University of Wisconsin School of Medicine and Public Health 9 articles published in JoVE Biology Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice Andrea Galmozzi1,2, Bernard P. Kok1, Enrique Saez1 1Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA, 2Department of Medicine, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, WI, USA This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture. Developmental Biology Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging Rebecca J. Salamon1, Ziheng Zhang1, Ahmed I. Mahmoud1 1Department of Cell and Regenerative Biology, University of Wisconsin-Madison School of Medicine and Public Health Cardiomyocyte proliferation following injury is a dynamic process that requires a symphony of extracellular cues from non-myocyte cell populations. Utilizing lineage tracing, passive CLARITY, and three-dimensional whole-mount confocal microscopy techniques, we can analyze the influence of a variety of cell types on cardiac repair and regeneration. Cancer Research Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad Lauren B. Shunkwiler1, Jill D. Haag2, Michael N. Gould2, Bart M. G. Smits1 1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2Department of Oncology, McArdle Laboratory for Cancer Research, University of Wisconsin-Madison School of Medicine and Public Health This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique. 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. Biochemistry Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes Megan E. Dowdle1, Susanne Blaser Imboden1, Sookhee Park1, Sean P. Ryder2, Michael D. Sheets1 1Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, 2Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis. Biology Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging Tyler M. Bauman1,2, Emily A. Ricke2, Sally A. Drew3, Wei Huang3,4, William A. Ricke2,4 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. 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. Developmental Biology Immunostaining to Visualize Murine Enteric Nervous System Development Amanda J. Barlow-Anacker1, Christopher S. Erickson1, Miles L. Epstein2, Ankush Gosain1,2,3 1Department of Surgery, University of Wisconsin School of Medicine and Public Health, 2Department of Neurosciences, University of Wisconsin School of Medicine and Public Health, 3Department of Pediatrics, University of Wisconsin School of Medicine and Public Health The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression. Medicine A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury Toshio Takayama1, Xudong Shi1, Bowen Wang1, Sarah Franco1, Yifan Zhou1, Daniel DiRenzo1, Alycia Kent1, Peter Hartig1, Joshua Zent1, Lian-Wang Guo1 1Department of Surgery, University of Wisconsin School of Medicine and Public Health The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent neointima for studies of restenosis.
