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81 ARTICLES PUBLISHED IN JoVE

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Biology

Murine Renal Transplantation Procedure
Jiao-Jing Wang 1, Sara Hockenheimer 2, Alice A. Bickerstaff 1, Gregg A. Hadley 1
1Department of Surgery, The Ohio State University, 2Integrated Biomedical Sciences Graduate Program, The Ohio State University

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

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Biology

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells
Rita Nokes Cook 1, Su Fen Ang *1, Richard Seung-on Kang *1, Heike Fölsch 1
1Department of Cell and Molecular Biology, Northwestern University

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

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Bioengineering

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System
Ariella Shikanov 1, Min Xu 2, Teresa K. Woodruff 2,3,4, Lonnie D. Shea 1,4,5
1Institute for BioNanotechnology in Advanced Medicine, Northwestern University, 2Department of Obstetrics and Gynecology, Northwestern University, Feinberg School of Medicine, 3Center for Reproductive Research, Northwestern University, 4The Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 5Department of Chemical and Biological Engineering, Northwestern University

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

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Biology

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice
Yu Chen 1,2, Tambudzai Shamu 2, Hui Chen 3, Peter Besmer 3, Charles L. Sawyers 2,4, Ping Chi 1,5
1Department of Medicine, Memorial Sloan Kettering Cancer Center, 2Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, 3Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 4Howard Hughes, Medical Institute, 5Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

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Neuroscience

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling
Norman Atkins 1, Charlie M. Miller 1, Joseph R. Owens 1, Fred W. Turek 1
1Center for Sleep and Circadian Biology, Northwestern University

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

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Bioengineering

Polymer Microarrays for High Throughput Discovery of Biomaterials
Andrew L. Hook 1, Chien-Yi Chang 2, Jing Yang 1, David J. Scurr 1, Robert Langer 3, Daniel G. Anderson 3, Steve Atkinson 2, Paul Williams 2, Martyn C. Davies 1, Morgan R. Alexander 1
1Laboratory of Biophysics and Surface Analysis, University of Nottingham , 2School of Molecular Medical Sciences, University of Nottingham , 3David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

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Medicine

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure
Evyn Loucks 1, Sara Ahlgren 1,2
1Program in Developmental Biology, Children's Memorial Research Center, 2Department of Pediatrics, Northwestern University

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

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Immunology and Infection

Colonization of Euprymna scolopes Squid by Vibrio fischeri
Lynn M. Naughton 1, Mark J. Mandel 1
1Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

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Bioengineering

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography
Wei Song *1,2, Qing Wei *1, Shuliang Jiao 3, Hao F. Zhang 1,4
1Department of Biomedical Engineering, Northwestern University, 2Department of Physics, Harbin Institute of Technology, 3Department of Ophthalmology, University of Southern California, 4Department of Ophthalmology, Northwestern University

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

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Medicine

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects
Liang Feng 1,2, Hui Chen 1, Genn Suyeoka 1, Xiaorong Liu 1,2
1Department of Ophthalmology, Northwestern University, 2Department of Neurobiology, Northwestern University

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

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Medicine

An Orthotopic Murine Model of Human Prostate Cancer Metastasis
Janet Pavese 1, Irene M. Ogden 1, Raymond C. Bergan 1,2,3
1Department of Medicine, Northwestern University, 2Robert H. Lurie Cancer Center, Northwestern University, 3Center for Molecular Innovation and Drug Discovery, Northwestern University

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

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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

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis
Baobing Zhao 1, Yang Mei 1, Jing Yang 1, Peng Ji 1
1Department of Pathology, Northwestern University

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

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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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Chemistry

Synthesis and Characterization of Functionalized Metal-organic Frameworks
Olga Karagiaridi 1, Wojciech Bury 1,2, Amy A. Sarjeant 1, Joseph T. Hupp 1, Omar K. Farha 1,3
1Department of Chemistry, Northwestern University, 2Department of Chemistry, Warsaw University of Technology, 3Department of Chemistry, Faculty of Science, King Abdulaziz University

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

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Biology

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans
Carmen I. Nussbaum-Krammer 1, Mário F. Neto 1, Renée M. Brielmann 1, Jesper S. Pedersen 1, Richard I. Morimoto 1
1Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

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Chemistry

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation
Lydia A. Finney 1, Qiaoling Jin 2
1X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, 2Department of Physics and Astronomy, Northwestern University

Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.

