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University of Wisconsin-Madison

64 ARTICLES PUBLISHED IN JoVE

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Biology

Using an EEG-Based Brain-Computer Interface for Virtual Cursor Movement with BCI2000
J. Adam Wilson 1, Gerwin Schalk 2, Léo M. Walton 1, Justin C. Williams 1
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.

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Biology

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices
Pedro J. Resto 1, Brian Mogen 2, Fan Wu 2, Erwin Berthier 2, David Beebe 2, Justin Williams 2
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.

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Biology

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos
Erica Andersen 1,2,3, Namrata Asuri 1,2,3, Matthew Clay 2,3,4, Mary Halloran 1,2,3,4
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.

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Neuroscience

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat
Kutluay Uluç 1, Amrendra Miranpuri 1, Gregory C. Kujoth 1, Erinç Aktüre 1, Mustafa K. Başkaya 1
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.

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Immunology and Infection

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells
Marina Durward 1, Jerome Harms 2, Gary Splitter 2
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.

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Neuroscience

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons
Christopher Viesselmann *1, Jason Ballweg *1, Derek Lumbard *1, Erik W. Dent 1
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.

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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.

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Biology

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract
Lisa L. Abler 1, Vatsal Mehta 1, Kimberly P. Keil 1, Pinak S. Joshi 1, Chelsea-Leigh Flucus 1, Heather A. Hardin 1, Christopher T. Schmitz 1, Chad M. Vezina 1
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.

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JoVE Journal

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping
Shih-Heng Su 1, Katie A. Clark 2, Nicole M. Gibbs 1, Susan M. Bush 1, Patrick J. Krysan 1
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.

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Medicine

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging
Yu Huang *1,2, Basheal Agrawal *3, Paul A. Clark 3, Justin C. Williams 1,2,3, John S. Kuo 3,4
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.

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Neuroscience

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals
Gregory J. Gage 1, Colin R. Stoetzner 1, Thomas Richner 2, Sarah K. Brodnick 2, Justin C. Williams 2, Daryl R. Kipke 1,3
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.

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Education

Voltage Biasing, Cyclic Voltammetry, & Electrical Impedance Spectroscopy for Neural Interfaces
Seth J. Wilks 1, Tom J. Richner 2, Sarah K. Brodnick 2, Daryl R. Kipke 3, Justin C. Williams 2, Kevin J. Otto 1,4
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.

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Bioengineering

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo
Anthony J. Sprangers 1, Brian T. Freeman 1, Nicholas A. Kouris 1, Brenda M. Ogle 2
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.

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Immunology and Infection

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence
Elizabeth Hussa 1, Heidi Goodrich-Blair 1
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.

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Biology

Visualizing Bacteria in Nematodes using Fluorescent Microscopy
Kristen E. Murfin 1, John Chaston 1, Heidi Goodrich-Blair 1
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.

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Biology

Trajectory Data Analyses for Pedestrian Space-time Activity Study
Feng Qi 1, Fei Du 2
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.

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Biology

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination
Joshua C. Neuman 1, Nathan A. Truchan 2, Jamie W. Joseph 3, Michelle E. Kimple 2
1Department of Nutrional Sciences, University of Wisconsin-Madison, 2Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Wisconsin-Madison, 3School of Pharmacy, University of Waterloo

Assaying in vitro β-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining β-cell function.

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Biology

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae
R. Scott McIsaac 1,2, Sanford J. Silverman 1, Lance Parsons 1, Ping Xu 1, Ryan Briehof 1, Megan N. McClean 1, David Botstein 1,3
1The Lewis-Sigler Institute for Integrative Genomics, Princeton University, 2Graduate Program in Quantitative and Computational Biology, Princeton University, 3Department of Molecular Biology, Princeton University

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

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Medicine

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia
Tristan M. Nicholson 1,2, Kristen S. Uchtmann 1, Conrad D. Valdez 1, Ashleigh B. Theberge 1,3, Tihomir Miralem 1, William A. Ricke 1
1Department of Urology, University of Wisconsin-Madison, 2Medical Scientist (MD/PhD) Training Program, University of Rochester School of Medicine & Dentistry, 3Molecular and Environmental Toxicology Center, University of Wisconsin-Madison

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

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Biology

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila
Daniel T. Babcock 1, Barry Ganetzky 1
1Department of Genetics, University of Wisconsin-Madison

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

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Biology

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules
Erin Gemperline 1, Lingjun Li 1,2
1Department of Chemistry, University of Wisconsin- Madison, 2School of Pharmacy, University of Wisconsin- Madison

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described. 

