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MPM-2 Mediated Immunoprecipitation of Proteins Undergoing Proline-directed Phosphorylation

Featured protocol,  Authors: Roberta Antonelli
Roberta AntonelliAffiliation 1: International School for Advanced Studies, Neurobiology Department, Trieste, Italy
Affiliation 2: Laboratory of Translational Research in Child and Adolescent Cancer, Vall d'Hebron Research Institute (VHIR)-UAB, Barcelona, Spain
For correspondence: roberta.antonelli@vhir.org
Bio-protocol author page: a3828
 and Paola Zacchi
Paola ZacchiAffiliation: International School for Advanced Studies, Neurobiology Department, Trieste, Italy
Bio-protocol author page: a3829
date: 12/5/2016, 26 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2046.

Brief version appeared in J Neurosci, May 2016
Immunoprecipitation (IP) represents a widely utilized biochemical method to isolate a specific protein from a complex mixture taking advantage of an antibody that specifically recognizes that particular target molecule. This procedure is extremely versatile and can be applied to concentrate a specific protein, to identify interacting partners in complex with it or to detect post-translational modifications. The mitotic protein monoclonal 2 (MPM-2) is an antibody originally raised against extracts of synchronized mitotic HeLa cells to identify proteins selectively present in mitotic, and not in interphase-cells (Davis et al., 1983). MPM-2 recognizes phosphorylated serine or threonine residues followed by proline (pS/T-P), consensus epitopes generated by the concerted action of proline-directed protein kinases and phosphatases (Lu et al., 2002). These reversible phosphorylation events have emerged to control various cellular processes by promoting conformational changes on the target that are not simply due to the phosphorylation event per se. These motifs, once phosphorylated, are able to recruit Pin1 (Peptidyl-prolyl Isomerase NIMA interacting protein 1) (Lu et al., 1996; Lu and Zhou, 2007), a chaperone which drives the cis/trans isomerization reaction on the peptide bond, switching the substrate between functionally diverse conformations (Lu, 2004; Wulf et al., 2005). This protocol describes a general MPM-2 based immunoprecipitation strategy using the scaffolding molecule postsynaptic density protein-95 (PSD-95) (Chen et al., 2005), a neuronal Pin1 target (Antonelli et al., 2016), as an example to illustrate the detailed procedure.

Delayed Spatial Win-shift Test on Radial Arm Maze

Featured protocol,  Authors: Simone N. De Luca
Simone N. De LucaAffiliation: School of Health and Biomedical Sciences, RMIT University, Melbourne, Vic, Australia
Bio-protocol author page: a3841
Luba Sominsky
Luba Sominsky Affiliation: School of Health and Biomedical Sciences, RMIT University, Melbourne, Vic, Australia
Bio-protocol author page: a3842
 and Sarah J. Spencer
Sarah J. SpencerAffiliation: School of Health and Biomedical Sciences, RMIT University, Melbourne, Vic, Australia
For correspondence: Sarah.Spencer@rmit.edu.au
Bio-protocol author page: a3843
date: 12/5/2016, 29 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2053.

Brief version appeared in J Neuroinflammation, May 2016
The radial arm maze (RAM) is used to assess reference and working memory in rodents. This task relies on the rodent’s ability to orientate itself in the maze using extra-maze visual cues. This test can be used to investigate whether a rodent’s cognition is improved or impaired under a variety of experimental conditions. Here, we describe one way to test spatial working and reference memory. This delayed spatial win-shift (DSWS) procedure on the RAM was adapted from Packard and White (1990). The win-shift component of the test refers to the alternation of baiting, or rewarding, arms during the trial and test phase. The rodent is required to hold spatial information both within the task and across a delay to obtain the food-pellet reward (Taylor et al., 2003b). This task measures the incidence and type of memory errors made by the rodent both in the training and test phases of the learning task. A working memory error (re-entry of an arm that has been baited) can occur in both phases of the task, whilst a reference memory error (entry into an arm that has been baited during the training phase and is no longer baited) can only occur during the test phase.

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.