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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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Developmental Biology

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart
Lisa D. Wilsbacher 1,2, Shaun R. Coughlin 2
1Feinberg Cardiovascular Research Institute, Northwestern University, 2Cardiovascular Research Institute, University of California, San Francisco

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

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Bioengineering

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development
Joseph S. Uzarski 1,2, Jimmy Su 1,2,3,4, Yan Xie 1,2, Zheng J. Zhang 1,2, Heather H. Ward 5, Angela Wandinger-Ness 6, William M. Miller 7,8, Jason A. Wertheim 1,2,3,4,8,9
1Comprehensive Transplant Center, Feinberg School of Medicine, Northwestern University, 2Department of Surgery, Feinberg School of Medicine, Northwestern University, 3Department of Biomedical Engineering, Northwestern University, 4Simpson Querrey Institute for BioNanotechnology in Medicine, Northwestern University, 5Department of Internal Medicine, University of New Mexico HSC, 6Department of Pathology, University of New Mexico HSC, 7Department of Chemical and Biological Engineering, Northwestern University, 8Chemistry of Life Processes Institute, Northwestern University, 9Department of Surgery, Jesse Brown VA Medical Center

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

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Engineering

Visualizing Hyporheic Flow Through Bedforms Using Dye Experiments and Simulation
Susa H. Stonedahl 1, Kevin R. Roche 2, Forrest Stonedahl 3, Aaron I. Packman 2
1Engineering and Physical Science, St. Ambrose University, 2Civil and Environmental Engineering, Northwestern University, 3Mathematics and Computer Science, Augustana College

This manuscript describes how to create regular bedforms in a flume, visualize flow through the bedforms, and use computer simulations to simulate the hyporheic flow. The computer simulations compare well with the experimental observations. This coupled simulation and experiment is well-suited for both research and educational purposes.

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Behavior

Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice
Carmen Lin 1, John Disterhoft 1, Craig Weiss 1
1Department of Physiology, Northwestern University

The preparation presented here for whisker-signaled eyeblink conditioning in head-fixed mice precisely stimulates specific whiskers while allowing mice to ambulate on a cylindrical treadmill. A whisker stimulation conditioned stimulus (CS) paired with a periorbital shock unconditioned stimulus (US) results in reliable associative learning on this apparatus.

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Biology

DNA Electroporation, Isolation and Imaging of Myofibers
Alexis R. Demonbreun 1, Elizabeth M. McNally 1
1Center for Genetic Medicine, Northwestern University

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

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Chemistry

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis
Rafael D. Melani *1,2, Henrique S. Seckler *1, Owen S. Skinner 1, Luis H. F. Do Vale 1,3, Adam D. Catherman 1, Pierre C. Havugimana 1, Marcelo Valle de Sousa 3, Gilberto B. Domont 2, Neil L. Kelleher 1, Philip D. Compton 1
1Departments of Chemistry and Molecular Biosciences, Chemistry of Life Processes Institute, Proteomics Center of Excellence, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 2Institute of Chemistry, Proteomics Unit, Federal University of Rio de Janeiro, 3Department of Cell Biology, Brazilian Center for Protein Research, Laboratory of Biochemistry and Protein Chemistry, University of Brasilia

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

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Neuroscience

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining
Siwei Zhang *1,2, Jingjing Li *1,3, Robert Lea 1, Enrique Amaya 1
1The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, 2Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, 3Department of Craniofacial Development and Stem Cell Biology, Dental Institute, King's College London

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

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Biology

Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method
Sally M. McFall 1, Mário F. Neto 1, Jennifer L. Reed 1, Robin L. Wagner 2
1Center for Innovation in Global Health Technologies (CIGHT), Department of Biomedical Engineering, Northwestern University, 2Pritzker School of Medicine, The University of Chicago

