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An ex vivo Perifusion Method for Quantitative Determination of Neuropeptide Release from Mouse Hypothalamic Explants

Featured protocol,  Authors: Ophélia Le Thuc
Ophélia Le ThucAffiliation 1: Université Côte d’Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France
Affiliation 2: Technische Universität München (TUM), Munich, Germany
Affiliation 3: Technische Universität München (TUM), Munich, Germany
Affiliation 4: Helmholtz Diabetes Center (HDC) & German Center for Diabetes Research (DZD), Munich, Germany
Bio-protocol author page: a5077
Jacques Noël
Jacques NoëlAffiliation: Université Côte d’Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France
Bio-protocol author page: a5078
 and Carole Rovère
Carole RovèreAffiliation: Université Côte d’Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France
For correspondence: rovere@ipmc.cnrs.fr
Bio-protocol author page: a5079
date: 8/20/2017, 27 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2521.

Brief version appeared in EMBO Rep, Dec 2016
The hypothalamus is a primary brain area which, in mammals, regulates several physiological functions that are all related to maintain general homeostasis, by linking the central nervous system (CNS) and the periphery. The hypothalamus itself can be considered an endocrine brain region of some sort as it hosts in its different nuclei several kinds of neuropeptide-producing and -secreting neurons. These neuropeptides have specific roles and participate in the regulation of homeostasis in general, which includes the regulation of energy metabolism, feeding behavior, water intake and body core temperature for example.

A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics

Featured protocol,  Authors: Julia F. Alterman
Julia F. AltermanAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a5012
Andrew H. Coles
Andrew H. ColesAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4672
Lauren M. Hall
Lauren M. HallAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a5013
Neil Aronin
Neil AroninAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4673
Anastasia Khvorova
Anastasia KhvorovaAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4674
 and Marie-Cécile Didiot
Marie-Cécile DidiotAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
For correspondence: marie.didiot@umassmed.edu
Bio-protocol author page: a4675
date: 8/20/2017, 22 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2501.

Brief version appeared in Mol Ther, Oct 2016
Primary neurons represent an ideal cellular system for the identification of therapeutic oligonucleotides for the treatment of neurodegenerative diseases. However, due to the sensitive nature of primary cells, the transfection of small interfering RNAs (siRNA) using classical methods is laborious and often shows low efficiency. Recent progress in oligonucleotide chemistry has enabled the development of stabilized and hydrophobically modified small interfering RNAs (hsiRNAs). This new class of oligonucleotide therapeutics shows extremely efficient self-delivery properties and supports potent and durable effects in vitro and in vivo. We have developed a high-throughput in vitro assay to identify and test hsiRNAs in primary neuronal cultures. To simply, rapidly, and accurately quantify the mRNA silencing of hundreds of hsiRNAs, we use the QuantiGene 2.0 quantitative gene expression assay. This high-throughput, 96-well plate-based assay can quantify mRNA levels directly from sample lysate. Here, we describe a method to prepare short-term cultures of mouse primary cortical neurons in a 96-well plate format for high-throughput testing of oligonucleotide therapeutics. This method supports the testing of hsiRNA libraries and the identification of potential therapeutics within just two weeks. We detail methodologies of our high throughput assay workflow from primary neuron preparation to data analysis. This method can help identify oligonucleotide therapeutics for treatment of various neurological diseases.

Assessment of Thermal Pain Sensation in Rats and Mice Using the Hargreaves Test

Featured protocol,  Authors: Menghon Cheah
Menghon CheahAffiliation 1: John van Geest Centre for Brain Repair, University of Cambridge, Cambridge, United Kingdom
Affiliation 2: Centre for Developmental Neurobiology, King’s College London, London, United Kingdom
Bio-protocol author page: a5025
James W Fawcett
James W FawcettAffiliation 1: John van Geest Centre for Brain Repair, University of Cambridge, Cambridge, United Kingdom
Affiliation 2: Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Bio-protocol author page: a5026
 and Melissa R Andrews
Melissa R AndrewsAffiliation: Biological Sciences, University of Southampton, Southampton, United Kingdom
For correspondence: M.R.Andrews@soton.ac.uk
Bio-protocol author page: a5027
date: 8/20/2017, 18 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2506.

Brief version appeared in J Neurosci, Jul 2016
The Hargreaves test is specifically designed to assess thermal pain sensation in rodents such as rats and mice. This test has been used in experiments involving pain sensitization or recovery of thermal pain response following neural injury and regeneration. We present here a step-by-step protocol highlighted with important notes to guide first-time users through the learning process. Additionally, we have also included representative data from a rat model of sensory denervation showing how the data can be analysed to obtain meaningful results. We hope that this protocol can also assist potential users in deciding whether the Hargreaves test is a suitable test for their experiment.