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Behavior

Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat
Daniel E. Bradford *1, Katherine P. Magruder *1, Rachel A. Korhumel 1, John J. Curtin 1
1Department of Psychology, University of Wisconsin-Madison

Potentiation of the startle reflex is measured via electromyography of the orbicularis oculi muscle during low (uncertain) and high (certain) probability electric shock threat in the Threat Probability Task. This provides an objective measure of distinct negative emotional states (fear/anxiety) for research on psychopathology, substance use/abuse, and broad affective science.

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JoVE Core

Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers
Sarah M. Collier 1, Matthew D. Ruark 2, Lawrence G. Oates 3,4, William E. Jokela 5, Curtis J. Dell 6
1Office of Sustainability, University of Wisconsin-Madison, 2Department of Soil Science, University of Wisconsin-Madison, 3Department of Agronomy, University of Wisconsin-Madison, 4Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, 5USDA-ARS Dairy Forage Research Center, 6USDA-ARS Pasture Systems Watershed Management Research Unit

This article showcases the static chamber-based method for measurement of greenhouse gas flux from soil systems. With relatively modest infrastructure investments, measurements may be obtained from multiple treatments/locations and over timeframes ranging from hours to years.

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Behavior

Methods to Test Visual Attention Online
Amanda Yung 1, Pedro Cardoso-Leite 2, Gillian Dale 3, Daphne Bavelier 2,4, C. Shawn Green 3
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.

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Immunology and Infection

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection
Elizabeth A. Harvie 1,2, Anna Huttenlocher 2,3
1Microbiology Doctoral Training Program, University of Wisconsin-Madison, 2Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, 3Department of Pediatrics, University of Wisconsin-Madison

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

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Neuroscience

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila
Rebeccah J. Katzenberger 1, Carin A. Loewen 2, R. Tayler Bockstruck 1, Mikal A. Woods 3, Barry Ganetzky 2, David A. Wassarman 1
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.

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Developmental Biology

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells
Irina Elcheva 1, Vera Brok-Volchanskaya 1, Igor Slukvin 2
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.

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Engineering

Iridium Oxide-reduced Graphene Oxide Nanohybrid Thin Film Modified Screen-printed Electrodes as Disposable Electrochemical Paper Microfluidic pH Sensors
Jiang Yang 1,2, Tae-Joon Kwak 3, Xiaodong Zhang 4, Robert McClain 5, Woo-Jin Chang *3,6, Sundaram Gunasekaran *1
1Department of Biological Systems Engineering, University of Wisconsin-Madison, 2Environment, Energy and Natural Resources Center, Department of Environmental Science and Engineering, Fudan University, 3Department of Mechanical Engineering, University of Wisconsin-Milwaukee, 4Department of Physics, School of Science, Tianjin University, 5Department of Chemistry, University of Wisconsin-Madison, 6School of Freshwater Sciences, University of Wisconsin-Milwaukee

The study demonstrates the growth of iridium oxide-reduced graphene oxide (IrO2-RGO) nanohybrid thin films on irregular and rough screen-printed carbon substrate through a green electrochemical synthesis, and their implementation as a pH sensor with a patterned paper-fluidic platform.