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

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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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Biochemistry

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b
Ye Han *1, Kyle A. Lyman *1, Matt Clutter 2, Gary E. Schiltz 3, Quratul-Ain Ismail 1, Xiangying Cheng 1, Chi-Hao Luan 4, Dane M. Chetkovich 1,5
1Davee Department of Neurology and Clinical Neurosciences, Feinberg School of Medicine, Northwestern University, 2Center for Molecular Innovation and Drug Discovery, Northwestern University, 3Department of Pharmacology, Feinberg School of Medicine, Northwestern University, 4High Throughput Analysis Laboratory, Department of Molecular Biosciences, Northwestern University, 5Department of Physiology, Feinberg School of Medicine, Northwestern University

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

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Biology

Activating Autophagy by Aerobic Exercise in Mice
Altea Rocchi 1, Congcong He 1
1Department of Cell and Molecular Biology, Northwestern University

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

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Neuroscience

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells
Lauren A. Laboissonniere 1, Takuma Sonoda 2, Seul Ki Lee 2, Jeffrey M. Trimarchi 1, Tiffany M. Schmidt 2
1Department of Genetics, Development, and Cell Biology, Iowa State University, 2Department of Neurobiology, Northwestern University

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

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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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Behavior

Experience is Instrumental in Tuning a Link Between Language and Cognition: Evidence from 6- to 7- Month-Old Infants' Object Categorization
Danielle R. Perszyk 1, Sandra R. Waxman 1
1Psychology Department, Northwestern University

At 3-4 months, listening to human and nonhuman primate vocalizations boosts infants' cognition; by 6 months, only human vocalizations exert this cognitive advantage. We describe an exposure manipulation that reveals the powerful shaping role of experience as infants specify which sounds to link to cognition and which to tune out.

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Developmental Biology

In Ovo Electroporation in the Chicken Auditory Brainstem
Ting Lu 1, Ariel Loren Cohen 1, Jason Tait Sanchez 1,2,3
1Roxelyn and Richard Pepper Department of Communication Sciences and Disorders, Northwestern University, 2Knowles Hearing Research Center, Northwestern University, 3Department of Neurobiology, Northwestern University

Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

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Environment

Production and Measurement of Organic Particulate Matter in a Flow Tube Reactor
Yue Zhang 1,2, Pengfei Liu 1, Zhaoheng Gong 1, Franz M. Geiger 3, Scot T. Martin 1,4
1School of Engineering and Applied Sciences, Harvard University, 2Department of Environmental Science and Engineering, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, 3Department of Chemistry, Northwestern University, 4Department of Earth and Planetary Sciences, Harvard University

This paper describes the operation procedure for the flow tube reactor and related data collection. It shows the protocols for setting the experiments, recording data and generating the number-diameter distribution as well as the particle mass information, which gives useful information about chemical and physical properties of the organic aerosols.

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Environment

Production and Measurement of Organic Particulate Matter in the Harvard Environmental Chamber
Yue Zhang 1,2, Zhaoheng Gong 1, Suzane de Sa 1, Adam P. Bateman 1, Yingjun Liu 1, Yongjie Li 1, Franz M. Geiger 3, Scot T. Martin 1,4
1School of Engineering and Applied Sciences, Harvard University, 2Department of Environmental Science and Engineering, Gillings School of Global Public Health, University of North Carolina, 3Department of Chemistry, Northwestern University, 4Department of Earth and Planetary Sciences, Harvard University

This paper describes operation procedures for the Harvard Environmental Chamber (HEC) and related instrumentation for measuring gaseous and particle species. The environmental chamber is used to produce and study secondary organic species produced from the organic precursors, especially related to atmospheric organic particulate matter.

