University of Wisconsin, Madison View Institution's Website 54 articles published in JoVE Behavior A Common Marmoset Model of Mother-Infant Intervention for Breastfeeding Disorders in the Presence of Paternal Inhibition and Maternal Neglect Mayuko Yoda1,2, Yoshimasa Kamei3, Hayato Sakurai4, Hiroko Kakei4, Ting Tao1,2,4, Hideo Yamanouchi4, Tetsuya Kunikata4, Masanori Hariyama5, Ricki Colman6, Mamiko Koshiba1,4,5 1Graduate School of Sciences and Technology for Innovation, Yamaguchi University, 2Faculty of Health Sciences and Nursing, Juntendo University, 3Obstetrician, Medical Department, Saitama Medical University, 4Department of Pediatrics, Saitama Medical University, 5Graduate School of Information Sciences, Tohoku University, 6Department of Cell and Regenerative Biology and Wisconsin National Primate Research Center, University of Wisconsin, Madison Here, we present a nonhuman primate model of mother-infant intervention for breastfeeding disorders in the presence of paternal inhibition and maternal neglect. The mate model video complements education to support primate and human caregivers with respect to infants with breastfeeding problems such as pain. Biochemistry Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows Bryan S. Sibert*1,2,3, Joseph Y. Kim*1,4, Jie E. Yang1,2,3, Elizabeth R. Wright1,2,3,5 1Department of Biochemistry, University of Wisconsin, Madison, 2Cryo-Electron Microscopy Research Center, Department of Biochemistry, University of Wisconsin, Madison, 3Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin, Madison, 4Department of Chemistry, University of Wisconsin, Madison, 5Morgridge Institute for Research The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding. Biology Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis Takuya Uemura1, Jiaqi Wang1, Yuri Aratani1, Simon Gilroy2, Masatsugu Toyota1,2 1Department of Biochemistry and Molecular Biology, Saitama University, 2Department of Botany, University of Wisconsin Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors. Engineering Building Langmuir Probes and Emissive Probes for Plasma Potential Measurements in Low Pressure, Low Temperature Plasmas Peixuan Li*1, Noah Hershkowitz*1, Gregory Severn*2 1Department of Engineering Physics, University of Wisconsin, 2Department of Physics & Biophysics, University of San Diego The main goal of this work is to make it easier for research groups unfamiliar with Langmuir probes and emissive probes to use them as plasma diagnostics, especially near plasma boundaries. We do this by demonstrating how to build the probes from readily available materials and supplies. Neuroscience Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics Caitlin A. Murphy1, Aeyal Raz1,2, Sean M Grady1, Matthew I Banks1 1Department of Anesthesiology, University of Wisconsin, 2Department of Anesthesiology, Rambam Health Care Campus, the Ruth and Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane. Neuroscience Translaminar Autonomous System Model for the Modulation of Intraocular and Intracranial Pressure in Human Donor Posterior Segments Tasneem P. Sharma1, Stacy M. Curry1, Husain Lohawala2, Colleen McDowell3 1North Texas Eye Research Institute, Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, 2Mechanical Engineer Consultant, 3Department of Ophthalmology and Visual Sciences, School of Medicine and Public Health, University of Wisconsin We describe and detail the use of the translaminar autonomous system. This system utilizes the human posterior segment to independently regulate the pressure inside the segment (intraocular) and surrounding the optic nerve (intracranial) to generate a translaminar pressure gradient that mimics features of glaucomatous optic neuropathy. Environment Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope Kenichi Yazaki1, Mayumi Y. Ogasa2, Katsushi Kuroda3, Yasuhiro Utsumi4, Peter Kitin5, Yuzou Sano6 1Department of Plant Ecology, Forestry and Forest Products Research Institute (FFPRI), 2Kansai Research Center, Forestry and Forest Products Research Institute (FFPRI), 3Department of Wood Properties and Processing, Forestry and Forest Products Research Institute (FFPRI), 4Faculty of Agriculture, Kyushu University, 5Department of Bacteriology, University of Wisconsin, 6Research Faculty of Agriculture, Hokkaido University Observing the water distribution within the xylem provides significant information regarding water flow dynamics in woody plants. In this study, we demonstrate