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Biology

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging
Tyler M. Bauman 1,2, Emily A. Ricke 2, Sally A. Drew 3, Wei Huang 3,4, William A. Ricke 2,4
1Division of Urologic Surgery, Washington University in St. Louis School of Medicine, 2Department of Urology, University of Wisconsin School of Medicine and Public Health, 3Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, 4O’Brien Urology Research Center, University of Wisconsin School of Medicine and Public Health

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

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Bioengineering

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo
Brian Burkel 1,2, Brett A. Morris 1, Suzanne M. Ponik 1, Kristin M. Riching 1, Kevin W. Eliceiri 2,3,4, Patricia J. Keely 1,2,5
1Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 2Laboratory for Optical and Computational Instrumentation, University of Wisconsin-Madison, 3Department of Biomedical Engineering, University of Wisconsin-Madison, 4Morgridge Institute for Research, University of Wisconsin-Madison, 5Paul P. Carbone Comprehensive Cancer center, University of Wisconsin-Madison

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

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Biochemistry

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes
Chen Ji 1, Xuelin Lou 1
1Department of Neuroscience, University of Wisconsin-Madison

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale.

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JoVE Journal

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts
Keren B. Turton 1, Stephane Esnault 2, Larissa P. Delain 2, Deane F. Mosher 1,2
1Department of Biomolecular Chemistry, University of Wisconsin-Madison, 2Department of Medicine, University of Wisconsin-Madison

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and α/β C-termini, can be quantified.

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Developmental Biology

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment
Destiny L. Baars 1, Kendra A. Takle *1,2, Jonathon Heier *1,3, Francisco Pelegri 1
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.

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JoVE Journal

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health
Shawn A. Steffan 1,2, Prarthana S. Dharampal 2, Luis Diaz-Garcia 3,4, Cameron R. Currie 5, Juan Zalapa 1,3, Chris Todd Hittinger 6,7,8
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.

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Bioengineering

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications
Cameron J. Stewart 1, Megan N. McClean 1
1Department of Biomedical Engineering, University of Wisconsin-Madison

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

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Developmental Biology

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish
Elaine L. Welch *1, Celeste C. Eno *1, Sreelaja Nair 2, Robin E. Lindeman 3, Francisco Pelegri 1
1Laboratory of Genetics, University of Wisconsin-Madison, 2Department of Biological Sciences, Tata Institute of Fundamental Research, 3Department of Genetics, Cell Biology, and Development, University of Minnesota

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

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Environment

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples
Lawrence G. Oates 1, Harry W. Read 2, Jessica L. M. Gutknecht 3, David S. Duncan 1, Teri B. Balser 4, Randall D. Jackson 1
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.

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Bioengineering

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae
Kayla Huemer *1,2, Jayne M. Squirrell *3, Robert Swader 2, Kirsten Pelkey 2, Danny C. LeBert 4, Anna Huttenlocher 5,6, Kevin W. Eliceiri 1,2,3
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.

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Cancer Research

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol
Tanner J. McArdle 1, Brenda M. Ogle 1,2,3,4,5, Felicite K. Noubissi 6
1Department of Biomedical Engineering, University of Minnesota - Twin Cities, 2Stem Cell Institute, University of Minnesota - Twin Cities, 3Masonic Cancer Center, University of Minnesota - Twin Cities, 4Lillehei Heart Institute, University of Minnesota - Twin Cities, 5Institute for Engineering in Medicine, University of Minnesota - Twin Cities, 6Department of Biology, Jackson State University

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

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Chemistry

Reactive Vapor Deposition of Conjugated Polymer Films on Arbitrary Substrates
Nongyi Cheng 1,2, Trisha L. Andrew 2,3
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.

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JoVE Core

Stress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray Diffraction
Cecilia S.N. Cheung 1,2, Donald J. Weidner 1, Li Li 1, Philip G. Meredith 3, Haiyan Chen 1, Matthew Whitaker 1, Xianyin Chen 4
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.