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Genetics

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents
Naemeh Pourshafie 1, Philip R. Lee 2, Ke-lian Chen 1, George G. Harmison 1, Laura C. Bott 1,3, Kenneth H. Fischbeck 1, Carlo Rinaldi 1,4
1Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2Section on Nervous System Development and Plasticity, The Eunice Kennedy Shriver National Institute of Child and Human Development, National Institutes of Health, 3Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University, 4Department of Physiology, Anatomy and Genetics, University of Oxford

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

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JoVE Journal

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development
Vishnu V. Krishnamurthy *1, Aurora J. Turgeon *2, John S. Khamo 1, Payel Mondal 1, Savanna R. Sharum 1, Wenyan Mei 2, Jing Yang 2, Kai Zhang 1,3,4
1Department of Biochemistry, University of Illinois at Urbana-Champaign, 2Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, 3Neuroscience Program, University of Illinois at Urbana-Champaign, 4Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

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Medicine

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model
Dou Yu 1, Gina Li 1, Maciej S. Lesniak 1, Irina V. Balyasnikova 1
1Department of Neurological Surgery, Feinberg School of Medicine, Northwestern University

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

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Behavior

Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat
Yingli Jing *1,2,3,4, Fan Bai *1,2,3,4, Hui Chen 1,2,3,4, Hao Dong 1,2,3,4
1China Rehabilitation Research Center, 2Institute of Rehabilitation Science of China, 3Center of Neural Injury and Repair, Beijing Institute for Brain Disorders, 4Beijing Key Laboratory of Neural Injury and Rehabilitation

Here we present a combination of laser Doppler perfusion imaging (LDPI) and laser Doppler perfusion monitoring (LDPM) to measure spinal cord local blood flows and oxygen saturation (SO2), as well as a standardized procedure for introducing spinal cord trauma on rat.

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Biology

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis
Mohammed A. Amin 1, Dileep Varma 1
1Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

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Bioengineering

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro
Corey M. Rountree 1, John B. Troy 2, Laxman Saggere 1
1Department of Mechanical and Industrial Engineering, University of Illinois at Chicago, 2Department of Biomedical Engineering, Northwestern University

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

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Immunology and Infection

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies
Ariel Finkielsztein 1, Lorraine Mascarenhas 1, Veronika Butin-Israeli 1, Ronen Sumagin 1
1Department of Pathology, Feinberg School of Medicine, Northwestern University

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

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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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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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Bioengineering

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation
Sean Allen 1, Michael Vincent 1, Evan Scott 1,2
1Interdisciplinary Biological Sciences, Northwestern University, 2Department of Biomedical Engineering, Northwestern University

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

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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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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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Chemistry

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology
Max Z. Levine *1,2, Nicole E. Gregorio *2,3, Michael C. Jewett 4,5,6,7, Katharine R. Watts 2,3, Javin P. Oza 2,3
1Department of Biological Sciences, California Polytechnic State University, San Luis Obispo, 2Center for Applications in Biotechnology, California Polytechnic State University, San Luis Obispo, 3Department of Chemistry and Biochemistry, California Polytechnic State University, 4Department of Chemical and Biological Engineering, Northwestern University, 5Chemistry of Life Processes Institute, Northwestern University, 6Center for Synthetic Biology, Northwestern University, 7Interdisciplinary Biological Sciences Program, Northwestern University

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

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Immunology and Infection

A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis
Guillermo J. Ares 1, Christie Buonpane 1, Carrie Yuan 1, Douglas Wood 1, Catherine J. Hunter 1,2
1Department of Pediatrics, Northwestern University, 2Division of Pediatric Surgery, Ann & Robert H. Lurie Children's Hospital of Chicago

Enteroids are emerging as a novel model in the study of human disease. The protocol describes how to simulate an enteroid model of human necrotizing enterocolitis using lipopolysaccharide (LPS) treatment of enteroids generated from neonatal tissue. Collected enteroids demonstrate inflammatory changes akin to those seen in human necrotizing enterocolitis.

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Behavior

Defining the Role Of Language in Infants' Object Categorization with Eye-tracking Paradigms
Alexander LaTourrette 1, Sandra R. Waxman 1,2
1Department of Psychology, Northwestern University, 2Institute for Policy Research, Northwestern University

Here we present a protocol for familiarization-test paradigms which provide a direct test of infant categorization and help to define the role of language in early category learning.