Microinjection of Virus into Lumbar Enlargement of Spinal Dorsal Horn in Mice

Featured protocol,  Authors: Zhi-Jun Zhang*
Zhi-Jun ZhangAffiliation 1: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Affiliation 2: Department of Human Anatomy, Nantong University, Jiangsu, China
Bio-protocol author page: a3741
Peng-Bo Jing*
Peng-Bo JingAffiliation: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Bio-protocol author page: a3742
 and Yong-Jing Gao
Yong-Jing GaoAffiliation 1: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Affiliation 2: Co-innovation Center of Neuroregeneration, Nantong University, Jiangsu, China
For correspondence: gaoyongjing@hotmail.com
Bio-protocol author page: a3743
 (*contributed equally to this work) date: 11/20/2016, 133 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2020.

Brief version appeared in J Clin Invest, Feb 2016
In order to explore the role of a specific gene/protein in the specific segment of spinal cord, the technique of intraspinal injection is particularly used to deliver viral vectors targeting the specific gene/protein. These viral vectors can knockdown or overexpress the specific gene/protein in specific cells (glial cells or neurons). In this protocol, lentivirus containing shRNA for CXCL13 were injected into dorsal horn of the spinal lumbar enlargement segment (Jiang et al., 2016). This technique allows the study of the role of CXCL13 in the ipsilateral dorsal horn in neuropathic pain without affecting DRG or contralateral dorsal horn.

An in vitro Model of Neuron-macrophage Interaction to Generate Macrophages with Neurite Outgrowth Properties

Featured protocol,  Authors: Hyeok Jun Yun
Hyeok Jun YunAffiliation 1: Department of Brain Science, Ajou University School of Medicine, Suwon, Korea
Affiliation 2: BK21 PLUS Program, Department of Biomedical Sciences, Neuroscience Graduate Program, Ajou University School of Medicine, Suwon, Korea
Bio-protocol author page: a3721
 and Byung S. Kim
Byung S. KimAffiliation 1: Department of Brain Science, Ajou University School of Medicine, Suwon, Korea
Affiliation 2: BK21 PLUS Program, Department of Biomedical Sciences, Neuroscience Graduate Program, Ajou University School of Medicine, Suwon, Korea
Affiliation 3: Department of Neurology, Ajou University School of Medicine, Suwon, Korea
For correspondence: kimbg@ajou.ac.kr
Bio-protocol author page: a1956
date: 11/20/2016, 101 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2012.

Brief version appeared in J Neurosci, Dec 2015
Macrophages are known to play beneficial roles in axon regeneration after nerve injury. To develop an in vitro model in which injury signals can elicit pro-regenerative macrophage activation, we established co-cultures consisting of adult dorsal root ganglia sensory neurons and peritoneal macrophages and added cAMP analogue dibutyryl cAMP. The conditioned medium collected from the co-cultures exhibited robust neurite outgrowth activities. The neurite outgrowth activities were almost completely abrogated by addition of minocycline, a macrophage deactivator, indicating that factors responsible for neurite outgrowth are produced by activated macrophages.

In vitro Brainstem-spinal Cord Preparation from Newborn Rat

Featured protocol,  Authors: Jean-Patrick Le Gal
Jean-Patrick Le GalAffiliation: Institut de Neurosciences Cognitives et Intégratives d’Aquitaine (INCIA), Université de Bordeaux, Bordeaux, France
Bio-protocol author page: a3699
Angelo Nicolosi
Angelo NicolosiAffiliation: Institut de Neurosciences Cognitives et Intégratives d’Aquitaine (INCIA), Université de Bordeaux, Bordeaux, France
Bio-protocol author page: a3700
Laurent Juvin
Laurent JuvinAffiliation: Institut de Neurosciences Cognitives et Intégratives d’Aquitaine (INCIA), Université de Bordeaux, Bordeaux, France
Bio-protocol author page: a3701
 and Didier Morin
Didier MorinAffiliation: Institut de Neurosciences Cognitives et Intégratives d’Aquitaine (INCIA), Université de Bordeaux, Bordeaux, France
For correspondence: didier.morin@u-bordeaux.fr
Bio-protocol author page: a3702
date: 11/20/2016, 111 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2003.

