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Various Modes of Spinal Cord Injury to Study Regeneration in Adult Zebrafish

Featured protocol,  Authors: Subhra Prakash Hui
Subhra Prakash HuiAffiliation 1: Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, Kolkata, India
Affiliation 2: Victor Chang Cardiac Research Institute, Lowy Packer Building, Darlinghurst, Australia
Bio-protocol author page: a3817
 and Sukla Ghosh
Sukla GhoshAffiliation: Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, Kolkata, India
For correspondence: suklagh2010@gmail.com
Bio-protocol author page: a3818
date: 12/5/2016, 42 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2043.

Brief version appeared in PLOS ONE, Dec 2015
Spinal cord injury (SCI) in mammals leads to failure of both sensory and motor functions, due to lack of axonal regrowth below the level of injury as well as inability to replace lost neural cells and to stimulate neurogenesis. In contrast, fish and amphibians are capable of regenerating a variety of their organs like limb/fin, jaw, heart and various parts of the central nervous system (CNS). Zebrafish embryo and adult has become a very popular model to study developmental biology, cell biology and regeneration for various reasons. Adult zebrafish, one of the most important vertebrate models to study regeneration, can regenerate many of their body parts like fin, jaw, heart and CNS. In the present article we provide information on how to inflict different injury modalities in adult fish spinal cord. Presently, the significant focus of mammalian SCI is to use crush and contusion injury. To generate an entity comparable to the mammalian mode of injury, we have introduced the crush model in adult zebrafish along with complete transection injury, which is also known to be a valuable model to study axonal regeneration. Here we provide full description of the highly reproducible surgical procedures including some representative results. This protocol has been adapted from our previous publications, viz. Hui et al., 2010 and Hui et al., 2014. Briefly, we have described the two different injury modalities, crush and complete transection, and demonstrated the outcome of inflicting these injuries in the adult zebrafish cord by histological analysis of the tissues.

Various Modes of Spinal Cord Injury to Study Regeneration in Adult Zebrafish

Authors: Subhra Prakash Hui
Subhra Prakash HuiAffiliation 1: Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, Kolkata, India
Affiliation 2: Victor Chang Cardiac Research Institute, Lowy Packer Building, Darlinghurst, Australia
Bio-protocol author page: a3817
 and Sukla Ghosh
Sukla GhoshAffiliation: Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, Kolkata, India
For correspondence: suklagh2010@gmail.com
Bio-protocol author page: a3818
date: 12/5/2016, 42 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2043.

[Abstract] Spinal cord injury (SCI) in mammals leads to failure of both sensory and motor functions, due to lack of axonal regrowth below the level of injury as well as inability to replace lost neural cells and to stimulate neurogenesis. In contrast, fish and amphibians are capable of regenerating a variety of ...

Allogeneic Transplantation of Testicular Hyperplasia in rag1 Mutant Zebrafish

Authors: Toshihiro Kawasaki
Toshihiro Kawasaki Affiliation: Genetic Strains Research Center, National Institute of Genetics, and Department of Genetics, School of Life Science, the Graduate University for Advanced Studies (SOKENDAI), Mishima, Japan
Bio-protocol author page: a3674
 and Noriyoshi Sakai
Noriyoshi SakaiAffiliation: Genetic Strains Research Center, National Institute of Genetics, and Department of Genetics, School of Life Science, the Graduate University for Advanced Studies (SOKENDAI), Mishima, Japan
For correspondence: nosakai@nig.ac.jp
Bio-protocol author page: a3675
date: 11/5/2016, 130 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1992.

[Abstract] Allogeneic organ transplantation is a powerful tool for clinical and basic research studies. However, the graft is often rejected by the host organism. Here, we describe a protocol that uses immunodeficient rag1 mutant zebrafish. These zebrafish escaped rejection, which made it possible to successfully ...

Detection of Reactive Oxygen Species Using MitoSOX and CellROX in Zebrafish

Authors: Alberto Rissone
Alberto RissoneAffiliation 1: Translational and Functional Genomics Branch, National Human Research Institute (NHGRI), National Institutes of Health (NIH), Bethesda, MD, USA
Affiliation 2: Disorders of Immunity Section, Genetics and Molecular Biology Branch, NHGRI, NIH, Bethesda, MD, USA
For correspondence: alberto.rissone@nih.gov
Bio-protocol author page: a3533
 and Fabio Candotti
Fabio CandottiAffiliation 1: Disorders of Immunity Section, Genetics and Molecular Biology Branch, NHGRI, NIH, Bethesda, MD, USA
Affiliation 2: Division of Immunology and Allergy, University Hospital of Lausanne, Lausanne, Switzerland
For correspondence: Fabio.Candotti@chuv.ch
Bio-protocol author page: a3534
date: 10/5/2016, 392 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1941.