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Medicine

A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity
Jiao-Jing Wang *1, Longhui Qiu *1, Ramiro Fernandez 2, Xin Yi Yeap 1, Charlie Xiaoying Lin 3, Zheng Jenny Zhang 1,2
1Comprehensive Transplant Center, Northwestern University, 2Department of surgery, Feinberg School of Medicine, Northwestern University, 3Weinberg art and science college, Northwestern University

This protocol details the surgical steps of a mouse model of vascularized heterotopic spleen transplantation, a technically challenging model that can serve as a powerful tool in studying the fate and longevity of spleen cells, the mechanisms of distinct spleen cell populations in disease progression, and transplant immunity.

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Behavior

Lexical Decision Task for Studying Written Word Recognition in Adults with and without Dementia or Mild Cognitive Impairment
Alexandre Nikolaev 1, Eve Higby 2,3, JungMoon Hyun 4, Sameer Ashaie 5,6
1Helsinki Collegium for Advanced Studies, University of Helsinki, 2University of California, Riverside, 3California State University, East Bay, 4Hunter College, City University of New York, 5Feinberg School of Medicine, Northwestern University, 6Shirley Ryan AbilityLab

This article describes how to implement a simple lexical decision experiment to assess written word recognition in neurologically healthy participants and in individuals with dementia and cognitive decline. We also provide a detailed description of reaction time analysis using principal components analysis (PCA) and mixed-effects modeling.

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Neuroscience

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures
Andrew M. Chiu 1, Levi Barse 1, Pavla Hubalkova 1,2, Antonio Sanz-Clemente 1
1Department of Pharmacology, Feinberg School of Medicine, Northwestern University, 2Department of Cellular Neurophysiology, Institute of Physiology CAS

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

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Neuroscience

Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury
Steven J Schwulst 1, Mecca B.A.R. Islam 1
1Department of Surgery, Northwestern University

Here we describe a protocol for the induction of murine traumatic brain injury via an open-head controlled cortical impact.

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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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Developmental Biology

Generation of Multicellular Human Primary Endometrial Organoids
Alina R. Murphy *1, Teerawat Wiwatpanit *2, Zhenxiao Lu 1, Batzaya Davaadelger 1, J. Julie Kim 1
1Department of Obstetrics and Gynecology, Northwestern University, 2National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA)

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

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Chemistry

Sample Preparation in Quartz Crystal Microbalance Measurements of Protein Adsorption and Polymer Mechanics
Gwen E. dePolo *1, Emily Schafer *2, Kazi Sadman 1, Jonathan Rivnay 2, Kenneth R. Shull 1
1Department of Materials Science and Engineering, Northwestern University, 2Department of Biomedical Engineering, Northwestern University

The quartz crystal microbalance can provide accurate mass and viscoelastic properties for films in the micron or submicron range, which is relevant for investigations in biomedical and environmental sensing, coatings, and polymer science. The sample thickness influences which information can be obtained from the material in contact with the sensor.

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Medicine

Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat
Feibo Zheng 1,3, Andy Tully 1,2, Kyle M Koss 1, Xiaomin Zhang 1, Longhui Qiu 1, Jiao-Jing Wang 1, Bilal A Naved 1,4, David Z Ivancic 1, James M Mathew 1, Jason A Wertheim 1, Zheng Jenny Zhang 1
1Comprehensive Transplant Center and Department of Surgery, Feinberg School of Medicine, Northwestern University, 2Department of Surgery, University of Illinois at Chicago, 3Department of Surgery, Tianjin Occupational Diseases Precaution and Therapeutic Hospital, 4Department of Biomedical Engineering, McCormick School of Engineering, Northwestern University

This protocol presents a robust, reproducible model of vascularized composite allotransplant (VCA) geared toward simultaneous study of immunology and functional recovery. The time invested in meticulous technique in a right mid-thigh hind limb orthotopic transplant with hand sewn vascular anastomoses and neural coaptation yields the ability to study functional recovery.