An ex vivo Perifusion Method for Quantitative Determination of Neuropeptide Release from Mouse Hypothalamic Explants

Authors: Ophélia Le Thuc
Ophélia Le ThucAffiliation 1: Université Côte d’Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France
Affiliation 2: Technische Universität München (TUM), Munich, Germany
Affiliation 3: Technische Universität München (TUM), Munich, Germany
Affiliation 4: Helmholtz Diabetes Center (HDC) & German Center for Diabetes Research (DZD), Munich, Germany
Bio-protocol author page: a5077
Jacques Noël
Jacques NoëlAffiliation: Université Côte d’Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France
Bio-protocol author page: a5078
 and Carole Rovère
Carole RovèreAffiliation: Université Côte d’Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France
For correspondence: rovere@ipmc.cnrs.fr
Bio-protocol author page: a5079
date: 8/20/2017, 27 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2521.

[Abstract] The hypothalamus is a primary brain area which, in mammals, regulates several physiological functions that are all related to maintain general homeostasis, by linking the central nervous system (CNS) and the periphery. The hypothalamus itself can be considered an endocrine brain region of some sort as it hosts in its different nuclei several kinds ...

A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics

Authors: Julia F. Alterman
Julia F. AltermanAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a5012
Andrew H. Coles
Andrew H. ColesAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4672
Lauren M. Hall
Lauren M. HallAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a5013
Neil Aronin
Neil AroninAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4673
Anastasia Khvorova
Anastasia KhvorovaAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4674
 and Marie-Cécile Didiot
Marie-Cécile DidiotAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
For correspondence: marie.didiot@umassmed.edu
Bio-protocol author page: a4675
date: 8/20/2017, 22 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2501.

[Abstract] Primary neurons represent an ideal cellular system for the identification of therapeutic oligonucleotides for the treatment of neurodegenerative diseases. However, due to the sensitive nature of primary cells, the transfection of small interfering RNAs (siRNA) using classical methods is laborious and often shows low efficiency. Recent progress in oligonucleotide ...

Assessment of Thermal Pain Sensation in Rats and Mice Using the Hargreaves Test

Authors: Menghon Cheah
Menghon CheahAffiliation 1: John van Geest Centre for Brain Repair, University of Cambridge, Cambridge, United Kingdom
Affiliation 2: Centre for Developmental Neurobiology, King’s College London, London, United Kingdom
Bio-protocol author page: a5025
James W Fawcett
James W FawcettAffiliation 1: John van Geest Centre for Brain Repair, University of Cambridge, Cambridge, United Kingdom
Affiliation 2: Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Bio-protocol author page: a5026
 and Melissa R Andrews
Melissa R AndrewsAffiliation: Biological Sciences, University of Southampton, Southampton, United Kingdom
For correspondence: M.R.Andrews@soton.ac.uk
Bio-protocol author page: a5027
date: 8/20/2017, 18 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2506.

[Abstract] The Hargreaves test is specifically designed to assess thermal pain sensation in rodents such as rats and mice. This test has been used in experiments involving pain sensitization or recovery of thermal pain response following neural injury and regeneration. We present here a step-by-step protocol highlighted with important notes to guide first-time ...

Preparation of Crude Synaptosomal Fractions from Mouse Brains and Spinal Cords

Author: Oliver Wirths
Oliver WirthsAffiliation: Molecular Psychiatry, Dept. of Psychiatry and Psychotherapy, University Medical Center Göttingen (UMG), Göttingen, Germany
For correspondence: owirths@gwdg.de
Bio-protocol author page: a4960
date: 8/5/2017, 95 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2423.

[Abstract] The current protocol describes the preparation of crude synaptosomal fractions from mouse brain or spinal cord samples. In detail, a sequential protocol yielding crude synaptosomal and light membrane fractions is provided. This fast and easy method might be sufficient to assess the amount of synaptic proteins in down-steam applications like Western-blot ...

Extraction of Soluble and Insoluble Protein Fractions from Mouse Brains and Spinal Cords

Author: Oliver Wirths
Oliver WirthsAffiliation: Molecular Psychiatry, Dept. of Psychiatry and Psychotherapy, University Medical Center Göttingen (UMG), Göttingen, Germany
For correspondence: owirths@gwdg.de
Bio-protocol author page: a4960
date: 8/5/2017, 103 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2422.

[Abstract] The current protocol details the preparation of soluble and insoluble protein lysates from mouse brain or spinal cord samples. In detail, tissue homogenization and sequential protein extraction are described. This procedure yields soluble and insoluble protein extracts that can be further processed in down-stream applications like ELISA or Western ...

Isolation and Culturing of Rat Primary Embryonic Basal Forebrain Cholinergic Neurons (BFCNs)

Authors: Wei Xu
Wei XuAffiliation 1: Department of Neurology and Institute of Neurology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
Affiliation 2: Department of Neurosciences, University of California San Diego, School of Medicine, La Jolla, USA
Bio-protocol author page: a4915
 and Chengbiao Wu
Chengbiao WuAffiliation: Department of Neurosciences, University of California San Diego, School of Medicine, La Jolla, USA
For correspondence: chw049@ucsd.edu
Bio-protocol author page: a4916
date: 7/20/2017, 198 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2413.

[Abstract] The basal forebrain is located close to the medial and ventral surfaces of the cerebral hemispheres that develop from the sub-pallium. It regulates multiple processes including attention, learning, memory and sleep. Dysfunction and degeneration of basal forebrain cholinergic neurons (BFCNs) is believed to be involved in many disorders of the brain ...

