Indiana University View Institution's Website 28 articles published in JoVE Developmental Biology Preparation and Immunofluorescence Staining of Bundles and Single Fiber Cells from the Cortex and Nucleus of the Eye Lens Michael P. Vu1, Catherine Cheng1 1School of Optometry and Vision Science Program, Indiana University This protocol describes methods to prepare peripheral, mature, and nuclear eye lens fiber cells for immunofluorescence staining to study complex cell-to-cell interdigitations and the membrane architecture. Biology Assessment of Chemical Toxicity in Adult Drosophila Melanogaster Jessica M. Holsopple*1,2, Shannon R. Smoot*1, Ellen M. Popodi1,2, John K. Colbourne3, Joseph R. Shaw4, Brian Oliver5, Thomas C. Kaufman1, Jason M. Tennessen1 1Department of Biology, Indiana University, 2Bloomington Drosophila Stock Center, Department of Biology, Indiana University, 3School of Biosciences, University of Birmingham, 4O'Neill School of Public and Environmental Affairs, Indiana University, 5Section of Developmental Genomics, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Kidney and Digestive Diseases, National Institutes of Health This protocol describes an efficient and inexpensive method that uses liquid media to assess the effects of chemical toxicants on the viability of adult Drosophila melanogaster. Biology Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae Gisela Cairo*1, Anne MacKenzie*1, Dai Tsuchiya1,2, Soni Lacefield1 1Department of Biology, Indiana University, 2Stowers Institute for Biomedical Research Time-lapse microscopy is a valuable tool for studying meiosis in budding yeast. This protocol describes a method that combines cell-cycle synchronization, time-lapse microscopy, and conditional depletion of a target protein to demonstrate how to study the function of a specific protein during meiotic chromosome segregation. Biology Artificial Intelligence Approaches to Assessing Primary Cilia Ruchi Bansal1, Staci E. Engle1, Tisianna K. Kamba1, Kathryn M. Brewer1, Wesley R. Lewis2, Nicolas F. Berbari1,3,4 1Department of Biology, Indiana University-Purdue University Indianapolis, 2Nikon Instruments Inc., 3Stark Neurosciences Research Institute, Indiana University, 4Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine The use of artificial intelligence (Ai) to analyze images is emerging as a powerful, less biased, and rapid approach compared with commonly used methods. Here we trained Ai to recognize a cellular organelle, primary cilia, and analyze properties such as length and staining intensity in a rigorous and reproducible manner. Biochemistry Screening for Phytoestrogens using a Cell-based Estrogen Receptor β Reporter Assay Emily M. Chester1, Emily Fender1, Michael D. Wasserman1 1Department of Anthropology, Indiana University We have optimized a commercially available estrogen receptor β reporter assay for screening human and nonhuman primate foods for estrogenic activity. We validated this assay by showing that the known estrogenic human food soy registers high, while other foods show no activity. Neuroscience Collection of Frozen Rodent Brain Regions for Downstream Analyses Jim Wager-Miller1, Michelle Murphy Green1, Hana Shafique1, Ken Mackie1 1Department of Psychological and Brain Sciences, Gill Center for Biomolecular Research, Indiana University This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools. Immunology and Infection Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins Asha A. Philip1, Jin Dai1, Sarah P. Katen1, John T. Patton1 1Department of Biology, Indiana University Generation of recombinant rotaviruses from plasmid DNA provides an essential tool for the study of rotavirus replication and pathogenesis, and the development of rotavirus expression vectors and vaccines. Herein, we describe a simplified reverse genetics approach for generating recombinant rotaviruses, including strains expressing fluorescent reporter proteins. Developmental Biology A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos Sanat Upadhyay1, Leoncio Vergara2, Pranjali Shah2, Jan-Åke Gustafsson3,4, Ioannis Kakadiaris1,4, Maria Bondesson5 1Computational Biomedicine Lab, Texas Institute of Measurement Evaluation and Statistics, University of Houston, 2Institute of Biosciences and Technology, Texas A&M Health Science Center, 3Department of Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, University of Houston, 4Department of Biosciences and Nutrition, Novum, Karolinska Institutet, 5Department of Intelligent Systems Engineering, Indiana University This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations. Bioengineering Spatial Temporal Analysis of Fieldwise Flow in Microvasculature Sherry G. Clendenon1,2, Xiao Fu1,3, Robert A. Von Hoene1, Jeffrey L. Clendenon4, James P. Sluka1,2, Seth Winfree5, Henry Mang5, Michelle Martinez5, Adele Filson5, James