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Immunology and Infection

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections
Dinesh Jaishankar 1, Cormac Cosgrove 1, Ryan J. Deaton 2, I. Caroline Le Poole 1,3
1Robert Lurie Comprehensive Cancer Center and Department of Dermatology, Feinberg School of Medicine, Northwestern University, 2Department of Pathology, University of Illinois at Chicago, 3Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

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Biochemistry

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking
Santosh Adhikari 1, Chiranjib Banerjee *1, Joe Moscatelli *1,2, Elias M. Puchner 1
1School of Physics and Astronomy, University of Minnesota, Twin Cities, Physics and Nanotechnology (PAN), 2Middlebury College

Conventional BODIPY conjugates can be used for live-cell single-molecule localization microscopy (SMLM) through exploitation of their transiently forming, red-shifted ground state dimers. We present an optimized SMLM protocol to track and resolve subcellular neutral lipids and fatty acids in living mammalian and yeast cells at the nanoscopic length scale.

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Neuroscience

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve
Nicholas P. Boyer 1, Maite Azcorra 1,2, Peter Jung 3,4, Anthony Brown 1
1Department of Neuroscience, The Ohio State University, 2Present address: Interdepartmental Neuroscience Graduate Program and Department of Neurobiology, Northwestern University, 3Quantitative Biology Institute, Ohio University, 4Department of Physics and Astronomy, Ohio University

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

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Immunology and Infection

Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes
Steven Droho 1, Carla M. Cuda *2, Jeremy A. Lavine *1
1Department of Ophthalmology, Feinberg School of Medicine, Northwestern University, 2Department of Medicine, Division of Rheumatology, Feinberg School of Medicine, Northwestern University

This protocol provides a method to digest whole eyes into a single cell suspension for the purpose of multi-parameter flow cytometric analysis in order to identify specific ocular mononuclear phagocytic populations, including monocytes, microglia, macrophages, and dendritic cells.

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Developmental Biology

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids
Maxwell E Edmonds 1, Micah D. Forshee 1, Teresa K. Woodruff 1
1Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

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Genetics

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations
Amanda O. Shaver 1, Goncalo J. Gouveia 2, Pamela S. Kirby 3, Erik C. Andersen 4, Arthur S. Edison 1,2,3
1Department of Genetics, University of Georgia, 2Department of Biochemistry and Molecular Biology, University of Georgia, 3Complex Carbohydrate Research Center, University of Georgia, 4Department of Molecular Biosciences, Northwestern University

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

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Kidney Organoid Generation at the Air-Liquid Interface
Ashwani Kumar Gupta 1,2, David Z. Ivancic 1, Bilal A. Naved 1,3, Jason A. Wertheim 1,2,3,4,5,6, Leif Oxburgh 7
1Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, 2Department of Surgery, Northwestern University Feinberg School of Medicine, 3Department of Biomedical Engineering, McCormick School of Engineering, Northwestern University, 4Simpson Querrey Institute for BioNanotechnology, Northwestern University, 5Chemistry of Life Processes Institute, Northwestern University, 6Department of Surgery, Jesse Brown VA Medical Center, 7The Rogosin Institute

This protocol describes asynchronous mixing of human embryonic stem cells derived kidney progenitors at the air-liquid interface to efficiently generate kidney organoids.

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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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JoVE Core

Obtaining Quality Extended Field-of-View Ultrasound Images of Skeletal Muscle to Measure Muscle Fascicle Length
Amy N. Adkins 1,4,5, Wendy M. Murray 1,2,3,4,5
1Department of Biomedical Engineering, Northwestern University, 2Department of Physical Medicine & Rehabilitation, Northwestern University Feinberg School of Medicine, 3Department of Physical Therapy and Human Movement Sciences, Northwestern University Feinberg School of Medicine, 4Shirley Ryan AbilityLab, 5Edward Hines, Jr. VA Hospital

This study describes how to obtain high quality musculoskeletal images using the extended field-of-view ultrasound (EFOV-US) method for the purpose of making muscle fascicle length measures. We apply this method to muscles with fascicles that extend past the field-of-view of common traditional ultrasound (T-US) probes.

