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20 ARTICLES PUBLISHED IN JoVE

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Biology

Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging
Melissa Paulite 1, Zahra Fakhraai 2, Boris B. Akhremitchev 3, Kerstin Mueller 1, Gilbert C. Walker 1
1Department of Chemistry, University of Toronto, 2Department of Chemistry, University of Wisconsin, 3Department of Chemistry, Duke University

The assembly of a nearfield infrared microscope for imaging protein aggregates is described.

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Immunology and Infection

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions
Crystal Tobin 1, Angela Pollard 1, Laura Knoll 1
1Department of Medical Microbiology and Immunology, University of Wisconsin

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

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Neuroscience

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats
Aaron M. Johnson 1,2, Emerald J. Doll 2, Laura M. Grant 2, Lauren Ringel 2, Jaime N. Shier 2, Michelle R. Ciucci 1,2
1Department of Surgery-Division of Otolaryngology, University of Wisconsin, 2Department of Communicative Disorders, University of Wisconsin

Voice disorders are debilitating in aging and Parkinson disease. The ultrasonic vocalizations of rats, also affected by these conditions, can be used to study these voice disorders, their neural substrates, and the nature of functional recovery with behavioral intervention.

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Neuroscience

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient
Pamela R. Westmark 1, Cara J. Westmark 1, Athavi Jeevananthan 2, James S. Malter 1
1Department of Pathology and Laboratory Medicine, Waisman Center for Developmental Disabilities, University of Wisconsin, 2Department of Biochemistry, Waisman Center for Developmental Disabilities, University of Wisconsin

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

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Biology

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA
Srikumar Sengupta 1, Jennifer M. Bolin 1, Victor Ruotti 1, Bao Kim Nguyen 1, James A. Thomson 1,2,3, Angela L. Elwell 1, Ron Stewart 1
1Regenerative Biology, Morgridge Institute for Research, 2Department of Cell & Regenerative Biology, University of Wisconsin, 3Department of Molecular, Cellular, & Regenerative Biology, University of California

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

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JoVE Core

Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine
Tinen L. Iles 1, Brian Howard 2, Stephen Howard 3, Stephen Quallich 2, Christopher Rolfes 2, Eric Richardson 4, Hanna R. Iaizzo 5, Paul A. Iaizzo 1
1Surgery, University of Minnesota, 2Biomedical Engineering, University of Minnesota, 3Medtronic, Inc., 4Bioengineering, Rice University, 5Pharmacy, University of Wisconsin

We have developed a swine model for the target delivery of pharmacological agents within the pericardial space/fluid. Using this approach, the relative benefits of administered agents on induced atrial fibrillation, relative refractory periods and/or ischemic protection can be investigated.

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Chemistry

Cooling Rate Dependent Ellipsometry Measurements to Determine the Dynamics of Thin Glassy Films
Ethan C. Glor 1, Zahra Fakhraai 1
1Department of Chemistry, University of Pennsylvania

Here, we present a protocol for cooling rate dependent ellipsometry experiments, which can determine the glass transition temperature (Tg), average dynamics, fragility and the expansion coefficient of the super-cooled liquid and glass for a variety of glassy materials.

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Neuroscience

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia
Vishal Chanana 1, Abdulfettah Tumturk 2, Douglas Kintner 1, Eshwar Udho 1, Peter Ferrazzano 1,3, Pelin Cengiz 1,3
1Waisman Center, University of Wisconsin, 2Department of Neurological Surgery, University of Wisconsin, 3Department of Pediatrics, University of Wisconsin

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

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Developmental Biology

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment
Destiny L. Baars 1, Kendra A. Takle *1,2, Jonathon Heier *1,3, Francisco Pelegri 1
1Laboratory of Genetics, University of Wisconsin, 2Department of Neurobiology, University of Massachusetts Medical School, 3Interdisciplinary Biomedical Graduate Program, University of Pittsburgh School of Medicine

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

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Developmental Biology

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish
Elaine L. Welch *1, Celeste C. Eno *1, Sreelaja Nair 2, Robin E. Lindeman 3, Francisco Pelegri 1
1Laboratory of Genetics, University of Wisconsin-Madison, 2Department of Biological Sciences, Tata Institute of Fundamental Research, 3Department of Genetics, Cell Biology, and Development, University of Minnesota

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

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Biology

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor
Thomas R. Vincent 1, James Canham 1, Masatsugu Toyota 2,3,4, Marieta Avramova 1, Sam T. Mugford 5, Simon Gilroy 2, Anthony J. Miller 1, Saskia Hogenhout 5, Dale Sanders 1
1Department of Metabolic Biology, John Innes Centre, Norwich Research Park, 2Department of Botany, University of Wisconsin, Madison, 3Department of Biochemistry and Molecular Biology, Saitama University, 4Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 5Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

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Genetics

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells
Hector Guillen-Ahlers 1,2, Prahlad K. Rao 1, Danu S. Perumalla 1, Maria J. Montoya 1, Avinash Y.L. Jadhav 1, Michael R. Shortreed 3, Lloyd M. Smith 3, Michael Olivier 1,2
1Department of Genetics, Texas Biomedical Research Institute, 2Department of Internal Medicine-Molecular Medicine, Wake Forest University School of Medicine, 3Department of Chemistry, University of Wisconsin

