University of Wisconsin-Madison

28 articles published in JoVE

• Genetics

  
  A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration Michael Landowski^1,2, Samuel Grindel¹, Ying Hao³, Sakae Ikeda^1,2, Catherine Bowes Rickman^3,4, Akihiro Ikeda^{1,2 1}Department 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.

• Genetics

  
  The Serial Anesthesia Array for the High-Throughput Investigation of Volatile Agents Using Drosophila melanogaster Zachariah P. G. Olufs¹, Dena Johnson-Schlitz¹, David A. Wassarman², Misha Perouansky^{1 1}Department 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.

• Behavior

  
  Four Temporary Waterslide Designs Adapted to Different Slope Conditions to Encourage Child Socialization in Playgrounds Zhudi Hua¹, Ting Tao¹, Risa Akita¹, Tomofusa Akita¹, Yoshiaki Hayakawa¹, Masanori Hariyama², Hayato Sakurai³, Ricki Colman⁴, Mamiko Koshiba^{1,2,3 1}Graduate 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.

• Biology

  
  Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology Martin K. Ventura¹, Valerie J. Stull², Susan M. Paskewitz^{1 1}Department 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.

• Chemistry

  
  Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus Ashley Phetsanthad*¹, Nhu Q. Vu*¹, Lingjun Li^{1,2 1}Department 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.

• Cancer Research

  
  A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment Brian M. Burkel¹, David R. Inman¹, María Virumbrales-Muñoz^1,2, Erica J. Hoffmann¹, Suzanne M. Ponik^{1,3 1}Department 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.

• Biochemistry

  
  A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues Dylan Nicholas Tabang¹, Danqing Wang¹, Lingjun Li^{1,2 1}Department 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.

• Genetics

  
  Determining the Role of Maternally-Expressed Genes in Early Development with Maternal Crispants Cara E. Moravec¹, Gabriella C. Voit¹, Francisco Pelegri^{1 1}Laboratory 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.

• Bioengineering

  
  Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools Yuming Liu¹, Kevin W. Eliceiri^{1,2,3 1}Laboratory 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.

• Immunology and Infection

  
  High Throughput Co-culture Assays for the Investigation of Microbial Interactions Mia I. Temkin¹, Caitlin M. Carlson¹, Aaron L. Stubbendieck², Cameron R. Currie¹, Reed M. Stubbendieck^{1 1}Department 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.

• Medicine

  
  Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing Teresa T. Liu*^1,2, Allison C. Rodgers*³, Tristan M. Nicholson⁴, 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. Lloyd¹², Timothy A. Hacker³, William A. Ricke^{1,6 1}Department 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).

• Genetics

  
  In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster Olivia Gevedon¹, Harris Bolus¹, Shu Hui Lye¹, Keaton Schmitz¹, Jesualdo Fuentes-González¹, Kathryn Hatchell², Lyndsey Bley², Jason Pienaar¹, Carin Loewen², Stanislava Chtarbanova^{1 1}Department 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.

• Bioengineering

  
  A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator Benjamin Kerzner¹, Hannah L. Martin², Michael Weiser¹, Gianluca Fontana¹, Nicholas Russell¹, William L. Murphy^1,2, Erik A. Lund³, Christopher J. Doro^{1 1}Department 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.

• Developmental Biology

  
  Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish Elaine L. Welch*¹, Celeste C. Eno*¹, Sreelaja Nair², Robin E. Lindeman³, Francisco Pelegri^{1 1}Laboratory 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.

• Bioengineering

  
  Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications Cameron J. Stewart¹, Megan N. McClean^{1 1}Department 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.

• Biology

  
  High-resolution Optical Mapping of the Mouse Sino-atrial Node Di Lang¹, Alexey V. Glukhov^{1 1}Department of Medicine, University of Wisconsin-Madison School of Medicine and Public Health Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.

• 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 Kwak³, Xiaodong Zhang⁴, Robert McClain⁵, Woo-Jin Chang*^3,6, Sundaram Gunasekaran*^{1 1}Department 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.

• Neuroscience

  
  Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia Vishal Chanana¹, Abdulfettah Tumturk², Douglas Kintner¹, Eshwar Udho¹, Peter Ferrazzano^1,3, Pelin Cengiz^{1,3 1}Waisman Center, University of Wisconsin, ²Department of Neurological Surgery, University of Wisconsin, ³Department of Pediatrics, University of Wisconsin Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

• Biochemistry

  
  Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes Chen Ji¹, Xuelin Lou^{1 1}Department 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.

• Genetics

  
  Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts Keren B. Turton¹, Stephane Esnault², Larissa P. Delain², Deane F. Mosher^{1,2 1}Department 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.

• Bioengineering

  
  Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo Brian Burkel^1,2, Brett A. Morris¹, Suzanne M. Ponik¹, Kristin M. Riching¹, Kevin W. Eliceiri^2,3,4, Patricia J. Keely^{1,2,5 1}Department 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.

• 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 1}Microbiology 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.

• 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. Korhumel¹, John J. Curtin^{1 1}Department 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.

• Environment

  
  Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers Sarah M. Collier¹, Matthew D. Ruark², Lawrence G. Oates^3,4, William E. Jokela⁵, Curtis J. Dell^{6 1}Office 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.

• Biology

  
  A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination Joshua C. Neuman¹, Nathan A. Truchan², Jamie W. Joseph³, Michelle E. Kimple^{2 1}Department 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.

• Biology

  
  MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules Erin Gemperline¹, Lingjun Li^{1,2 1}Department 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. 

• Biology

  
  An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila Daniel T. Babcock¹, Barry Ganetzky^{1 1}Department 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.

• 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. Uchtmann¹, Conrad D. Valdez¹, Ashleigh B. Theberge^1,3, Tihomir Miralem¹, William A. Ricke^{1 1}Department 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.