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23 ARTICLES PUBLISHED IN JoVE

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Medicine

Cell Population Analyses During Skin Carcinogenesis
Dongsheng Gu 1, Qipeng Fan 1, Jingwu Xie 1
1Department of Pediatrics, Wells Center for Pediatric Research, IU Simon Cancer Center, Indiana University

Carcinogenesis is a process involving interactions between cancer cells and the cancer microenvironment. To dissect the molecular events, one needs to isolate different cell populations at different stages during cancer development. Using a mouse model for basal cell carcinoma, we describe a protocol for cell analyses during carcinogenesis.

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Biology

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster
Carrie M. Spratford 1, Justin P. Kumar 1
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.

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Neuroscience

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders
Soumya Narayan 1, Charlee McLean 2, Akira Sawa 1, Sandra Y. Lin 3, Narayan Rai 2, MariaMananita S. Hipolito 2, Nicola Cascella 4, John J.I. Nurnberger, Jr. 5, Koko Ishizuka 1, Evaristus A. Nwulia 2
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.

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Biology

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays
Nydia Morales-Soto 1,2, Morgen E. Anyan 1, Anne E. Mattingly 1, Chinedu S. Madukoma 1, Cameron W. Harvey 3, Mark Alber 3, Eric Déziel 4, Daniel B. Kearns 5, Joshua D. Shrout 1,2,6
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.

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Genetics

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae
Sebastian Grünberg 1, Gabriel E. Zentner 2
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.

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Developmental Biology

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro
Kaela M. Varberg 1,2, Seth Winfree 3,4, Kenneth W. Dunn 3,4, Laura S. Haneline 1,2,5,6,7
1Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 2Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 3Indiana Center for Biological Microscopy, Indiana University School of Medicine, 4Department of Medicine, Indiana University School of Medicine, 5Department of Pediatrics, Indiana University School of Medicine, 6Department of Microbiology and Immunology, Indiana University School of Medicine, 7Indiana University Simon Cancer Center, Indiana University School of Medicine

Here, we present a protocol for time-lapse imaging and analysis of vasculogenesis in vitro using phase contrast microscopy coupled with the open source software, Kinetic Analysis of Vasculogenesis. This protocol can be applied to quantitatively assess the vasculogenic potential of numerous cell types or experimental conditions that model vascular disease.

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Developmental Biology

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics
Hongde Li 1, Jason M. Tennessen 1
1Department of Biology, Indiana University

This protocol describes how to prepare Drosophila larvae for GC-MS-based metabolomic analysis.

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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 Bambach 2, Lauren K. Slone 1, Drew H. Abney 1, David J. Crandall 2, Linda B. Smith 1
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.

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Behavior

Gaze in Action: Head-mounted Eye Tracking of Children's Dynamic Visual Attention During Naturalistic Behavior
Lauren K. Slone 1, Drew H. Abney 1, Jeremy I. Borjon 1, Chi-hsin Chen 2, John M. Franchak 3, Daniel Pearcy 1, Catalina Suarez-Rivera 1, Tian Linger Xu 1, Yayun Zhang 1, Linda B. Smith 1, Chen Yu 1
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.

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Behavior

Measuring Biophysical and Psychological Stress Levels Following Visitation to Three Locations with Differing Levels of Nature
Yun Chang 1, Alan Ewert 2, Lisa M. Kamendulis 3, Barbara A. Hocevar 3
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. 

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Behavior

In Vivo Protocol of Controlled Subconcussive Head Impacts for the Validation of Field Study Data
Zachary W. Bevilacqua 1, Megan E. Huibregtse 1, Keisuke Kawata 1,2
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.

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Developmental Biology

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos
Sanat Upadhyay 1, Leoncio Vergara 2, Pranjali Shah 2, Jan-Åke Gustafsson 3,4, Ioannis Kakadiaris 1,4, Maria Bondesson 5
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.

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Neuroscience

Collection of Frozen Rodent Brain Regions for Downstream Analyses
Jim Wager-Miller 1, Michelle Murphy Green 1, Hana Shafique 1, Ken Mackie 1
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.