the practical approach to observe xylem water distribution in situ by using a cryostat and cryo-SEM, which eliminates artifactual changes in the water status during sample preparation. Environment In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors Prarthana S. Dharampal1, Caitlin M. Carlson2, Luis Diaz-Garcia3,4, Shawn A. Steffan1,5 1Department of Entomology, University of Wisconsin-Madison, 2Department of Bacteriology, University of Wisconsin-Madison, 3Department of Horticulture, University of Wisconsin-Madison, 4Instituto Nacional de Investigaciones Forestales Agricolas y Pecuarias, 5USDA-ARS, Vegetable Crop Research Unit Fungicide sprays on flowering plants may expose solitary bees to high concentrations of pollen-borne fungicide residues. Using laboratory-based experiments involving in vitro-reared bee larvae, this study investigates the interactive effects of consuming fungicide-treated pollen derived from host and non-host plants. Genetics Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells Hector Guillen-Ahlers1,2, Prahlad K. Rao1, Danu S. Perumalla1, Maria J. Montoya1, Avinash Y.L. Jadhav1, Michael R. Shortreed3, Lloyd M. Smith3, Michael Olivier1,2 1Department of Genetics, Texas Biomedical Research Institute, 2Department of Internal Medicine-Molecular Medicine, Wake Forest University School of Medicine, 3Department of Chemistry, University of Wisconsin This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells. Bioengineering Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications Michal Adamski1, Gianluca Fontana2, Joshua R. Gershlak4, Glenn R. Gaudette4, Hau D. Le1, William L. Murphy2,3 1Department of Surgery, University of Wisconsin-Madison, 2Department of Orthopedics and Rehabilitation, University of Wisconsin School of Medicine and Public Health, 3Department of Biomedical Engineering, University of Wisconsin College of Engineering, 4Department of Biomedical Engineering, Worcester Polytechnic Institute Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications. Environment Stress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray Diffraction Cecilia S.N. Cheung1,2, Donald J. Weidner1, Li Li1, Philip G. Meredith3, Haiyan Chen1, Matthew Whitaker1, Xianyin Chen4 1Mineral Physics Institute, Department of Geoscience, Stony Brook University, 2Geological Engineering, Department of Civil and Environmental Engineering, University of Wisconsin-Madison, 3Rock and Ice Physics Laboratory, Department of Earth Sciences, University College London, 4Department of Chemistry, Stony Brook University We report detailed procedures for compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus coupled with synchrotron X-radiation. Such experiments allow quantification of the stress distribution within samples, that ultimately sheds light on compaction processes in geomaterials. Chemistry Reactive Vapor Deposition of Conjugated Polymer Films on Arbitrary Substrates Nongyi Cheng1,2, Trisha L. Andrew2,3 1Department of Chemistry, University of Wisconsin-Madison, 2Department of Chemistry, University of Massachusetts Amherst, 3Department of Polymer Science and Engineering, University of Massachusetts Amherst This paper presents a protocol for reactive vapor deposition of poly(3,4-ethylenedioxythiophene), poly(3,4-propylenedioxythiophene), and poly(thieno[3,2-b]thiophene) films on glass slides and rough substrates, such as textiles and paper. Bioengineering Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae Kayla Huemer*1,2, Jayne M. Squirrell*3, Robert Swader2, Kirsten Pelkey2, Danny C. LeBert4, Anna Huttenlocher5,6, Kevin W. Eliceiri1,2,3 1Department of Biomedical Engineering, University of Wisconsin-Madison, 2Morgridge Institute for Research, University of Wisconsin-Madison, 3Laboratory for Optical and Computational Instrumentation, University of Wisconsin-Madison, 4Cellular and Molecular Pathology Graduate Program, University of Wisconsin-Madison, 5Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, 6Department of Pediatrics, University of Wisconsin-Madison This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration. Environment Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health Shawn A. Steffan1,2, Prarthana S. Dharampal2, Luis Diaz-Garcia3,4, Cameron R. Currie5, Juan Zalapa1,3, Chris Todd Hittinger6,7,8 1Vegetable Crop Research Unit, USDA-ARS, 2Department of Entomology, University of Wisconsin-Madison, 3Department of Horticulture, University of Wisconsin-Madison, 4Instituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias, 5Department of Bacteriology, University of Wisconsin-Madison, 6Laboratory of Genetics, Genome Center of Wisconsin, 7DOE Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, 8J.F. Crow Institute for the Study of Evolution, University of Wisconsin-Madison Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss. Biology Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor Thomas R. Vincent1, James Canham1, Masatsugu Toyota2,3,4, Marieta Avramova1, Sam T. Mugford5, Simon Gilroy2, Anthony J. Miller1, Saskia Hogenhout5, Dale Sanders1 1Department of Metabolic Biology, John Innes Centre, Norwich Research Park, 2Department of Botany, University of Wisconsin, Madison, 3Department of Biochemistry and Molecular Biology, Saitama University, 4Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 5Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution. Environment A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples Lawrence G. Oates1, Harry W. Read2, Jessica L. M. Gutknecht3, David S. Duncan1, Teri B. Balser4, Randall D. Jackson1 1Department of Agronomy and Great Lakes Bioenergy Research Center, University of Wisconsin - Madison, 2Department of Soil Science, University of Wisconsin - Madison, 3Department of Soil, Water, and Climate, University of Minnesota, 4Faculty of Science and Engineering, Curtin University The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. Developmental Biology Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment Destiny L. Baars1, Kendra A. Takle*1,2, Jonathon Heier*1,3, Francisco Pelegri1 1Laboratory of Genetics, University of Wisconsin, 2Department of Neurobiology, University of Massachusetts Medical School, 3Interdisciplinary Biomedical Graduate Program, University of Pittsburgh School of Medicine A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species. Medicine Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine Tinen L. Iles1, Brian Howard2, Stephen Howard3, Stephen Quallich2, Christopher Rolfes2, Eric Richardson4, Hanna R. Iaizzo5, Paul A. Iaizzo1 1Surgery, University of Minnesota, 2Biomedical Engineering, University of Minnesota, 3Medtronic, Inc., 4Bioengineering, Rice University, 5Pharmacy, University of Wisconsin We have developed a swine model for the target delivery of pharmacological agents within the pericardial space/fluid. Using this approach, the relative benefits of administered agents on induced atrial fibrillation, relative refractory periods and/or ischemic protection can be investigated. Biology Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin) Graham S. Erwin1, Matthew P. Grieshop1, Devesh Bhimsaria1,2, Asuka Eguchi3, José A. Rodríguez-Martínez1, Aseem Z. Ansari1,4 1Department of Biochemistry, University of Wisconsin–Madison, 2Department of Electrical and Computer Engineering, University of Wisconsin–Madison, 3Graduate Program in Cellular and Molecular Biology, University of Wisconsin–Madison, 4The Genome Center, University of Wisconsin–Madison Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC). Developmental Biology Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells Irina Elcheva1, Vera Brok-Volchanskaya1, Igor Slukvin2 1Primate Research Center, University of Wisconsin-Madison, 2Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors. Neuroscience A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila Rebeccah J. Katzenberger1, Carin A. Loewen2, R. Tayler Bockstruck1, Mikal A. Woods3, Barry Ganetzky2, David A. Wassarman1 1Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 2Department of Genetics, University of Wisconsin-Madison, 3Department of Natural Sciences, University of Puerto Rico-Aguadilla Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies. Medicine Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay Christopher J. Johnson1, Christina M. Carlson2, Aaron R. Morawski3, Alyson Manthei4, Neil R. Cashman5 1USGS National Wildlife Health Center, 2Department of Soil Science, University of Wisconsin–Madison, 3Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 4Merial Veterinary Scholars Program, School of Veterinary Medicine, University of Wisconsin–Madison, 5Department of Neurology, University of British Columbia Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers. Behavior Methods to Test Visual Attention Online Amanda Yung1, Pedro Cardoso-Leite2, Gillian Dale3, Daphne Bavelier2,4, C. Shawn Green3 1Center for Visual Science, University