[Abstract] The production of free radicals is the result of normal cellular metabolism. Free radicals are involved in innumerable different cellular and biological functions such as signaling, proliferation, cell death, aging, inflammation, etc. Under physiological conditions, the levels of reactive oxygen species ...

Thirty-Second Net Stressor Task in Adult Zebrafish

Authors: Steven Tran
Steven TranAffiliation: Department of Cell and Systems Biology, University of Toronto Mississauga, Mississauga, Canada
For correspondence: stevenhuy.tran@mail.utoronto.ca
Bio-protocol author page: a2034
 and Robert Gerlai
Robert GerlaiAffiliation: Department of Psychology, University of Toronto Mississauga, Mississauga, Canada
For correspondence: robert_gerlai@yahoo.com
Bio-protocol author page: a2035
date: 3/5/2015, 1399 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1413.

[Abstract] Zebrafish have become a popular animal model for behavioral neuroscience (Gerlai, 2014). Recent studies have demonstrated that brief experimental handling prior to euthanizing animals can subsequently alter biological measures quantified post-mortem (e.g. cortisol levels) (Ramsay et al., 2009; Tran ...

Fluorescence Microscopy for Cilia in Cultured Cells and Zebrafish Embryos

Authors: Jingli Cao
Jingli CaoAffiliation: State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai, China
Bio-protocol author page: a1497
Xueliang Zhu
Xueliang Zhu Affiliation: State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai, China
Bio-protocol author page: a1498
 and Xiumin Yan
Xiumin YanAffiliation: State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai, China
For correspondence: yanx@sibcb.ac.cn
Bio-protocol author page: a1499
date: 7/20/2014, 3829 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1188.

[Abstract] Cilia are microtubule-based hair-like projections found in organisms, ranging from protozoa to mammals. This protocol provides methods for immunofluorescence staining of cilia in cultured cells and zebrafish embryos....

Motility Assay for Zebrafish Embryos

Authors: Michelle F. Goody
Michelle F. Goody Affiliation: Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, USA
Bio-protocol author page: a619
 and Clarissa A. Henry
Clarissa A. HenryAffiliation: School of Biology and Ecology, University of Maine, Orono, USA
For correspondence: clarissa.henry@umit.maine.edu
Bio-protocol author page: a620
date: 6/5/2013, 3580 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.787.

[Abstract] Analyzing the swimming ability of 2 days post fertilization zebrafish embryos can be a useful technique to study neuromuscular function. Here is a protocol for determining the time it takes for zebrafish embryos to swim a predetermined distance....

Phalloidin Staining and Immunohistochemistry of Zebrafish Embryos

Authors: Michelle F. Goody
Michelle F. Goody Affiliation: Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, USA
Bio-protocol author page: a619
 and Clarissa A. Henry
Clarissa A. HenryAffiliation: School of Biology and Ecology, University of Maine, Orono, USA
For correspondence: clarissa.henry@umit.maine.edu
Bio-protocol author page: a620
date: 6/5/2013, 8764 views, 1 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.786.

[Abstract] Fluorescent conjugated Phalloidin is a stain that allows for visualization of F-actin. In immunohistochemistry, primary antibodies and fluorescent conjugated secondary antibodies can be used to visualize subcellular localization and relative amounts of proteins of interest. Here is a protocol for Phalloidin ...

[Bio101] Cell Cycle Analysis and GFP Expression of Zebrafish Embryos by FACS (Originally by Narie Storer)

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 2/20/2012, 8740 views, 2 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.185.

[Abstract] Cell cycle analysis in combination of GFP expression has been well used to study the cell cycle distribution of GFP labeled cells. This protocol is a method to analyze the cell cycles of GFP marked cells from zebrafish embryos.
...

[Bio101] Zebrafish Embryo DNA Preparation

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 2/20/2012, 9581 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.184.

[Abstract] This protocol explains how to extract DNA from a single zebrafish embryo....