E. Klaunig6, James A. Glazier1,2, Kenneth W. Dunn5 1Biocomplexity Institute, Indiana University, 2Department of Intelligent Systems Engineering, Indiana University, 3Department of Physics, Indiana University, 4Scientific Designs, 5Department of Medicine, Indiana University, 6School of Public Health, Indiana University To quantify microvascular flow from high speed capillary flow image sequences, we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow) software. Across the full image field and over time, STAFF evaluates flow velocities and generates a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses. Behavior Measuring Biophysical and Psychological Stress Levels Following Visitation to Three Locations with Differing Levels of Nature Yun Chang1, Alan Ewert2, Lisa M. Kamendulis3, Barbara A. Hocevar3 1School of Kinesiology and Recreation, Illinois State University, 2Department of Recreation, Park, and Tourism Studies, Indiana University, 3Department pf Environmental and Occupational Health, School of Public Health-Bloomington, Indiana University The purpose of this paper is to identify changes in stress levels after visitation to three different settings and to describe the methods used in identifying stress levels based on measures of salivary cortisol, α-amylase, and a psychological self-report instrument. Immunology and Infection Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth Beatriz H D Panariello1, Bruna A Garcia2, Simone Duarte1 1Department of Cariology, Operative Dentistry and Dental Public Health, Indiana University School of Dentistry, 2Department of Restorative Dentistry, Ceará Federal University, School of Dentistry Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J·cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h. Behavior In Vivo Protocol of Controlled Subconcussive Head Impacts for the Validation of Field Study Data Zachary W. Bevilacqua1, Megan E. Huibregtse1, Keisuke Kawata1,2 1Department of Kinesiology, School of Public Health-Bloomington, Indiana University, 2Program in Neuroscience, College of Arts and Sciences, Indiana University The subconcussive soccer heading model is a safe and concise methodological approach to isolate and measure the effects of subconcussive head impacts. Developmental Biology Thawing, Culturing, and Cryopreserving Drosophila Cell Lines Arthur Luhur1, Kristin M. Klueg1, Johnny Roberts1, Andrew C. Zelhof1 1Drosophila Genomics Resource Center, Department of Biology, Indiana University Bloomington Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research. Behavior Gaze in Action: Head-mounted Eye Tracking of Children's Dynamic Visual Attention During Naturalistic Behavior Lauren K. Slone1, Drew H. Abney1, Jeremy I. Borjon1, Chi-hsin Chen2, John M. Franchak3, Daniel Pearcy1, Catalina Suarez-Rivera1, Tian Linger Xu1, Yayun Zhang1, Linda B. Smith1, Chen Yu1 1Department of Psychological and Brain Sciences, Indiana University, 2Department of Otolaryngology-Head and Neck Surgery, The Ohio State University, 3Department of Psychology, University of California, Riverside Young children do not passively observe the world, but rather actively explore and engage with their environment. This protocol provides guiding principles and practical recommendations for using head-mounted eye trackers to record infants' and toddlers' dynamic visual environments and visual attention in the context of natural behavior. Behavior A View of Their Own: Capturing the Egocentric View of Infants and Toddlers with Head-Mounted Cameras Jeremy I. Borjon*1, Sara E. Schroer*1, Sven Bambach2, Lauren K. Slone1, Drew H. Abney1, David J. Crandall2, Linda B. Smith1 1Department of Psychological and Brain Sciences, Indiana University, 2School of Informatics, Computing, and Engineering, Indiana University Infants and toddlers view the world in a fundamentally different way from their parents. Head-mounted cameras provide a tractable mechanism to understand the infant visual environment. This protocol provides guiding principles for experiments in the home or laboratory to capture the egocentric view of toddlers and infants. Developmental Biology Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics Hongde Li1, Jason M. Tennessen1 1Department of Biology, Indiana University This protocol describes how to prepare Drosophila larvae for GC-MS-based metabolomic analysis. Genetics Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae Sebastian Grünberg1, Gabriel E. Zentner2 1Basic Sciences Division, Fred Hutchinson Cancer Research Center, 2Department of Biology, Indiana University We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins. Biology Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays Nydia Morales-Soto1,2, Morgen E. Anyan1, Anne E. Mattingly1, Chinedu S. Madukoma1, Cameron W. Harvey3, Mark Alber3, Eric Déziel4, Daniel B. Kearns5, Joshua