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Developmental Biology

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes
Jiayin Yang *1,2,6, Lai-Yung Wong *2, Xiao-Yu Tian *3, Rui Wei 1,2, Wing-Hon Lai 2, Ka-Wing Au 2, Zhiwei Luo 4,5, Carl Ward 4,5, Wai-In Ho 2, David P. Ibañez 4,5, Hao Liu 4,5, Xichen Bao 4,5, Baoming Qin 4,5, Yu Huang 3, Miguel A. Esteban 4,5,7, Hung-Fat Tse 1,2,6,7
1Department of Medicine, University of Hong Kong-Shenzhen Hospital, 2The Cardiology Division, Department of Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, 3School of Biomedical Sciences, Institute of Vascular Medicine, Li Ka Shing Institute of Health Sciences, Chinese University of Hong Kong, 4Key Laboratory of Regenerative Biology of the Chinese Academy of Sciences, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health and Guangzhou Medical University, 5Laboratory of RNA, Chromatin, and Human Disease, CAS Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 6Research Centre of Heart, Brain, Hormone, and Healthy Ageing, Li Ka Shing Faculty of Medicine, University of Hong Kong, 7Hong Kong-Guangdong Stem Cell and Regenerative Medicine Research Centre, University of Hong Kong and Guangzhou Institutes of Biomedicine and Health

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

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Bioengineering

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications
Michal Adamski 1, Gianluca Fontana 2, Joshua R. Gershlak 4, Glenn R. Gaudette 4, Hau D. Le 1, William L. Murphy 2,3
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.

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Environment

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors
Prarthana S. Dharampal 1, Caitlin M. Carlson 2, Luis Diaz-Garcia 3,4, Shawn A. Steffan 1,5
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.

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Bioengineering

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator
Benjamin Kerzner 1, Hannah L. Martin 2, Michael Weiser 1, Gianluca Fontana 1, Nicholas Russell 1, William L. Murphy 1,2, Erik A. Lund 3, Christopher J. Doro 1
1Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, 2Department of Biomedical Engineering, University of Wisconsin-Madison, 3Summit Orthopedics

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

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Genetics

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster
Olivia Gevedon 1, Harris Bolus 1, Shu Hui Lye 1, Keaton Schmitz 1, Jesualdo Fuentes-González 1, Kathryn Hatchell 2, Lyndsey Bley 2, Jason Pienaar 1, Carin Loewen 2, Stanislava Chtarbanova 1
1Department of Biological Sciences, University of Alabama, 2Department of Genetics, University of Wisconsin-Madison

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

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Medicine

Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing
Teresa T. Liu *1,2, Allison C. Rodgers *3, Tristan M. Nicholson 4, Jill A. Macoska 5,6, Paul C. Marker 6,7, Chad M. Vezina 6,8, Dale E. Bjorling 6,8, Alejandro Roldan-Alzate 2,9,10, Diego Hernando 10,11, Granville L. Lloyd 12, Timothy A. Hacker 3, William A. Ricke 1,6
1Department of Urology, University of Wisconsin-Madison, 2K12 Kure, University of Wisconsin-Madison, 3Cardiovascular Research Center, Department of Medicine, University of Wisconsin-Madison, 4Department of Urology, University of Washington, 5University of Massachusetts Boston, 6U54 George M. O'Brien Center, University of Wisconsin-Madison, 7College of Pharmacy, University of Wisconsin-Madison, 8School of Veterinary Medicine, University of Wisconsin-Madison, 9Department of Mechanical Engineering, University of Wisconsin-Madison, 10Department of Radiology, University of Wisconsin-Madison, 11Department of Medical Physics, University of Wisconsin-Madison, 12Rocky Mountain Regional VA Medical Center, University of Colorado

We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD).

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A Balanced Test of Comprehension Versus Production Practice using Artificial Language Learning
Elise W. M. Hopman 1, Mackenzie Ludin 1, Maryellen C. MacDonald 1
1Department of Psychology, University of Wisconsin-Madison

The goal of this protocol is to evaluate comprehension versus production language learning training through a computer-based experiment in a way that balances inherent task differences between comprehension and production.