Brief version appeared in J Neurosci, Jan 2016
The brainstem-spinal cord preparation of newborn rat contains neural networks able to produce motor output in absence of sensory feedback. These neural structures, commonly called central pattern generators (CPGs), are involved in many vital functions such as respiration (Morin and Viala, 2002; Giraudin et al., 2008) or locomotion (Juvin et al., 2005). Here we describe a procedure for the isolation of the brainstem-spinal cord tissue of neonatal rat (0-2 days old). A surgical method under binocular microscope allows the brainstem and the spinal cord to be isolated in vitro and the motor outputs to be recorded. This preparation can then be used for diverse experimental approaches, such as electrophysiology, pharmacology or anatomical studies, and constitutes a useful model to study the interaction between CPGs (Juvin et al., 2007; 2012; Giraudin et al., 2012; Le Gal et al., 2014; 2016).

MPM-2 Mediated Immunoprecipitation of Proteins Undergoing Proline-directed Phosphorylation

Authors: Roberta Antonelli
Roberta AntonelliAffiliation 1: International School for Advanced Studies, Neurobiology Department, Trieste, Italy
Affiliation 2: Laboratory of Translational Research in Child and Adolescent Cancer, Vall d'Hebron Research Institute (VHIR)-UAB, Barcelona, Spain
For correspondence: roberta.antonelli@vhir.org
Bio-protocol author page: a3828
 and Paola Zacchi
Paola ZacchiAffiliation: International School for Advanced Studies, Neurobiology Department, Trieste, Italy
Bio-protocol author page: a3829
date: 12/5/2016, 26 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2046.

[Abstract] Immunoprecipitation (IP) represents a widely utilized biochemical method to isolate a specific protein from a complex mixture taking advantage of an antibody that specifically recognizes that particular target molecule. This procedure is extremely versatile and can be applied to concentrate a specific protein, to identify interacting partners in complex ...

Delayed Spatial Win-shift Test on Radial Arm Maze

Authors: Simone N. De Luca
Simone N. De LucaAffiliation: School of Health and Biomedical Sciences, RMIT University, Melbourne, Vic, Australia
Bio-protocol author page: a3841
Luba Sominsky
Luba Sominsky Affiliation: School of Health and Biomedical Sciences, RMIT University, Melbourne, Vic, Australia
Bio-protocol author page: a3842
 and Sarah J. Spencer
Sarah J. SpencerAffiliation: School of Health and Biomedical Sciences, RMIT University, Melbourne, Vic, Australia
For correspondence: Sarah.Spencer@rmit.edu.au
Bio-protocol author page: a3843
date: 12/5/2016, 29 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2053.

[Abstract] The radial arm maze (RAM) is used to assess reference and working memory in rodents. This task relies on the rodent’s ability to orientate itself in the maze using extra-maze visual cues. This test can be used to investigate whether a rodent’s cognition is improved or impaired under a variety of experimental conditions. Here, we describe one way to ...

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 their organs like limb/fin, jaw, heart and various ...

Microinjection of Virus into Lumbar Enlargement of Spinal Dorsal Horn in Mice

Authors: Zhi-Jun Zhang*
Zhi-Jun ZhangAffiliation 1: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Affiliation 2: Department of Human Anatomy, Nantong University, Jiangsu, China
Bio-protocol author page: a3741
Peng-Bo Jing*
Peng-Bo JingAffiliation: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Bio-protocol author page: a3742
 and Yong-Jing Gao
Yong-Jing GaoAffiliation 1: Institute of Nautical Medicine, Nantong University, Jiangsu, China
Affiliation 2: Co-innovation Center of Neuroregeneration, Nantong University, Jiangsu, China
For correspondence: gaoyongjing@hotmail.com
Bio-protocol author page: a3743
 (*contributed equally to this work) date: 11/20/2016, 133 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2020.

[Abstract] In order to explore the role of a specific gene/protein in the specific segment of spinal cord, the technique of intraspinal injection is particularly used to deliver viral vectors targeting the specific gene/protein. These viral vectors can knockdown or overexpress the specific gene/protein in specific cells (glial cells or neurons). In this protocol, ...

