia 1
1School of Kinesiology, Brain Behavior Laboratory, University of Michigan

This article describes new approaches to measure and strengthen functionally specific neural pathways with transcranial magnetic stimulation. These advanced noninvasive brain stimulation methodologies can provide new opportunities for the understanding of brain-behavior relations and development of new therapies to treat brain disorders.

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Neuroscience

Long-range Channelrhodopsin-assisted Circuit Mapping of Inferior Colliculus Neurons with Blue and Red-shifted Channelrhodopsins
David Goyer 1, Michael T. Roberts 1
1Kresge Hearing Research Institute, Department of Otolaryngology - Head and Neck Surgery, University of Michigan

Channelrhodopsin-assisted circuit mapping (CRACM) is a precision technique for functional mapping of long-range neuronal projections between anatomically and/or genetically identified groups of neurons. Here, we describe how to utilize CRACM to map auditory brainstem connections, including the use of a red-shifted opsin, ChrimsonR.

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JoVE Core

International Expert Consensus and Recommendations for Neonatal Pneumothorax Ultrasound Diagnosis and Ultrasound-guided Thoracentesis Procedure
Jing Liu 1,2, Dalibor Kurepa 3, Francesco Feletti 4,5, Almudena Alonso-Ojembarrena 6, Jovan Lovrenski 7, Roberto Copetti 8, Erich Sorantin 9, Javier Rodriguez-Fanjul 10, Karishma Katti 3, Andrea Aliverti 4, Huayan Zhang 11,12, Misun Hwang 13, Tsu F. Yeh 14, Cai-Bao Hu 15, Xing Feng 16, Ru-Xin Qiu 1,2, Jing-Han Chi 17, Li-Li Shang 18, Guo-Rong Lyu 19, Shao-Zheng He 20, Yan-Fen Chai 21, Zhan-Jun Qiu 22, Hai-Ying Cao 2,23, Yue-Qiao Gao 1,2, Xiao-Ling Ren 1,2, Guo Guo 1,24, Li Zhang 1,2, Ying Liu 1,2, Wei Fu 1,2, Zu-Lin Lu 1,2, Hong-Lei Li 1,2
1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The National Neonatal Lung Ultrasound Training Base, 3Division of Neonatal-Perinatal Medicine, Cohen Children's Medical Center, 4Department of Electronics, Information and Bioengineering, Politecnico di Milano, 5Dipartimento di Diagnostica per Immagini, Ausl della Romagna, S. Maria delle Croci Hospital, 6Neonatal Intensive Care Unit, Puerta del Mar University Hospital, 7Faculty of Medicine, University of Novi Sad, Serbia, Institute for Children and Adolescents Health Care of Vojvodina, 8Emergency Department, University Hospital of Cattinara, 9Division of Pediatric Radiology, Department of Radiology, Medical University Graz, 10Pediatric Intensive Care Unit, Pediatric Service Hospital Joan XXIII Tarragona, University Rovira i Virgil, 11Center for Newborn Care, Guangzhou Women and Children's Medical Center, 12Division of Neonatology, Children's Hospital of Philadelphia, 13Section of Neonatal Imaging, Department of Radiology, Children's Hospital of Philadelphia, 14Maternal Child Health Research institute, Taipei Medical University and China Medical University, 15Intensive Care Unit, Zhejiang Hospital, 16Department of Neonatology, Children's Hospital of Soochow University, 17Department of Neonatology and NICU, Bayi Children's Hospital Affiliated to the Seventh Medical Center of Chinese PLA General Hospital, 18Intensive Care Unit, Second Affiliated Hospital of Heilongjiang University of Chinese Medicine, 19Collaborative Innovation Center for Maternal and Infant Health Service Application Technology, Quanzhou Medical College, 20Department of Ultrasound, Second Affiliated Hospital of Fujian Medical University, 21Department of Emergency Medicine, Tianjin Medical University General Hospital, 22Department of Emergency and Critical Care Medicine, Affiliated Hospital of Traditional Chinese Medicine, 23Department of Ultrasound, GE Healthcare, 24The Neonatal Intensive Care Unit, Fifth Medical Center of Chinese PLA General Hospital

Pneumothorax is a common emergency and critical disease in newborn infants that needs rapid, clear diagnosis and timely treatment. Diagnosis and treatment based on chest X-rays are associated with delayed management and radiation damage. Lung ultrasound (US) provides useful guidance for rapid, accurate diagnosis and the precise thoracentesis of pneumothorax.

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Engineering

Simulating Imaging of Large Scale Radio Arrays on the Lunar Surface
Alexander M. Hegedus 1
1Department of Climate and Space Sciences and Engineering, University of Michigan

A simulation framework for testing the imaging capabilities of large-scale radio arrays on the lunar surface is presented. Major noise components are discussed, and a software pipeline is walked through with details on how to customize it for novel scientific uses.