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Bioengineering

Spatial Temporal Analysis of Fieldwise Flow in Microvasculature
Sherry G. Clendenon 1,2, Xiao Fu 1,3, Robert A. Von Hoene 1, Jeffrey L. Clendenon 4, James P. Sluka 1,2, Seth Winfree 5, Henry Mang 5, Michelle Martinez 5, Adele Filson 5, James E. Klaunig 6, James A. Glazier 1,2, Kenneth W. Dunn 5
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.

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Biochemistry

Screening for Phytoestrogens using a Cell-based Estrogen Receptor β Reporter Assay
Emily M. Chester 1, Emily Fender 1, Michael D. Wasserman 1
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.

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Immunology and Infection

Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins
Asha A. Philip 1, Jin Dai 1, Sarah P. Katen 1, John T. Patton 1
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.

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Biology

Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue
Daria Barwinska 1, Michael J. Ferkowicz 1, Ying-Hua Cheng 1, Seth Winfree 1,2, Kenneth W. Dunn 1, Katherine J. Kelly 1, Timothy A. Sutton 1, Brad H. Rovin 3, Samir V. Parikh 3, Carrie L. Phillips 4, Pierre C. Dagher 1, Tarek M. El-Achkar 1, Michael T. Eadon 1,
1Department of Medicine, Indiana University School of Medicine, 2Department of Cellular & Integrative Physiology, Indiana University School of Medicine, 3Division of Nephrology, Department of Medicine, Ohio State University Wexner Medical Center, 4Division of Pathology, Indiana University School of Medicine

We describe a protocol for laser microdissection of sub-segments of the human kidney, including the glomerulus, proximal tubule, thick ascending limb, collecting duct and interstitium. The RNA is then isolated from the obtained compartments and RNA sequencing is carried out to determine changes in the transcriptomic signature within each sub-segment.

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Biology

Artificial Intelligence Approaches to Assessing Primary Cilia
Ruchi Bansal 1, Staci E. Engle 1, Tisianna K. Kamba 1, Kathryn M. Brewer 1, Wesley R. Lewis 2, Nicolas F. Berbari 1,3,4
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.

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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 Tsuchiya 1,2, Soni Lacefield 1
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.

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Biology

Assessment of Chemical Toxicity in Adult Drosophila Melanogaster
Jessica M. Holsopple *1,2, Shannon R. Smoot *1, Ellen M. Popodi 1,2, John K. Colbourne 3, Joseph R. Shaw 4, Brian Oliver 5, Thomas C. Kaufman 1, Jason M. Tennessen 1
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.

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Developmental Biology

Preparation and Immunofluorescence Staining of Bundles and Single Fiber Cells from the Cortex and Nucleus of the Eye Lens
Michael P. Vu 1, Catherine Cheng 1
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.

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Biology

Whole Mount Imaging to Visualize and Quantify Peripheral Lens Structure, Cell Morphology, and Organization
Grace Emin *1, Sadia T. Islam *1, Rylee E. King 1, Velia M. Fowler 1, Catherine Cheng 2, Justin Parreno 1,3
1Department of Biological Sciences, University of Delaware, 2School of Optometry and Vision Science Program, Indiana University, 3Department of Biomedical Engineering, University of Delaware

The present protocols describe novel whole mount imaging for the visualization of peripheral structures in the ocular lens with methods for image quantification. These protocols can be used in studies to better understand the relationship between lens microscale structures and lens development/function.

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Biology

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors
Laura C. Brown 1, Jay Chopra 2, Rachel E. Horness 3, Julia C. van Kessel 4
1Department of Chemistry, Indiana University, 2Indiana University School of Medicine, 3Department of Chemistry, Carleton College, 4Department of Biology, Indiana University

Thiophenesulfonamide compounds are potent and specific inhibitors of Vibrio quorum sensing regulators LuxR/HapR that block their activity in vivo, thus preventing transcription of genes for virulence, motility, and biofilms. This protocol details how these compounds are synthesized, modeled in silico, and assayed in vivo for activity against LuxR/HapR.

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