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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

Automated, High-Throughput Detection of Bacterial Adherence to Host Cells
Jing Yang 1, Qing-Ming Qin 1, Erin Van Schaik 1, James E. Samuel 1, Paul de Figueiredo 1,2
1Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, 2College of Veterinary Medicine, Texas A&M University

Detection of host-bacterial pathogen interactions based on phenotypic adherence using high-throughput fluorescence labeling imaging along with automated statistical analysis methods enables rapid evaluation of potential bacterial interactions with host cells.

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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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Neuroscience

Implantation and Control of Wireless, Battery-free Systems for Peripheral Nerve Interfacing
Hongkai Wang 1,2, Dom D’Andrea 1, Yeon Sik Choi 3,4, Yasmine Bouricha 1, Grace Wickerson 3,4, Hak-Young Ahn 3, Hexia Guo 3,4, Yonggang Huang 3,4,5,6, Milap S. Sandhu 7, Sumanas W. Jordan 8, John A. Rogers 3,4,6,9,10,11,12, Colin K. Franz 1,3,13
1Laboratory of Regenerative Rehabilitation, Shirley Ryan AbilityLab, Department of Physical Medicine and Rehabilitation, Northwestern University Feinberg School of Medicine, 2Northwestern University Interdepartmental Neuroscience Program, 3Center for Bio-integrated Electronics, Querrey Simpson Institute for Bioelectronics, Northwestern University, 4Department of Materials Science and Engineering, Northwestern University, 5Department of Civil and Environmental Engineering, Northwestern University, 6Department of Mechanical Engineering, Northwestern University, 7Arms and Hands Lab, Shirley Ryan AbilityLab, Department of Physical Medicine and Rehabilitation, Northwestern University Feinberg School of Medicine, 8Division of Plastic and Reconstructive Surgery, Biologics, Shirley Ryan AbilityLab, Northwestern University, 9Department of Biomedical Engineering, Northwestern University, 10Department of Neurological Surgery, Northwestern University, 11Department of Chemistry, Northwestern University, 12Department of Electrical and Computer Engineering, Northwestern University, 13The Ken and Ruth Davee Department of Neurology, Northwestern University Feinberg School of Medicine

This is a protocol for the surgical implantation and operation of a wirelessly powered interface for peripheral nerves. We demonstrate the utility of this approach with examples from nerve stimulators placed on either the rat sciatic or phrenic nerve.

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Neuroscience

Slicing the Embryonic Chicken Auditory Brainstem to Evaluate Tonotopic Gradients and Microcircuits
Sandesh Mohan 1, Abhijit Roy 1, George Ordiway 1, Jason Tait Sanchez 1,2,3
1Roxelyn and Richard Department of Communication Sciences and Disorders, Northwestern University, 2Knowles Hearing Research Center, Northwestern University, 3Department of Neurobiology, Northwestern University

Here we present a protocol for obtaining non-coronal auditory brainstem slices of the chicken embryo for the investigation of tonotopic properties and developmental trajectories within one brainstem slice. These slices include sagittal, horizontal, and horizontal/transverse sections encompassing larger tonotopic regions within an individual slice plane that the traditional coronal sections.

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Neuroscience

Evaluation of Auditory Brainstem Response in Chicken Hatchlings
George Ordiway 1, Miranda McDonnell 1, Sandesh Mohan 1, Jason Tait Sanchez 1,2,3
1Roxelyn and Richard Pepper Department of Communication Sciences and Disorders, Northwestern University, 2Knowles Hearing Research Center, Northwestern University, 3Department of Neurobiology, Northwestern University

We have used standard auditory brainstem response (ABR) techniques and applied them to hatchling chickens, a precocious avian model for auditory function. The protocol outlines animal preparation and ABR acquisition techniques in detail, with steps that could translate to other avian or rodent models.