of Rochester, 2Faculty of Psychology and Educational Sciences, University of Geneva, 3Department of Psychology, University of Wisconsin-Madison, 4Department of Brain and Cognitive Sciences, University of Rochester To replicate laboratory settings, online data collection methods for visual tasks require tight control over stimulus presentation. We outline methods for the use of a web application to collect performance data on two tests of visual attention. Engineering Patterning via Optical Saturable Transitions - Fabrication and Characterization Precious Cantu1, Trisha L. Andrew2, Rajesh Menon1 1Department of Electrical and Computer Engineering, The University of Utah, 2Department of Chemistry, The University of Wisconsin-Madison We report that the diffraction limit of conventional optical lithography can be overcome by exploiting the transitions of organic photochromic derivatives induced by their photoisomerization at low light intensities.1-3 This paper outlines our fabrication technique and two locking mechanisms, namely: dissolution of one photoisomer and electrochemical oxidation. Bioengineering Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-µCP) and Sequential Nucleophilic Substitution Gavin T. Knight*1, Tyler Klann*1, Jason D. McNulty1,2, Randolph S. Ashton1 1Department of Biomedical Engineering, University of Wisconsin, Madison, 2Department of Mechanical Engineering, University of Wisconsin, Madison Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands. Biology Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4 Jinfeng Teng1, Stephen Loukin1, Xinliang Zhou1, Ching Kung1,2 1Laboratory of Cell and Molecular Biology, University of Wisconsin – Madison, 2Department of Genetics, University of Wisconsin – Madison To complement commonly used methods to study TRPV4’s, two methods are described: Its mechanosensitivity can be studied by Ca2+-aequorin luminometry in transgenic yeast upon hypo-osmotic challenge. It can also be examined in TRPV4-RNA injected Xenopus oocytes by whole-cell two-electrode voltage clamp or patch clamp in on-cell or excised mode. Chemistry Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples Diego Fajardo1, Sastry S. Jayanty2, Shelley H. Jansky1 1USDA-ARS and Department of Horticulture, University of Wisconsin - Madison, 2Department of Horticulture and Landscape Architecture, Colorado State University This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones. Immunology and Infection Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation Ewa Jankowska-Gan1, Subramanya Hegde1, William J. Burlingham1 1Department of Surgery, University of Wisconsin-Madison, School of Medicine and Public Health We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients. Biology Trajectory Data Analyses for Pedestrian Space-time Activity Study Feng Qi1, Fei Du2 1School of Environmental and Life Sciences, Kean University, 2Department of Geography, University of Wisconsin-Madison A suite of spatiotemporal processing methods are presented to analyze human trajectory data, such as that collected using a GPS device, for the purpose of modeling pedestrian space-time activities. Biology Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy Kathryn Norby1, Ya-Fang Chiu1, Bill Sugden1 1Department of Oncology, University of Wisconsin - Madison A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time. Immunology and Infection Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence Elizabeth Hussa1, Heidi Goodrich-Blair1 1Department of Bacteriology, University of Wisconsin-Madison The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners. Biology Visualizing Bacteria in Nematodes using Fluorescent Microscopy Kristen E. Murfin1, John Chaston1, Heidi Goodrich-Blair1 1Department of Bacteriology, University of Wisconsin-Madison To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode. Biology GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil Chao Liang1,2, Harry W. Read2, Teri C. Balser2,3 1DOE-Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, 2Department of Soil Science, University of Wisconsin, Madison, 3Department of Soil and Water Science, University of Florida We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization. Immunology and Infection Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens Hana T. Millen1, Jordan C. Gonnering1, Ryan K. Berg2, Susan K. Spencer3, William E. Jokela3, John M. Pearce4, Jackson S. Borchardt1, Mark A. Borchardt3 1Wisconsin Water