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Chemistry

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials
Andrew J. Chetwynd *1, Wei Zhang *2, Klaus Faserl *3, James A. Thorn 4, Iseult Lynch 1, Rawi Ramautar 2, Herbert H. Lindner 3
1School of Geography Earth and Environmental Sciences, University of Birmingham, 2Biomedical Microscale Analytics, Leiden University, 3Institute of Medical Biochemistry, Medical University of Innsbruck, 4AB Sciex UK Ltd

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis – mass spectrometry approach.

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Developmental Biology

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo
Wei Zhang *1, Xinjuan Lei *1, Xin Zhou *2,3, Boling He 1, Liqin Xiao 1, Huimin Yue 1, Shulin Wang 1, Yuting Sun 1, Yajun Wu 1, Liyang Wang 1,4, George Ghartey-Kwansah 1, Odell D. Jones 5, Joseph L. Bryant 6, MengMeng Xu 7, Jianjie Ma 3, Xuehon Xu 1
1National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest of China/CGDB, Shaanxi Normal University College of Life Sciences, 2Shaanxi Key Laboratory of Ischemic Cardiovascular Disease, Shaanxi Key Laboratory of Brain disorders, Institute of Basic & Translational Medicine, Xi'an Medical University, 3Ohio State University College of Medicine, 4Hematology-Oncology Division, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 5University of Pennsylvania ULAR, 6University of Maryland School of Medicine, 7Columbia University Medical Center

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

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Immunology and Infection

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model
Kevin Boerner 1, Anny-Claude Luissint 1, Charles A. Parkos 1
1Department of Pathology, University of Michigan, Ann Arbor

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

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Immunology and Infection

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection
Zhe Zhang Ryan Lew *1, Jing Liu *1, Hsiao Hui Ong 1, Vivian Jiayi Tan 2, Annika Luukkainen 3, Yew Kwang Ong 1,4, Mark Thong 1,4, Kia Joo Puan 5, Vincent Tak Kwong Chow 2,6, Kai Sen Tan 1,2,6, De Yun Wang 1,6
1Department of Otolaryngology, Yong Loo Lin School of Medicine, National University of Singapore, 2Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 3Haartman Institute, Medicum, University of Helsinki, 4Department of Otolaryngology, Head and Neck Surgery, National University Hospital, National University Health System, 5Agency for Science, Technology and Research (A*STAR), Singapore Immunology Network (SIgN), 6NUHS Infectious Diseases Translational Research Program, Yong Loo Lin School of Medicine, National University of Singapore

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.

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Immunology and Infection

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model
Vicky Garcia-Hernandez 1, Philipp-Alexander Neumann 2, Stefan Koch 3,4, Renae Lyons 1, Asma Nusrat 1, Charles A. Parkos 1
1Department of Pathology, University of Michigan, 2Department of Surgery, Klinikum Rechts der Isar, Technische Universität München, 3Wallenberg Centre for Molecular Medicine, Linköping University, 4Department of Biomedical and Clinical Sciences (BKV), Linköping University

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

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Biology

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts
Chithra K Muraleedharan 1, Jay Mierzwiak 1, Darius Feier 1, Asma Nusrat 1, Miguel Quiros 1
1Department of Pathology, School of Medicine, University of Michigan

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

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Engineering

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope
Kinga A. Unocic 1, Dale K. Hensley 1, Franklin S. Walden 2, Wilbur C. Bigelow 3, Michael B. Griffin 4, Susan E. Habas 4, Raymond R. Unocic 1, Lawrence F. Allard 5
1Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, 2Protochips Inc., 3Department of Materials Science & Engineering, University of Michigan, 4Catalytic Carbon Transformation & Scale-up, National Renewable Energy Laboratory, 5Materials Science & Technology Division, Oak Ridge National Laboratory

Here, we present a protocol for performing in situ TEM closed-cell gas reaction experiments while detailing several commonly used sample preparation methods.

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Biochemistry

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor
Sadia Sultana 1, Greg M. Anderson 1, Kevin Pierre Hoffmann 1, Jan-Ulrik Dahl 1
1School of Biological Sciences, Illinois State University

This protocol describes the extraction and visualization of aggregated and soluble proteins from Escherichia coli after treatment with a proteotoxic antimicrobial. Following this procedure allows a qualitative comparison of protein aggregate formation in vivo in different bacterial strains and/or between treatments.

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Medicine

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter
Zhaokai Li *1,2, Liu Liu *1, Zhong Wang 1
1Department of Cardiac Surgery, Cardiovascular Center, The University of Michigan, Ann Arbor, 2Department of Cardiovascular Medicine, Xiangya Hospital, Central South University

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as αMHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux.