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Immunology and Infection

High Throughput Co-culture Assays for the Investigation of Microbial Interactions
Mia I. Temkin 1, Caitlin M. Carlson 1, Aaron L. Stubbendieck 2, Cameron R. Currie 1, Reed M. Stubbendieck 1
1Department of Bacteriology, University of Wisconsin-Madison, 2Independent Scholar

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

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Neuroscience

Cochlear Surface Preparation in the Adult Mouse
Qiao-Jun Fang 1,2, Fan Wu 1, Renjie Chai 2, Su-Hua Sha 1
1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2MOE Key Laboratory of Developmental Genes and Human Disease, Institute of Life Sciences, Southeast University

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

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Neuroscience

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration
Jessica M. Sidisky 1, Daniel T. Babcock 1
1Department of Biological Sciences, Lehigh University

The goal of this procedure is to dissect the dorsal longitudinal muscle (DLM) tissue to assess the structural integrity of DLM neuromuscular junctions (NMJs) in neurodegenerative disease models using Drosophila melanogaster.

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Bioengineering

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools
Yuming Liu 1, Kevin W. Eliceiri 1,2,3
1Laboratory for Optical and Computational Instrumentation, Center for Quantitative Cell Imaging, University of Wisconsin-Madison, 2Departments of Medical Physics and Biomedical Engineering, University of Wisconsin-Madison, 3Morgridge Institute for Research, Madison, Wisconsin, 4test, test

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

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Neuroscience

Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence
Christopher S. Morrow 1, Amani A. Gillette 2,3, Melissa C. Skala 2,3, Darcie L. Moore 1
1Department of Neuroscience, University of Wisconsin-Madison, 2Morgridge Institute for Research, 3Department of Biomedical Engineering, University of Wisconsin-Madison

This protocol describes strategies to identify and enrich for cell-state in primary adult mouse neural stem cell cultures by autofluorescence imaging using i) a confocal microscope, ii) a fluorescent activated cell sorter to perform intensity imaging, or iii) a multiphoton microscope to perform fluorescence lifetime imaging.

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Genetics

Determining the Role of Maternally-Expressed Genes in Early Development with Maternal Crispants
Cara E. Moravec 1, Gabriella C. Voit 1, Francisco Pelegri 1
1Laboratory of Genetics, University of Wisconsin-Madison

Early development is dependent on maternally-inherited products, and the role of many of these products is currently unknown. Herein, we described a protocol that uses CRISPR-Cas9 to identify maternal-effect phenotypes in a single generation.

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Biology

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology
Martin K. Ventura 1, Valerie J. Stull 2, Susan M. Paskewitz 1
1Department of Entomology, University of Wisconsin-Madison, 2Global Health Institute, University of Wisconsin-Madison

This paper outlines basic methods to standardize important factors such as density, feed availability, hydration source, and environmental controls for the long-term rearing of laboratory cultures of the edible cricket, Gryllus bimaculatus.

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Chemistry

Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus
Ashley Phetsanthad *1, Nhu Q. Vu *1, Lingjun Li 1,2
1Department of Chemistry, University of Wisconsin-Madison, 2School of Pharmacy, University of Wisconsin-Madison

Mass spectrometric characterization of neuropeptides provides sequence, quantitation, and localization information. This optimized workflow is not only useful for neuropeptide studies, but also other endogenous peptides. The protocols provided here describe sample preparation, MS acquisition, MS analysis, and database generation of neuropeptides using LC-ESI-MS, MALDI-MS spotting, and MALDI-MS imaging.

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Cancer Research

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment
Brian M. Burkel 1, David R. Inman 1, María Virumbrales-Muñoz 1,2, Erica J. Hoffmann 1, Suzanne M. Ponik 1,3
1Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 2Department of Pathology, University of Wisconsin-Madison, 3Carbone Cancer Center, University of Wisconsin-Madison

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

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Biochemistry

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues
Dylan Nicholas Tabang 1, Danqing Wang 1, Lingjun Li 1,2
1Department of Chemistry, University of Wisconsin-Madison, 2School of Pharmacy, University of Wisconsin-Madison

Post-translational modifications (PTMs) change protein structures and functions. Methods for the simultaneous enrichment of multiple PTM types can maximize coverage in analyses. We present a protocol using dual-functional Ti(IV)-immobilized metal affinity chromatography followed by mass spectrometry for the simultaneous enrichment and analysis of protein N-glycosylation and phosphorylation in pancreatic tissues.