[Bio101] Flourescent Immunostaining Protocol for a-Bungorotoxin (AChRs) in Zebrafish

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 2/20/2012, 6518 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.183.

[Abstract] Zebrafish embryo is convenient for studying neuromuscular system due to its transparency. This protocol is a rapid method to visualize the AChR clusters on the postsynaptic muscle membrane.
...
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[Bio101] Dig RNA Probe Synthesis and Purification

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 1/20/2012, 14463 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.173.

[Abstract] In situ hybridization is an effective method to examine the expression level and location of gene of interest on tissues or cells. The method provided in this protocol is a detailed description of synthesizing antisense probe for in situ hybridization....

[Bio101] pH3 Antibody Staining Protocol: For Zebrafish

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 1/20/2012, 12519 views, 2 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.175.

[Abstract] Phosphorylation of histone H3 is tightly correlated with chromosome condensation during both mitosis and meiosis. Staining for phosphor histone H3 is a good indicator for the proliferation of the interested cells. This protocol provides a method to stain p-Histone H3 in zebrafish embryos.
...

[Bio101] Genotyping for Single Zebrafish (Fin Clip) or Zebrafish Embryo

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 2/5/2012, 11871 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.182.

[Abstract] Zebrafish is increasingly used a genetic model organism in biomedical studies. This protocol provides a detailed procedure about the identification of the genotype of an adult zebrafish or a zebrafish embryo.
...

[Bio101] Preparation of Torula Yeast RNA for Hybe Solutions (Originally by Caroline Burns)

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 2/5/2012, 10162 views, 5 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.180.

[Abstract] In situ hybridization in zebrafish embryos frequently suffers from high background signal. Torular yeast RNA is often added to reduce the non-specific binding that leads to the high background signal. Without appropriate preparation, torular yeast RNA can also add background to the staining. This method ...

[Bio101] Zebrafish Embryo DNA Preparation

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 2/20/2012, 9581 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.184.

[Abstract] This protocol explains how to extract DNA from a single zebrafish embryo....

Phalloidin Staining and Immunohistochemistry of Zebrafish Embryos

Authors: Michelle F. Goody
Michelle F. Goody Affiliation: Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, USA
Bio-protocol author page: a619
 and Clarissa A. Henry
Clarissa A. HenryAffiliation: School of Biology and Ecology, University of Maine, Orono, USA
For correspondence: clarissa.henry@umit.maine.edu
Bio-protocol author page: a620
date: 6/5/2013, 8764 views, 1 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.786.

[Abstract] Fluorescent conjugated Phalloidin is a stain that allows for visualization of F-actin. In immunohistochemistry, primary antibodies and fluorescent conjugated secondary antibodies can be used to visualize subcellular localization and relative amounts of proteins of interest. Here is a protocol for Phalloidin ...

[Bio101] Cell Cycle Analysis and GFP Expression of Zebrafish Embryos by FACS (Originally by Narie Storer)

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 2/20/2012, 8740 views, 2 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.185.

[Abstract] Cell cycle analysis in combination of GFP expression has been well used to study the cell cycle distribution of GFP labeled cells. This protocol is a method to analyze the cell cycles of GFP marked cells from zebrafish embryos.
...

[Bio101] Injecting Zebrafish Embryos

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 1/20/2011, 8679 views, 1 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.22.

[Abstract] Zebrafish is convenient for transient genetic manipulations such as morpholino-mediated gene knockdown, DNA/mRNA-based genetic overexpression. These genetic approaches do not involve the challenge of making transgenic animals, and their effect can last up to a week. This protocol describes the detailed ...

[Bio101] Genotyping Transgenic Zebrafish Using Genomic DNA Extracted from Clutch of Embryos (Originally by J. Lefebvre)

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 2/5/2012, 7351 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.181.

[Abstract] Transgenic zebrafish are very useful genetic tools to study various biological processes. Identification the right transgene founder and the subsequent transgenic animals are always tedious and time consuming. This protocol provides a relatively rapid and easy method to identify the founder parent using ...

[Bio101] Double In Situ Hybridization (the Fish Method)

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvani, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 2/5/2012, 7071 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.178.

[Abstract] Double in situ hybridization I very useful to examine the relationship between the expression of two genes. But it is tricky because of the cross reaction of two different antibodies. This protocol is a valid method to do double color in situ hybridization in zebrafish embryos.
...
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