Auburn University View Institution's Website 18 articles published in JoVE Biology Isolate Cell-Type-Specific RNAs from Snap-Frozen Heterogeneous Tissue Samples without Cell Sorting Huifei Sophia Zheng1, Chen-Che Jeff Huang1 1Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University This protocol aims to isolate cell-type-specific translating ribosomal mRNAs using the NuTRAP mouse model. Biology Development of a Mobile Mitochondrial Physiology Laboratory for Measuring Mitochondrial Energetics in the Field Hailey A. Parry1, Kang Nian Yap2, Geoffrey E. Hill2, Wendy R. Hood2, L. Bruce Gladden1, Michael Eddy3, Andreas N. Kavazis1 1School of Kinesiology, Auburn University, 2Department of Biological Sciences, Auburn University, 3Department of Electrical and Computer Engineering, Auburn University We designed and constructed a mobile laboratory to measure respiration rates in isolated mitochondria of wild animals captured at field locations. Here, we describe the design and outfitting of a mobile mitochondrial laboratory and the associated laboratory protocols. Genetics Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus Michelle Bui1, Ming Li1, Robyn R Raban1, Nannan Liu2, Omar S Akbari1,3 1Section of Cell and Developmental Biology, University of California, San Diego, 2Department of Entomology and Plant Patholog, Auburn University, 3Tata Institute for Genetics and Society, University of California, San Diego This protocol describes microinjection procedures for Culex quinquefasciatus embryos that are optimized to work with CRISPR/Cas9 gene editing tools. This technique can efficiently generate site-specific, heritable, germline mutations that can be used for building genetic technologies in this understudied disease vector. Developmental Biology Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals − Using Unconjugated Primary Antibodies from the Same Host Species Qiongxia Lyu1,2, Huifei Sophia Zheng2, Karly Laprocina2, Chen-Che Jeff Huang2 1College of Animal Science & Technology, Henan University of Science and Technology, 2Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University Several different methods have been established for multiplex immunostaining using primary antibodies from the same host species. Here, we describe the use of microwave-mediated antibody stripping and of fluorophore-tyramide to block antibody cross-reactivity during multiplex immunostaining on formalin-fixed paraffin-embedded mouse adrenal sections. Behavior Training Dogs for Awake, Unrestrained Functional Magnetic Resonance Imaging Lily R. Strassberg*1, L. Paul Waggoner*2, Gopikrishna Deshpande*1,3,4,5, Jeffrey S. Katz*1,3,4,5 1Department of Psychology, Auburn University, 2Canine Performance Sciences Program, College of Veterinary Medicine, Auburn University, 3AU MRI Research Center, Department of Electrical & Computer Engineering, Auburn University, 4Alabama Advanced Imaging Consortium, 5Center for Neuroscience, Auburn University Magnetic resonance imaging (MRI) on unrestrained awake dogs is a new method with several advantages over imaging with physical or chemical restraint. This protocol introduces a cost-effective training method that minimizes training in the MRI environment, which can be expensive, and maximizes the subject pool available for canine functional MRI. Environment Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids Qichen Wang1, Haixin Peng1, Brendan T. Higgins1 1Department of Biosystems Engineering, Auburn University Here, we present a protocol to construct lab-scale bubble column photobioreactors and use them to culture microalgae. It also provides a method for the determination of the culture growth rate and neutral lipid content. Biochemistry Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica Xuechun Feng1, Nannan Liu1 1Department of Entomology and Plant Pathology, Auburn University Here, we present a protocol to produce house fly carboxylesterase proteins in vitro with a baculovirus mediated insect cell expression system and later functionally characterize their roles in metabolizing permethrin, thereby, conferring pyrethroid resistance by conducting cell-based MTT assay and in vitro metabolic studies. Immunology and Infection Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy Peter B. Rogers1, Elizabeth Hiltbold Schwartz1 1Department of Biological Sciences, Auburn University Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression. Genetics Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing Ahmed Elaswad*1,2, Karim Khalil*1,3, David Cline1, Patrick Page-McCaw4, Wenbiao Chen4, Maximilian Michel5, Roger Cone5, Rex Dunham1 1School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, 2Department of Animal Wealth Development, Faculty of Veterinary Medicine, Suez Canal University, 3Anatomy and Embryology Department, Faculty of Veterinary Medicine, Cairo University, 4Department of Molecular Physiology and Biophysics, Vanderbilt University, 5Life Science Institute, University of Michigan A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes. Neuroscience Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models Holly C. Hunsberger1, Sharay E. Setti2, Ryan T. Heslin2, Jorge E. Quintero3, Greg A. Gerhardt3, Miranda N. Reed2 1Department of Psychology, West Virginia University, 2Department of Drug Discovery & Development, Auburn University, 3Department of Neuroscience, University of Kentucky Medical Center Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA). Environment Using Pharmacological Manipulation and High-precision Radio Telemetry to Study the Spatial Cognition in Free-ranging Animals Timothy C. Roth*1, Aaron R. Krochmal*2, William B. Gerwig, IV1, Sage Rush3, Nathaniel T. Simmons2, Jeffery D. Sullivan4, Katrina Wachter5 1Department of Psychology, Franklin and Marshall College, 2Department of Biology, Washington College, 3University of Pennsylvania, 4School of Forestry and Wildlife Sciences, Auburn University, 5Morsani College of Medicine, University of South Florida This paper describes a novel protocol that combines the pharmacological manipulation of memory and radio telemetry to document and quantify the role of cognition in navigation. Immunology and Infection Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain Iryna Sorokulova1, Ludmila Globa1, Oleg Pustovyy1, Vitaly Vodyanoy1 1Department of Anatomy, Physiology and Pharmacology, Auburn University We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds. Environment Sediment Core Sectioning and Extraction of Pore Waters under Anoxic Conditions Alison R. Keimowitz1, Yan Zheng2, Ming-Kuo Lee3, Michael Natter3, Jeffrey Keevan3 1Department of Chemistry, Vassar College, 2Division of Geochemistry, Lamont-Doherty Earth Observatory, 3Department of Geosciences, Auburn University A protocol for sectioning sediment cores and extracting pore waters under anoxic conditions in order to permit analysis of redox sensitive species in both solids and fluids is presented. Neuroscience Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals Feng Liu1, Nannan Liu1 1Department of Entomology and Plant Pathology, Auburn University Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved. Behavior Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice Michael L. Seibenhener1, Michael C. Wooten1 1Department of Biological Sciences, Auburn University A protocol is provided to use an Open Field Maze to access general locomotor activity, anxiety and emotionality in a laboratory mouse model. Immunology and Infection Assays for the Identification of Novel Antivirals against Bluetongue Virus Linlin Gu1, Stewart W. Schneller2, Qianjun Li1 1Division of Infectious Diseases, Department of Medicine, University of Alabama at Birmingham, 2Molette Laboratory for Drug Discovery, Department of Chemistry and Biochemistry, Auburn University Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals. Bioengineering Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria Rajesh Guntupalli1, Iryna Sorokulova1, Eric Olsen2, Ludmila Globa1, Oleg Pustovyy1, Vitaly Vodyanoy1 1Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, 2Clinical Research Laboratory, 81st Medical Group, Keesler Air Force Base Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA. Neuroscience Isolation and Culture of Hippocampal Neurons from Prenatal Mice Michael L. Seibenhener1, Marie W. Wooten1 1Department of Biological Sciences, Auburn University We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.