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

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Environment

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope
Kenichi Yazaki 1, Mayumi Y. Ogasa 2, Katsushi Kuroda 3, Yasuhiro Utsumi 4, Peter Kitin 5, Yuzou Sano 6
1Department of Plant Ecology, Forestry and Forest Products Research Institute (FFPRI), 2Kansai Research Center, Forestry and Forest Products Research Institute (FFPRI), 3Department of Wood Properties and Processing, Forestry and Forest Products Research Institute (FFPRI), 4Faculty of Agriculture, Kyushu University, 5Department of Bacteriology, University of Wisconsin, 6Research Faculty of Agriculture, Hokkaido University

Observing the water distribution within the xylem provides significant information regarding water flow dynamics in woody plants. In this study, we demonstrate the practical approach to observe xylem water distribution in situ by using a cryostat and cryo-SEM, which eliminates artifactual changes in the water status during sample preparation.

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Neuroscience

Translaminar Autonomous System Model for the Modulation of Intraocular and Intracranial Pressure in Human Donor Posterior Segments
Tasneem P. Sharma 1, Stacy M. Curry 1, Husain Lohawala 2, Colleen McDowell 3
1North Texas Eye Research Institute, Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, 2Mechanical Engineer Consultant, 3Department of Ophthalmology and Visual Sciences, School of Medicine and Public Health, University of Wisconsin

We describe and detail the use of the translaminar autonomous system. This system utilizes the human posterior segment to independently regulate the pressure inside the segment (intraocular) and surrounding the optic nerve (intracranial) to generate a translaminar pressure gradient that mimics features of glaucomatous optic neuropathy.

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Neuroscience

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics
Caitlin A. Murphy 1, Aeyal Raz 1,2, Sean M Grady 1, Matthew I Banks 1
1Department of Anesthesiology, University of Wisconsin, 2Department of Anesthesiology, Rambam Health Care Campus, the Ruth and Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

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Engineering

Building Langmuir Probes and Emissive Probes for Plasma Potential Measurements in Low Pressure, Low Temperature Plasmas
Peixuan Li *1, Noah Hershkowitz *1, Gregory Severn *2
1Department of Engineering Physics, University of Wisconsin, 2Department of Physics & Biophysics, University of San Diego

The main goal of this work is to make it easier for research groups unfamiliar with Langmuir probes and emissive probes to use them as plasma diagnostics, especially near plasma boundaries. We do this by demonstrating how to build the probes from readily available materials and supplies.

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JoVE Journal

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis
Takuya Uemura 1, Jiaqi Wang 1, Yuri Aratani 1, Simon Gilroy 2, Masatsugu Toyota 1,2
1Department of Biochemistry and Molecular Biology, Saitama University, 2Department of Botany, University of Wisconsin

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

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Genetics

Determining the Role of Maternally-Expressed Genes in Early Development with Maternal Crispants
Cara E. Moravec 1, Gabriella C. Voit 1, Francisco Pelegri 1
1Laboratory of Genetics, University of Wisconsin-Madison

Early development is dependent on maternally-inherited products, and the role of many of these products is currently unknown. Herein, we described a protocol that uses CRISPR-Cas9 to identify maternal-effect phenotypes in a single generation.

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Bioengineering

Multimodal Cross-Device and Marker-Free Co-Registration of Preclinical Imaging Modalities
Mirko Thamm 1,2, Justin J. Jeffery 3, Yapei Zhang 4, Bryan R. Smith 4, Stephen Marchant 5, Fabian Kiessling 1,6, Felix Gremse 1,2
1Institute for Experimental Molecular Imaging, RWTH Aachen University, 2Gremse-IT GmbH, 3Carbone Cancer Center, University of Wisconsin, 4Department of Biomedical Engineering and Institute for Quantitative Health Science and Engineering, Michigan State University, 5MediLumine Inc., 6Fraunhofer Institute for Digital Medicine MEVIS

The combination of multiple imaging modalities is often necessary to gain a comprehensive understanding of pathophysiology. This approach utilizes phantoms to generate a differential transformation between the coordinate systems of two modalities, which is then applied for co-registration. This method eliminates the need for fiducials in production scans.

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Medicine

Heterotopic Auxiliary Whole Liver Rat Transplant Model Utilizing a Hepaticoureterostomy for Allograft Rejection Studies
Bret Verhoven 1, Weifeng Zeng 2, Peter Chlebeck 1, Kristina Matkowskyj 3,4,5, Heather Jennings 1, Samuel Poore 2, David Al-Adra 1
1Department of Surgery, School of Medicine and Public Health, University of Wisconsin-Madison, 2Department of Surgery, Division of Plastic and Reconstructive Surgery, University of Wisconsin, 3Department of Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin-Madison, 4UW Carbone Cancer Center, 5William S. Middleton Memorial Veterans' Hospital

The rat heterotopic auxiliary liver transplant protocol described here offers a practical investigational tool for exploring mechanisms of hepatic allograft rejection. This model helps to alleviate the surgical hurdles and animal stress of orthotopic liver transplantation in rats.

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