An in vitro Model of Neuron-macrophage Interaction to Generate Macrophages with Neurite Outgrowth Properties

Authors: Hyeok Jun Yun
Hyeok Jun YunAffiliation 1: Department of Brain Science, Ajou University School of Medicine, Suwon, Korea
Affiliation 2: BK21 PLUS Program, Department of Biomedical Sciences, Neuroscience Graduate Program, Ajou University School of Medicine, Suwon, Korea
Bio-protocol author page: a3721
 and Byung S. Kim
Byung S. KimAffiliation 1: Department of Brain Science, Ajou University School of Medicine, Suwon, Korea
Affiliation 2: BK21 PLUS Program, Department of Biomedical Sciences, Neuroscience Graduate Program, Ajou University School of Medicine, Suwon, Korea
Affiliation 3: Department of Neurology, Ajou University School of Medicine, Suwon, Korea
For correspondence: kimbg@ajou.ac.kr
Bio-protocol author page: a1956
date: 11/20/2016, 101 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2012.

[Abstract] Macrophages are known to play beneficial roles in axon regeneration after nerve injury. To develop an in vitro model in which injury signals can elicit pro-regenerative macrophage activation, we established co-cultures consisting of adult dorsal root ganglia sensory neurons and peritoneal macrophages and added cAMP analogue dibutyryl cAMP. The conditioned ...

In vitro Brainstem-spinal Cord Preparation from Newborn Rat

Authors: Jean-Patrick Le Gal
Jean-Patrick Le GalAffiliation: Institut de Neurosciences Cognitives et Intégratives d’Aquitaine (INCIA), Université de Bordeaux, Bordeaux, France
Bio-protocol author page: a3699
Angelo Nicolosi
Angelo NicolosiAffiliation: Institut de Neurosciences Cognitives et Intégratives d’Aquitaine (INCIA), Université de Bordeaux, Bordeaux, France
Bio-protocol author page: a3700
Laurent Juvin
Laurent JuvinAffiliation: Institut de Neurosciences Cognitives et Intégratives d’Aquitaine (INCIA), Université de Bordeaux, Bordeaux, France
Bio-protocol author page: a3701
 and Didier Morin
Didier MorinAffiliation: Institut de Neurosciences Cognitives et Intégratives d’Aquitaine (INCIA), Université de Bordeaux, Bordeaux, France
For correspondence: didier.morin@u-bordeaux.fr
Bio-protocol author page: a3702
date: 11/20/2016, 111 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2003.

[Abstract] The brainstem-spinal cord preparation of newborn rat contains neural networks able to produce motor output in absence of sensory feedback. These neural structures, commonly called central pattern generators (CPGs), are involved in many vital functions such as respiration (Morin and Viala, 2002; Giraudin et al., 2008) or locomotion (Juvin et al., 2005). ...

Apparatus and General Methods for Exposing Rats to Audiogenic Stress

Author: Serge Campeau
Serge CampeauAffiliation: Department of Psychology and Neuroscience, Center for Neuroscience, University of Colorado Boulder, Boulder, Colorado, USA
For correspondence: Serge.Campeau@colorado.edu
Bio-protocol author page: a3678
date: 11/5/2016, 131 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1994.

[Abstract] Most organisms react innately to the sudden onset of environmental stimulation. Audiogenic or loud noise in rodents provides an effective threatening signal to study the central nervous circuits responsible for the elaboration of various responses typically elicited by threatening/stressful environmental stimulation. Audiogenic stress offers many advantages ...

Protocol for Primary Microglial Culture Preparation

Authors: Hong Lian
Hong LianAffiliation: Institute of Neuroscience, Zhejiang University School of Medicine, Hangzhou, China
For correspondence: honglian@zju.edu.cn
Bio-protocol author page: a3663
Ethan Roy
Ethan RoyAffiliation: Huffington Center on Aging, Baylor College of Medicine, Houston, Texas, USA
Bio-protocol author page: a3664
 and Hui Zheng
Hui ZhengAffiliation: Huffington Center on Aging, Baylor College of Medicine, Houston, Texas, USA
Bio-protocol author page: a3665
date: 11/5/2016, 200 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1989.

[Abstract] Primary microglia, in either mono-culture or co-culture with neurons or astrocytes, are a powerful tool for studying mechanisms underlying microglial inflammatory responses and cell type-specific interactions in the central nervous system (CNS). This protocol provides the details of how to prepare high purity primary microglia from newborn mouse pups. ...