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Behavior

Four Temporary Waterslide Designs Adapted to Different Slope Conditions to Encourage Child Socialization in Playgrounds
Zhudi Hua 1, Ting Tao 1, Risa Akita 1, Tomofusa Akita 1, Yoshiaki Hayakawa 1, Masanori Hariyama 2, Hayato Sakurai 3, Ricki Colman 4, Mamiko Koshiba 1,2,3
1Graduate School of Sciences and Technology for Innovation, Yamaguchi University, 2Graduate School of Information Sciences, Tohoku University, 3Pediatrics, Saitama Medical University Hospital, 4Department of Cell and Regenerative Biology, University of Wisconsin-Madison

Early life social learning is enhanced by interactions with effectively designed environments. Four events were held at different city parks using inexpensive, temporary waterslides to stimulate social learning. This study describes the prototypes used and the evaluation of the children's interactions.

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Genetics

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration
Michael Landowski 1,2, Samuel Grindel 1, Ying Hao 3, Sakae Ikeda 1,2, Catherine Bowes Rickman 3,4, Akihiro Ikeda 1,2
1Department of Medical Genetics, University of Wisconsin-Madison, 2McPherson Eye Research Institute, University of Wisconsin-Madison, 3Department of Ophthalmology, Duke University, 4Department of Cell Biology, Duke University

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

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Genetics

The Serial Anesthesia Array for the High-Throughput Investigation of Volatile Agents Using Drosophila melanogaster
Zachariah P. G. Olufs 1, Dena Johnson-Schlitz 1, David A. Wassarman 2, Misha Perouansky 1
1Department of Anesthesiology, University of Wisconsin-Madison, 2Department of Medical Genetics, University of Wisconsin-Madison

The fruit fly (Drosophila melanogaster) is widely used for biological and toxicological research. To expand the utility of flies, we developed an instrument, the serial anesthesia array, that simultaneously exposes multiple fly samples to volatile general anesthetics (VGAs), making it possible to investigate the collateral effects (toxic and protective) of VGAs.

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Bioengineering

High-Throughput Optogenetics Experiments in Yeast Using the Automated Platform Lustro
Zachary P. Harmer 1, Megan N. McClean 1,2
1Department of Biomedical Engineering, University of Wisconsin-Madison, 2University of Wisconsin Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health

This protocol outlines the steps for utilizing the automated platform Lustro to perform high-throughput characterization of optogenetic systems in yeast.

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Medicine

Heterotopic Auxiliary Whole Liver Rat Transplant Model Utilizing a Hepaticoureterostomy for Allograft Rejection Studies
Bret Verhoven 1, Weifeng Zeng 2, Peter Chlebeck 1, Kristina Matkowskyj 3,4,5, Heather Jennings 1, Samuel Poore 2, David Al-Adra 1
1Department of Surgery, School of Medicine and Public Health, University of Wisconsin-Madison, 2Department of Surgery, Division of Plastic and Reconstructive Surgery, University of Wisconsin, 3Department of Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin-Madison, 4UW Carbone Cancer Center, 5William S. Middleton Memorial Veterans' Hospital

The rat heterotopic auxiliary liver transplant protocol described here offers a practical investigational tool for exploring mechanisms of hepatic allograft rejection. This model helps to alleviate the surgical hurdles and animal stress of orthotopic liver transplantation in rats.

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Genetics

Monochrome Multiplex Quantitative PCR Telomere Length Measurement
Noelle A. Martin *1, Lauren W. Y. McLester-Davis *2,3,4, Trevor R. Roy 1, Madeline G. Magruder 5, Waylon J. Hastings 1, Stacy S. Drury 6
1School of Medicine, Tulane University, 2Native American Center for Health Professions, University of Wisconsin-Madison, 3Department of Medicine, University of Wisconsin-Madison, 4Department of Biochemistry, University of Wisconsin-Madison, 5School of Science and Engineering, Tulane University, 6Department of Psychiatry and Behavioral Sciences, Boston Children’s Hospital

Here we present a protocol for the measurement of relative telomere length (TL) using the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay. The MMqPCR assay is a repeatable, efficient, and cost-effective method for measuring TL from human DNA in population-based studies.

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