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Cancer Research

Longitudinal Intravital Imaging Through Clear Silicone Windows
Laura Maiorino *1,2,3, Margaret Shevik *1,4,5, José M. Adrover 1, Xiao Han 1,6, Elias Georgas 7,8, John Erby Wilkinson 9, Harrison Seidner 1, Leonie Foerschner 1, David A. Tuveson 1, Yi-Xian Qin 8, Mikala Egeblad 1
1Cold Spring Harbor Laboratory, 2Cold Spring Harbor Laboratory School of Biological Sciences, 3Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 4Medical Scientist Training Program, School of Medicine, Stony Brook University, 5Graduate Program in Pharmacology, Stony Brook University, 6Graduate Program in Genetics, Stony Brook University, 7Graduate Program in Biomedical Engineering, Stony Brook University, 8Department of Biomedical Engineering, Stony Brook University, 9Department of Pathology, University of Michigan

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin. These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

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Biochemistry

Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases
Olivia Roscow 1, Wei Zhang 1
1Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph

Ubiquitination is a critical protein post-translational modification, dysregulation of which has been implicated in numerous human diseases. This protocol details how phage display can be utilized to isolate novel ubiquitin variants that can bind and modulate the activity of E3 ligases that control the specificity, efficiency, and patterns of ubiquitination.

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Bioengineering

Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles
Yashar Bashirzadeh *1, Nadab Wubshet *1, Thomas Litschel 2, Petra Schwille 3, Allen P. Liu 1,4,5,6
1Department of Mechanical Engineering, University of Michigan, Ann Arbor, 2John A. Paulson School of Engineering and Applied Sciences, Harvard University, 3Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, 4Department of Biomedical Engineering, University of Michigan, Ann Arbor, 5Department of Biophysics, University of Michigan, Ann Arbor, 6Cellular and Molecular Biology Program, University of Michigan, Ann Arbor

This article introduces a simple method for expeditious production of giant unilamellar vesicles with encapsulated cytoskeletal proteins. The method proves to be useful for bottom-up reconstitution of cytoskeletal structures in confinement and cytoskeleton-membrane interactions.

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Developmental Biology

In Ovo Intravascular Injection in Chicken Embryos
Kai Jin 1,2, Jing Zhou 1,2, Gaoyuan Wu 1,2, Ziyi Lian 1,2, Zongyi Zhao 1,2, Shujian Zhou 1,2, Chen Chen 1,2, Hongyan Sun 1,2, Yingjie Niu 1,2, Qishenng Zuo 1,2, Yani Zhang 1,2, Jiuzhou Song 3, Guohong Chen 1,2, Bichun Li 1,2,4
1Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, 2Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, 3Animal & Avian Sciences, University of Maryland, College Park, 4College of Biotechnology, Jiangsu University of Science and Technology

The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos.

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Neuroscience

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster
Yangbo Xiao *1, Ye Yuan *2, Swathi Yadlapalli 1,2,3
1Department of Cell and Developmental Biology, University of Michigan, 2Cellular and Molecular Biology Program, University of Michigan, 3Michigan Neuroscience Institute Affiliate, University of Michigan

Here, we present a simplified endogenous gene tagging protocol for Drosophila, which utilizes a PCR-based technique for marker-free identification of successful genetic modifications, facilitating the development of stable knock-in lines.

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Bioengineering

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform
Ting Zhang 1, Vinitha Dhamotharan 1, Yu Xiao 1,2, Sydne Ballengee 1,3, Jill Pollon 1,4, George K. Gittes 1
1Division of Pediatric Surgery, Department of Surgery, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, 2North Allegheny High School, 3Wright State University Boonshoft School of Medicine, 4College of Engineering, University of Michigan

As the field of gene therapy continues to evolve, there is a growing need for innovative methods that can address these challenges. Here, a unique method is presented, which streamlines the process of generating high-yield and high-purity AAV vectors using a cell factory platform, meeting the quality standards for in vivo studies.

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Medicine

Rodent Inferior Vena Cava Venoplasty Balloon Model
Oscar Moreno 1, Catherine E. Luke 1, Amber Clay 1, Laura Durham 1, Sabrina Rocco 1, Kiran Kumar 1, Nathaniel Parchment 1, David E. Babcock 2, Daniel D. Myers 1, Thomas W. Wakefield 1, Peter Henke 1, Andrea T. Obi 1
1Section of Vascular Surgery, Department of Surgery, University of Michigan, 2Surmodics, Inc.

The protocol presents a survival rodent model to test venoplasty balloon (VB) interventions in non-thrombotic, thrombotic, and post-thrombotic deep veins.

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Cancer Research

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures
Natalie Ap. Rodrigues Fernandes 1, Camyla Rodrigues Nascimento 1, Laura Andrea González Maldonado 1,2, Carlos Rossa Junior 1
1Department of Diagnosis and Surgery - São Paulo State University (UNESP), School of Dentistry, Araraquara, 2Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan

Invasion is a major biological phenomenon in the development and progression of cancer. This process is influenced by non-neoplastic cells and components of the tumor microenvironment. The purpose of this study is to describe an alternative method for analyzing tumor cell invasion in vitro using three-dimensional (3D) cultures.

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