Microglial Phagocytosis Assay

Authors: Hong Lian
Hong LianAffiliation: Institute of Neuroscience, Zhejiang University School of Medicine, Hangzhou, China
For correspondence: honglian@zju.edu.cn
Bio-protocol author page: a3663
Ethan Roy
Ethan RoyAffiliation: Huffington Center on Aging, Baylor College of Medicine, Houston, Texas, USA
Bio-protocol author page: a3664
 and Hui Zheng
Hui ZhengAffiliation: Huffington Center on Aging, Baylor College of Medicine, Houston, Texas, USA
Bio-protocol author page: a3665
date: 11/5/2016, 214 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1988.

[Abstract] Phagocytosis is essential for microglial clearance of apoptotic cells, extracellular protein aggregates, and infectious bacteria in the central nervous system (CNS). While the preparation of primary microglial culture has been described elsewhere, this protocol describes the microglial phagocytosis experimental procedure and the subsequent measurement ...

Artificial Optogenetic TRN Stimulation of C. elegans

Authors: Ithai Rabinowitch
Ithai RabinowitchAffiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, USA
For correspondence: ithairab@gmail.com
Bio-protocol author page: a3605
Millet Treinin
Millet TreininAffiliation: Department of Medical Neurobiology, Hadassah Medical School, Hebrew University of Jerusalem, Jerusalem, Israel
Bio-protocol author page: a3606
 and Jihong Bai
Jihong BaiAffiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, USA
Bio-protocol author page: a3607
date: 10/20/2016, 148 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1966.

[Abstract] Optogenetics is a powerful tool for manipulating neuronal activity with high temporal and spatial precision. In the nematode C. elegans optogentics is especially useful and easy to apply. This is because C. elegans is translucent, so its neurons are highly accessible to optic stimulation. In addition, many of its neurons can be exclusively targeted ...
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Stereotaxic Injection of LPS into Mouse Substantia Nigra

Author: Huiming Gao
Huiming GaoAffiliation: National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA
For correspondence: gao2@niehs.nih.gov
Bio-protocol author page: a17
date: 4/20/2012, 10523 views, 4 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.153.

[Abstract] Stereotaxic injection is an attractive approach for studying genetic, cellular and circuit functions in the brain. Injection of anatomical tracers, site-targeted lesions and gene delivery by recombinant adeno-associated viruses and lentiviruses in mice are powerful tools to study nervous system development ...

Immunofluorescence Staining on Mouse Embryonic Brain Sections

Author: Xuecai Ge
Xuecai GeAffiliation 1: Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), Cambridge, USA
Affiliation 2: , Howard Hughes Medical Institute, Cambridge, USA
For correspondence: xuecaige@stanford.edu
Bio-protocol author page: a46
date: 6/5/2012, 10481 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.192.

[Abstract] This protocol comprises the entire process of immunofluorescence staining on mouse embryonic brains, starting from tissue preparation to mounting of the tissue sections....

c-Fos and Arc Immunohistochemistry on Rat Cerebellum

Author: Soyun Kim
Soyun KimAffiliation: Neuroscience Program, University of Southern California, Los Angeles, USA
For correspondence: soyunkimucsd@gmail.com
Bio-protocol author page: a45
date: 5/20/2012, 9977 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.191.

[Abstract] This protocol aims to introduce methods for sacrificing rats by transcardial perfusion and extracting the brain, and introduce methods for staining the rat brain tissue with c-Fos and Arc antibodies. Please note the expression of the proteins is very sensitive to behavioral paradigm that triggers neural ...

[Bio101] Microglia Cultures and Mixed Glial Culture

Author: Huiming Gao
Huiming GaoAffiliation: Neuropharmacology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA
For correspondence: gao2@niehs.nih.gov
Bio-protocol author page: a17
date: 11/5/2011, 9855 views, 3 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.149.

[Abstract] Primary rodent microglia-enriched cultures are the most popular model to study microglial biology in vitro and to explore immune signaling pathways. Mixed glial cultures that contain microglia and astroglia are very useful for investigating the precise mechanisms of microglia-astroglia interaction during ...

Mouse Cochlear Whole Mount Immunofluorescence

Authors: Omar Akil
Omar AkilAffiliation: Department Of Otolaryngology-HNS, University of California, San Francisco, USA
For correspondence: oakil@ohns.ucsf.edu
Bio-protocol author page: a238
 and Lawrence R. Lustig
Lawrence R. LustigAffiliation: Department Of Otolaryngology-HNS, University of California, San Francisco, USA
Bio-protocol author page: a239
date: 3/5/2013, 9578 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.332.

