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University of Bath

10 ARTICLES PUBLISHED IN JoVE

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Biology

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations
Iwan R. Evans 1, Jennifer Zanet 2, Will Wood 1, Brian M. Stramer 2
1Department of Biology and Biochemistry, University of Bath, 2Randall Division of Cell and Molecular Biophysics, King's College London

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

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Biology

Microinjection of Medaka Embryos for use as a Model Genetic Organism
Sean R. Porazinski 1, Huijia Wang 1, Makoto Furutani-Seiki 1
1Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

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Biology

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras
Sean R. Porazinski 1, Huijia Wang 1, Makoto Furutani-Seiki 1
1Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

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Immunology and Infection

Invasion of Human Cells by a Bacterial Pathogen
Andrew M. Edwards 1, Ruth C. Massey 1
1Department of Biology and Biochemistry, University of Bath

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

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JoVE Journal

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities
Sadahiro Iwabuchi 1, Yasuhiro Kakazu 1, Jin-Young Koh 1, Kirsty M. Goodman 2, N. Charles Harata 1
1Department of Molecular Physiology & Biophysics, University of Iowa Carver College of Medicine, 2Department of Biology & Biochemistry, University of Bath

We describe the use of styryl FM dyes to image synaptic vesicle recycling in functional nerve terminals. This protocol can be applied not only to evoked, but also spontaneous and miniature synaptic activities. The protocol expands the variety of synaptic events that can be effectively evaluated.

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Neuroscience

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons
Megan N. Evilsizor 1,2, Helen F. Ray-Jones 1,3, Jonathan Lifshitz 1,2,4,5, Jenna Ziebell 1,2
1Department of Child Health, University of Arizona College of Medicine - Phoenix, 2BARROW Neurological Institute, Phoenix Children's Hospital, 3Department of Biology and Biochemistry, University of Bath, 4Neuroscience Program, Arizona State University, 5Phoenix VA Healthcare System

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

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Bioengineering

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture
Michael P. Storm 1, Ian Sorrell 2, Rebecca Shipley 3, Sophie Regan 2, Kim A. Luetchford 1, Jean Sathish 2, Steven Webb 4, Marianne J. Ellis 1
1Department of Chemical Engineering and Centre for Regenerative Medicine, University of Bath, 2MRC Centre for Drug Safety Science and Institute of Translational Medicine, University of Liverpool, 3Mechanical Engineering, University College London, 4Department of Applied Mathematics, Liverpool John Moores University

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

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Immunology and Infection

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae
Helen Weavers 1,2, Anna Franz 1, Will Wood 3, Paul Martin 1,4
1School of Biochemistry, Biomedical Sciences, University of Bristol, 2School of Cellular and Molecular Medicine, Biomedical Sciences, University of Bristol, 3MRC Centre for Inflammation Research, University of Edinburgh, Queens Medical Research Institute, 4School of Physiology, Pharmacology, and Neuroscience, Biomedical Sciences, University of Bristol

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

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Neuroscience

Simultaneous Cryosectioning of Multiple Rodent Brains
Tabitha R.F. Green 1,2,3, J. Bryce Ortiz 1, Jordan L. Harrison 5, Jonathan Lifshitz 1,3,4, Rachel K. Rowe 1,3,4
1Department of Child Health, University of Arizona College of Medicine, 2Department of Biological Sciences, University of Bath, 3BARROW Neurological Institute at Phoenix Children's Hospital, 4Phoenix Veteran Affairs Healthcare System, 5Department of Basic Medical Sciences, University of Arizona College of Medicine

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

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Biology

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana
Yui-Leung Lau 1, Ludi Wang 1, Mutian Yang 1, James Doughty 1
1Department of Life Sciences, University of Bath

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

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