D. Shrout1,2,6 1Department of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame, 2Eck Institute for Global Health, University of Notre Dame, 3Department of Applied and Computational Mathematics and Statistics, University of Notre Dame, 4INRS-Institut Armand-Frappier, 5Department of Biology, Indiana University, 6Department of Biological Sciences, University of Notre Dame Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques. Neuroscience Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders Soumya Narayan1, Charlee McLean2, Akira Sawa1, Sandra Y. Lin3, Narayan Rai2, MariaMananita S. Hipolito2, Nicola Cascella4, John J.I. Nurnberger, Jr.5, Koko Ishizuka1, Evaristus A. Nwulia2 1Department of Psychiatry, Johns Hopkins University, 2Department of Psychiatry and Behavioral Sciences, Howard University, 3Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, 4Department of Psychiatry, Sheppard Pratt Hospital, 5Department of Psychiatry, Indiana University In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD. Biology Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster Carrie M. Spratford1, Justin P. Kumar1 1Department of Biology, Indiana University The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy. Medicine Controlled Cervical Laceration Injury in Mice Yi Ping Zhang1, Melissa J. Walker2, Lisa B. E. Shields1, Xiaofei Wang2, Chandler L. Walker2, Xiao-Ming Xu2, Christopher B. Shields1 1Norton Neuroscience Institute, Norton Healthcare, 2Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Department of Neurological Surgery and Goodman and Campbell Brain and Spine, Medical Neuroscience Graduate Program, and Department of Anatomy and Cell Biology, Indiana University School of Medicine A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of ±0.01 mm. Medicine Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats Ruben M. Sandoval1, Bruce A. Molitoris1 1Medicine/Nephrology, Indiana University School of Medicine A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states. Biology Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease Natalie D. Stull1, Andrew Breite2, Robert McCarthy2, Sarah A. Tersey1, Raghavendra G. Mirmira1,3,4 1Department of Pediatrics and the Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 2VITACYTE, LLC, 3Department of Medicine, Indiana University School of Medicine, 4Department of Cellular and Integrative Physiology, Indiana University School of Medicine A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease. Medicine Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation Hidemi Suzuki1,2, Lin Fan1,2, David S. Wilkes1,2 1Departments of Medicine, Microbiology and Immunology, Indiana University School of Medicine, 2Center for Immunobiology, Indiana University School of Medicine Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation. Medicine Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets Patrick T. Fueger1,2, Angelina M. Hernandez2, Yi-Chun Chen2, E. Scott Colvin1,2 1Department of Pediatrics, Indiana University School of Medicine, 2Department of Cellular & Integrative Physiology, Indiana University School of Medicine This protocol allows one to identify factors that modulate functional beta cell mass to find potential therapeutic targets for the treatment of diabetes. The protocol consists of a streamlined method to assess islet replication and beta cell function in isolated rat islets following manipulation of gene expression with adenoviruses. Biology Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood Nutan Prasain1, J. Luke Meador1, Mervin C. Yoder1 1Herman B Wells Center for Pediatric Research, Indiana University School of Medicine Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues. Medicine Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice Julio E. Ayala1, Deanna P. Bracy2,3, Carlo Malabanan3, Freyja D. James2,3, Tasneem Ansari3, Patrick T. Fueger4, Owen P. McGuinness2,3, David H. Wasserman2,3 1Diabetes and Obesity Research Center, Sanford-Burnham Medical Research Institute at Lake Nona, 2Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 3Vanderbilt Mouse Metabolic Phenotyping Center, Vanderbilt University School of Medicine, 4Department of Pediatrics and Cellular and Integrative Physiology, Indiana University School of Medicine The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress. Neuroscience Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents Wenhui Xiong1, Xingjie Ping1, Jianhua Gao1, Xiaoming Jin1 1Department of Anatomy and Cell Biology, Department of Neurosurgery, Stark Neuroscience Research Institute, Indiana University School of Medicine Partially isolated cortex (“undercut”) is an efficient animal model of posttraumatic epileptogenesis. Here we demonstrate how to make a novel surgical device and use it to make more precise and consistent lesions to generate this model.