[Abstract] This protocol comprises the entire process of immunofluorescence staining on mouse cochlea whole mount, starting from tissue preparation to the mounting of the tissue. This technique provides “three-dimensional” views of the stained components in order to determine the localization of a protein of interest ...

In utero Electroporation of Mouse Embryo Brains

Author: Xuecai Ge
Xuecai GeAffiliation 1: Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology (MIT), Cambridge, USA
Affiliation 2: , Howard Hughes Medical Institute, Cambridge, USA
For correspondence: xuecaige@stanford.edu
Bio-protocol author page: a46
date: 7/20/2012, 9170 views, 1 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.231.

[Abstract] This is a non-invasive technique to introduce transgenes into developing brains. In this technique, DNA is injected into the lateral ventricle of the embryonic brains, and is incorporated into the cells through electroporation. Embryos then continue their development in normal conditions in vivo. The ...

A Protocol for Electrophoretic Mobility Shift Assay (EMSA) from Primary Neuron

Author: Jiali Li
Jiali LiAffiliation: Department of Cell Biology and Neuroscience, Nelson Biological Laboratories, Rutgers University, Piscataway, NJ, USA
For correspondence: jli@dls.rutgers.edu
Bio-protocol author page: a179
date: 12/5/2012, 9044 views, 3 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.300.

[Abstract] The interaction of transcriptional or co-transcriptional factors with DNA is crucial for changes of neuronal gene expression during normal brain development as well as neurodegeneration. The electrophoretic mobility shift assay (EMSA) is a very powerful technique for studying changes of neuronal gene ...

Novel Object Recognition for Studying Memory in Mice

Authors: Tzyy-Nan Huang
Tzyy-Nan HuangAffiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan
For correspondence: eugene02@gate.sinica.edu.tw
Bio-protocol author page: a1680
 and Yi-Ping Hsueh
Yi-Ping HsuehAffiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan
For correspondence: yph@gate.sinica.edu.tw
Bio-protocol author page: a1681
date: 10/5/2014, 8505 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1249.

[Abstract] Memory tests are important indexes of the brain functions for rodents behavior assay. Many memory tasks require external forces (e.g. electric shocks) or intrinsic forces (e.g. hunger and thirsty) to trigger the responses. Under those conditions, rodents are under stresses, such as pain, tired, malnutrition ...

Optical Clearing Using SeeDB

Authors: Meng-Tsen Ke
Meng-Tsen KeAffiliation: Laboratory for Sensory Circuit Formation, RIKEN Center for Developmental Biology, Kobe, Japan
Bio-protocol author page: a1144
Satoshi Fujimoto
Satoshi FujimotoAffiliation: Laboratory for Sensory Circuit Formation, RIKEN Center for Developmental Biology, Kobe, Japan
Bio-protocol author page: a1145
 and Takeshi Imai
Takeshi ImaiAffiliation: Laboratory for Sensory Circuit Formation, RIKEN Center for Developmental Biology, Kobe, Japan
For correspondence: imai@cdb.riken.jp
Bio-protocol author page: a1146
date: 2/5/2014, 8430 views, 1 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1042.

[Abstract] We describe a water-based optical clearing agent, SeeDB (See Deep Brain), which clears fixed brain samples in a few days without quenching many types of fluorescent dyes, including fluorescent proteins and lipophilic neuronal tracers. SeeDB is a saturated solution of fructose (80.2% w/w) in water with ...

Hippocampal Neuron Dissociation Transfection and Culture in Microfluidics Chambers

Author: Yang Geng
Yang GengAffiliation: Department of Pediatrics and Bioengineering, Stanford University School of Medicine, Stanford, USA
For correspondence: yanggeng@stanford.edu
Bio-protocol author page: a64
date: 7/20/2012, 6880 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.235.

[Abstract] Microfluidics chamber is an ideal tool to study local events that occurring in neuronal projections by perfectly compartmentalizing the cell soma from certain branches. It is very well suited for live cell imaging or immunohistochemistry staining. This protocol has been carefully modified in detail ...
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