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Biology

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes
Timothy A. Crombie 1, Robyn E. Tanny 1, Claire M. Buchanan 1, Nicole M. Roberto 1, Erik C. Andersen 1
1Department of Molecular Biosciences, Northwestern University

This protocol can be used to perform large-scale ecological surveys of selfing Caenorhabditis nematodes. The primary advantage of this method is the efficient organization and analysis of ecological and molecular data associated with the nematodes collected from nature.

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Immunology and Infection

Determining Intestinal Permeability Using Lucifer Yellow in an Apical-Out Enteroid Model
Heather Liebe 1, Camille Schlegel 2, Xue Cai 2, Alena Golubkova 1, Tyler Leiva 1, William L. Berry 3, Catherine J. Hunter 1
1Division of Pediatric Surgery, Oklahoma Children's Hospital, 2Oklahoma University Health Sciences Center, 3Department of Surgery, University of Oklahoma Health Sciences Center

The present protocol outlines a method that utilizes lucifer yellow in an apical-out enteroid model to determine intestinal permeability. This method can be used to determine paracellular permeability in enteroids that model inflammatory bowel diseases such as necrotizing enterocolitis.

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Biochemistry

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET
Chiranjib Banerjee 1, Brandon Wey-Hung Liauw 1, Reza Vafabakhsh 1
1Department of Molecular Biosciences, Northwestern University

This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis.

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Neuroscience

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina
Shichu Chang 1, Wenjin Xu 1, Weijia Fan 2, John A. McDaniel 1, Marta Grannonico 1, David A. Miller 2, Mingna Liu 1, Hao F. Zhang 2, Xiaorong Liu 1,3,4,5
1Department of Biology, University of Virginia, 2Department of Biomedical Engineering, Northwestern University, 3Department of Ophthalmology, University of Virginia, 4Program in Fundamental Neuroscience, University of Virginia, 5Department of Psychology, University of Virginia

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

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Environment

Quantifying Elastic Properties of Environmental Biofilms using Optical Coherence Elastography
Evan Dieppa 1, Hannah Schmitz 2, Ziwei Wang 3, Fabrizio Sabba 4, George Wells 2, Oluwaseyi Balogun 1,2,3
1Theoretical and Applied Mechanics Program, Northwestern University, 2Civil and Environmental Engineering Department, Northwestern University, 3Mechanical Engineering Department, Northwestern University, 4Black & Veatch

This paper highlights the optical coherence elastography (OCE) technique's efficacy in rapidly and non-destructively characterizing biofilm elastic properties. We elucidate critical OCE implementation procedures for accurate measurements and present Young's modulus values for two granular biofilms.

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Neuroscience

Recording Forelimb Muscle Activity in Head-Fixed Mice with Chronically Implanted EMG Electrodes
Amy Claire Kristl 1, Turgay Akay 2, Andrew Miri 1
1Department of Neurobiology, Northwestern University, 2Department of Medical Neuroscience, Dalhousie University

This protocol describes the hand fabrication and surgical implantation of electromyographic (EMG) electrodes in the forelimb muscles of mice to record muscle activity during head-fixed behavior experiments.

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Medicine

Determination of Continuity Index Values in Atrial Fibrillation Ablation with Proactive Esophageal Cooling
Catherine Lazarus 1, Jacob Sherman 2, Natalie Putzel 3, Tiffany Sharkoski 4, William Zagrodzky 4, Erik Kulstad 4,5, Alex Ro 6, Jose Nazari 6, Westby Fisher 6, Mark Metzl 6
1Northwestern University, 2Washington University in St. Louis, 3University of Southern California, 4Attune Medical, 5University of Texas Southwestern Medical Center, 6NorthShore University Hospital

This protocol describes the methods used to determine the continuity index in patients undergoing pulmonary vein isolation procedures using radiofrequency ablation and demonstrates the differences in continuity index between ablation procedures using proactive esophageal cooling as compared to procedures using traditional luminal esophageal temperature monitoring.

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