Science Center, United States Geological Survey, 2University of Wisconsin – Madison, 3Agricultural Research Service, United States Department of Agriculture, 4Alaska Science Center, United States Geological Survey Glass wool filters have been used to concentrate waterborne viruses by a number of research groups around the world. Here we show a simple approach for constructing glass wool filters and demonstrate the filters are also effective in concentrating waterborne viral, bacterial and protozoan pathogens. Neuroscience Voltage Biasing, Cyclic Voltammetry, & Electrical Impedance Spectroscopy for Neural Interfaces Seth J. Wilks1, Tom J. Richner2, Sarah K. Brodnick2, Daryl R. Kipke3, Justin C. Williams2, Kevin J. Otto1,4 1Weldon School of Biomedical Engineering, Purdue University, 2Biomedical Engineering, University of Wisconsin-Madison, 3Biomedical Engineering, University of Michigan, 4Department of Biological Sciences, Purdue University The electrode-tissue interface of neural recording electrodes can be characterized with electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Application of voltage biasing changes the electrochemical properties of the electrode-tissue interface and can improve recording capability. Voltage biasing, EIS, CV, and neural recordings are complementary. Neuroscience Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals Gregory J. Gage1, Colin R. Stoetzner1, Thomas Richner2, Sarah K. Brodnick2, Justin C. Williams2, Daryl R. Kipke1,3 1Biomedical Engineering, University of Michigan, 2Biomedical Engineering, University of Wisconsin-Madison, 3NeuroNexus Technologies We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model. Bioengineering A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo Anthony J. Sprangers1, Brian T. Freeman1, Nicholas A. Kouris1, Brenda M. Ogle2 1Department of Biomedical Engineering, University of Wisconsin-Madison, 2Department of Biomedical Engineering, Materials Science Program, Laboratory for Optical and Computational Instrumentation, University of Wisconsin-Madison A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of ˜1,000 cells in peripheral tissues. Medicine Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging Yu Huang*1,2, Basheal Agrawal*3, Paul A. Clark3, Justin C. Williams1,2,3, John S. Kuo3,4 1Department of Biomedical Engineering, University of Wisconsin-Madison, 2Materials Science Program, University of Wisconsin-Madison, 3Department of Neurological Surgery, University of Wisconsin-Madison, 4Carbone Comprehensive Cancer Center and Center for Stem Cell and Regenerative Medicine, University of Wisconsin-Madison A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies. Biology Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping Shih-Heng Su1, Katie A. Clark2, Nicole M. Gibbs1, Susan M. Bush1, Patrick J. Krysan1 1Horticulture Department, University of Wisconsin-Madison, 2Department of Zoology, Oregon State University The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings. Biology Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA Srikumar Sengupta1, Jennifer M. Bolin1, Victor Ruotti1, Bao Kim Nguyen1, James A. Thomson1,2,3, Angela L. Elwell1, Ron Stewart1 1Regenerative Biology, Morgridge Institute for Research, 2Department of Cell & Regenerative Biology, University of Wisconsin, 3Department of Molecular, Cellular, & Regenerative Biology, University of California Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts. Neuroscience Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient Pamela R. Westmark1, Cara J. Westmark1, Athavi Jeevananthan2, James S. Malter1 1Department of Pathology and Laboratory Medicine, Waisman Center for Developmental Disabilities, University of Wisconsin, 2Department of Biochemistry, Waisman Center for Developmental Disabilities, University of Wisconsin A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs. Biology A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract Lisa L. Abler1, Vatsal Mehta1, Kimberly P. Keil1, Pinak S. Joshi1, Chelsea-Leigh Flucus1, Heather A. Hardin1, Christopher T. Schmitz1, Chad M. Vezina1 1Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin-Madison Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species. Neuroscience Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats Aaron M. Johnson1,2, Emerald J. Doll2, Laura M. Grant2, Lauren Ringel2, Jaime N. Shier2, Michelle R. Ciucci1,2 1Department of Surgery-Division of Otolaryngology, University of