University of Wyoming 13 articles published in JoVE Neuroscience Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging Rashmi Thapa1, Bo Liang2, Rongsong Liu3, Yun Li1 1Department of Zoology and Physiology, University of Wyoming, 2School of Electrical Engineering & Computer Science, University of North Dakota, 3Department of Mathematics and Statistics, University of Wyoming Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope. Developmental Biology Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography Todd A. Schoborg1 1Department of Molecular Biology, University of Wyoming A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography. Engineering Microfluidic Fabrication Techniques for High-Pressure Testing of Microscale Supercritical CO2 Foam Transport in Fractured Unconventional Reservoirs Hooman Hosseini1, Feng Guo2, Reza Barati Ghahfarokhi1, Saman A. Aryana2 1Department of Chemical and Petroleum Engineering, University of Kansas, 2Department of Chemical Engineering, University of Wyoming This paper describes a protocol along with a comparative study of two microfluidic fabrication techniques, namely photolithography/wet-etching/thermal-bonding and Selective Laser-induced Etching (SLE), that are suitable for high-pressure conditions. These techniques constitute enabling platforms for direct observation of fluid flow in surrogate permeable media and fractured systems under reservoir conditions. Biology Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination Gordon F. Custer*1,2, Reilly R. Dibner*3,4 1Department of Ecosystem Science and Management, University of Wyoming, 2Program in Ecology, University of Wyoming, 3EPSCoR-IDEA, University of Wyoming, 4Department of Botany, University of Wyoming Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination. Environment Detection of Viruses from Bioaerosols Using Anion Exchange Resin Joshua W. Schaeffer1, Jeffrey C. Chandler2, Margaret Davidson1,3, Sheryl L. Magzamen1, Alma Pérez-Méndez4, Stephen J. Reynolds1, Lawrence D. Goodridge5, John Volckens6, Alan B. Franklin2, Susan A. Shriner2, Bledar Bisha7 1High Plains Intermountain Center for Agricultural Health and Safety, Department of Environmental and Radiological Health Sciences, Colorado State University, 2National Wildlife Research Center, Wildlife Services, Animal and Plant Health Inspection Service, United States Department of Agriculture, 3Western Sydney University, 4Leprino Foods, Inc, 5Department of Food Science and Agricultural Chemistry, McGill University, 6Department of Mechanical Engineering, Colorado State University, 7Department of Animal Science, University of Wyoming An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols. Neuroscience Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology Adel B. Ghnenis1, Richard E. Czaikowski1, Zhaojie J. Zhang2, Jared S. Bushman1 1School of Pharmacy, University of Wyoming, 2Department of Zoology and Physiology, University of Wyoming Here we present a protocol to visualize fine structures of peripheral nerves by obtaining and staining 1-2 µm sections with toluidine blue Neuroscience Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons Zhenyu Liu1, Katelynne B. Donnelly1, Kara G. Pratt1 1Department of Zoology and Physiology and Program in Neuroscience, University of Wyoming In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function. Immunology and Infection Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material Yo Ching Cheng*1, Samia Hannaoui*1, Theodore Ralph John2, Sandor Dudas3, Stefanie Czub3, Sabine Gilch1 1Dept. of Ecosystem and Public Health, Calgary Prion Research Units, Faculty of Veterinary Medicine, University of Calgary, 2Dept. of Molecular Biology, University of Wyoming, 3Canadian Food Inspection Agency, Lethbridge Laboratories Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method. Biochemistry Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads Padmamalini Baskaran1, Baskaran Thyagarajan1 1School of Pharmacy, University of Wyoming This protocol describes the method of determining basal and forskolin-stimulated lipolysis in inguinal fat pads obtained from normal chow diet (NCD) or high fat diet (HFD) ± capsaicin fed wild type mice. As an index for lipolysis, glycerol release was measured from inguinal adipose fat pads. Biochemistry Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development Robyn M. Johnston1, Anne W. Sylvester2, Michael J. Scanlon1 1Plant Biology Section, School of Integrative Plant Science, Cornell University, 2Department of Developmental Genetics, University of Wyoming Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena. Biology A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation Lisa J. Edens1, Daniel L. Levy1 1Department of Molecular Biology, University of Wyoming Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified. Environment Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability Jodi R. Schilz1, K. J. Reddy2, Sreejayan Nair3, Thomas E. Johnson4, Ronald B. Tjalkens5, Kem P. Krueger3, Suzanne Clark6 1Division of Physical Therapy, Department of Orthopedics & Rehabilitation, University of New Mexico, 2Department of Ecosystem Science and Management, University of Wyoming, 3School of Pharmacy, University of Wyoming, 4Department of Environmental and Radiological Health Sciences, Colorado State University, 5Center for Environmental Medicine, Colorado State University, 6College of Pharmacy, California Northstate University Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment. Environment Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water Bledar Bisha1, Jaclyn A. Adkins2, Jana C. Jokerst3, Jeffrey C. Chandler1, Alma Pérez-Méndez4, Shannon M. Coleman4, Adrian O. Sbodio5, Trevor V. Suslow5, Michelle D. Danyluk6, Charles S. Henry2, Lawrence D. Goodridge7 1Department of Animal Science, University of Wyoming, 2Department of Chemistry, Colorado State University, 3Department of Environmental and Radiological Health Sciences, Colorado State University, 4Department of Animal Sciences, Colorado State University, 5Department of Plant Sciences, University of California, Davis, 6Department of Food Science and Human Nutrition, Citrus Research and Education Center, University of Florida, 7Department of Food Science and Agricultural Chemistry, McGill University A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (µPAD) imbedded with bacterial-indicative colorimetric substrates.