Wisconsin, 2Department of Communicative Disorders, University of Wisconsin Voice disorders are debilitating in aging and Parkinson disease. The ultrasonic vocalizations of rats, also affected by these conditions, can be used to study these voice disorders, their neural substrates, and the nature of functional recovery with behavioral intervention. Biology Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow Rebecca R. Vanderpool1, Naomi C. Chesler1 1Department of Biomedical Engineering, University of Wisconsin – Madison The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload. Medicine Thermal Ablation for the Treatment of Abdominal Tumors Christopher L. Brace*1,2, J. Louis Hinshaw*2, Meghan G. Lubner*2 1Department of Biomedical Engineering, University of Wisconsin-Madison, 2Department of Radiology, University of Wisconsin-Madison A thermal tumor ablation procedure is described. The entire procedure is detailed, including pretreatment planning and imaging studies, anesthesia, adjuvant techniques to facilitate a percutaneous approach, imaging guidance of the ablation device to the tumor, thermal treatment, post-treatment care and follow-up. Neuroscience Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat Kutluay Uluç1, Amrendra Miranpuri1, Gregory C. Kujoth1, Erinç Aktüre1, Mustafa K. Başkaya1 1Department of Neurological Surgery, School of Medicine and Public Health, University of Wisconsin-Madison Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown. Neuroscience Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons Christopher Viesselmann*1, Jason Ballweg*1, Derek Lumbard*1, Erik W. Dent1 1Department of Anatomy, University of Wisconsin-Madison This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis. Biology Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development Katie E. Holmes1, Matthew J. Wyatt2, Yu-chi Shen2, Deborah A. Thompson2,3, Kate F. Barald2,4 1Department of Veterinary Science, University of Wisconsin, Madison, 2Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, 3Present address: Department of Pulmonary Medicine, University of Michigan, Ann Arbor, MI, 4Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear. Immunology and Infection Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells Marina Durward1, Jerome Harms2, Gary Splitter2 1Pathology and Laboratory Medicine, University of Wisconsin-Madison, 2Pathobiological Sciences, University of Wisconsin-Madison Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4. Immunology and Infection Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions Crystal Tobin1, Angela Pollard1, Laura Knoll1 1Department of Medical Microbiology and Immunology, University of Wisconsin Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells. Biology Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos Erica Andersen1,2,3, Namrata Asuri1,2,3, Matthew Clay2,3,4, Mary Halloran1,2,3,4 1Genetics Training Program, University of Wisconsin-Madison, 2Department of Anatomy, University of Wisconsin-Madison, 3Department of Zoology, University of Wisconsin-Madison, 4Cell and Molecular Biology Training Program, University of Wisconsin-Madison This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy. Biology Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging Melissa Paulite1, Zahra Fakhraai2, Boris B. Akhremitchev3, Kerstin Mueller1, Gilbert C. Walker1 1Department of Chemistry, University of Toronto, 2Department of Chemistry, University of Wisconsin, 3Department of Chemistry, Duke University The assembly of a nearfield infrared microscope for imaging protein aggregates is described. Biology High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices Pedro J. Resto1, Brian Mogen2, Fan Wu2, Erwin Berthier2, David Beebe2, Justin Williams2 1Materials Science Program, University of Wisconsin-Madison, 2Department of Biomedical Engineering, University of Wisconsin-Madison A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation. Biology Using an EEG-Based Brain-Computer Interface for Virtual Cursor Movement with BCI2000 J. Adam Wilson1, Gerwin Schalk2, Léo M. Walton1, Justin C. Williams1 1Department of Biomedical Engineering, University of Wisconsin-Madison, 2Wadsworth Center, New York State Dept. of Health In this video, we demonstrate the steps required to run a brain-computer interface experiment, including setting up the EEG cap, calibrating the system, and training the user to move a cursor in two dimensions using imagined movements.