Aldicarb-induced Paralysis Assay to Determine Defects in Synaptic Transmission in Caenorhabditis elegans

Authors: Kelly H. Oh
Kelly H. OhAffiliation: Department of Cell Biology and Anatomy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, USA
Bio-protocol author page: a4871
 and Hongkyun Kim
Hongkyun KimAffiliation: Department of Cell Biology and Anatomy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, USA
For correspondence: Hongkyun.kim@rosalindfranklin.edu
Bio-protocol author page: a4870
date: 7/20/2017, 186 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2400.

[Abstract] Aldicarb treatment causes an accumulation of acetylcholine in the synaptic cleft of the neuromuscular junction, resulting in sustained muscle activation and eventually paralysis. Aldicarb-induced paralysis assay is an easy and fast method to determine whether synaptic transmission of a C. elegans mutant of interest is altered. This assay is based on ...

Ciberial Muscle 9 (CM9) Electrophysiological Recordings in Adult Drosophila melanogaster

Authors: Benjamin A. Eaton
Benjamin A. EatonAffiliation 1: Department of Cellular and Integrative Physiology, UT Health San Antonio, San Antonio, TX, USA
Affiliation 2: The Sam and Ann Barshop Institute for Longevity and Aging Studies, UT Health San Antonio, San Antonio, TX, USA
Bio-protocol author page: a4872
 and Rebekah E. Mahoney
Rebekah E. MahoneyAffiliation 1: The Sam and Ann Barshop Institute for Longevity and Aging Studies, UT Health San Antonio, San Antonio, TX, USA
Affiliation 2: Department of Cell Systems and Anatomy, UT Health San Antonio, San Antonio, TX, USA
For correspondence: mahoneyr@uthscsa.edu
Bio-protocol author page: a4873
date: 7/20/2017, 175 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2401.

[Abstract] The complexity surrounding presynaptic recordings in mammals is a significant barrier to the study of presynaptic mechanisms during neurotransmission in the mammalian central nervous system (CNS). Here we describe an adult fly neuromuscular junction (NMJ), the ciberial muscle 9 (CM9) NMJ, which allows for the recording of both evoked (EPSPs) and spontaneous ...

Measurement of the Intracellular Calcium Concentration with Fura-2 AM Using a Fluorescence Plate Reader

Authors: Magdiel Martínez
Magdiel MartínezAffiliation: Department of Physiology and Biophysics, Medical Sciences Campus, University of Puerto Rico, San Juan, Puerto Rico
Bio-protocol author page: a4908
Namyr A. Martínez
Namyr A. MartínezAffiliation: Department of Physiology and Biophysics, Medical Sciences Campus, University of Puerto Rico, San Juan, Puerto Rico
Bio-protocol author page: a4909
 and Walter I. Silva
Walter I. SilvaAffiliation: Department of Physiology and Biophysics, Medical Sciences Campus, University of Puerto Rico, San Juan, Puerto Rico
For correspondence: walter.silva@upr.edu
Bio-protocol author page: a4910
date: 7/20/2017, 284 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2411.

[Abstract] Intracellular calcium elevation triggers a wide range of cellular responses. Calcium responses can be affected or modulated by membrane receptors mutations, localization, exposure to agonists/antagonists, among others (Burgos et al., 2007; Martínez et al., 2016). Changes in intracellular calcium concentration can be measured using the calcium sensitive ...

Live Imaging of Myogenesis in Indirect Flight Muscles in Drosophila

Author: Dagan Segal
Dagan SegalAffiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
For correspondence: dagansegal@gmail.com
Bio-protocol author page: a4782
date: 7/5/2017, 286 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2377.

[Abstract] The indirect flight muscles (IFMs) are the largest muscles in the fly, making up the bulk of the adult thorax. IFMs in Drosophila are generated during pupariation by fusion of hundreds of muscle precursor cells (myoblasts) with larval muscle templates (myotubes). Prominent features, including the large number of fusion events, the structural similarity ...
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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, 12289 views, 2 Q&A
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 ...

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, 12287 views, 0 Q&A
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 ...

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, 12130 views, 0 Q&A
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....

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, 12080 views, 4 Q&A
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 ...

[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, 11668 views, 2 Q&A
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 ...

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, 11637 views, 0 Q&A
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 ...

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, 11277 views, 3 Q&A
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 ...

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, 11173 views, 1 Q&A
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 ...

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, 10676 views, 1 Q&A
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 ...

Organotypic Slice Culture of Embryonic Brain Sections

Author: Froylan Calderon de Anda
Froylan Calderon de AndaAffiliation: Center For Molecular Neurobiology Hamburg (ZMNH), University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany
For correspondence: froylan.calderon@zmnh.uni-hamburg.de
Bio-protocol author page: a227
date: 2/5/2013, 8374 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.327.

[Abstract] This technique will allow using brain slices to study several aspects of cortical development (i.e. neurogenesis), as well as neuronal differentiation (i.e. neuronal migration, axon and dendrite formation) in situ. This protocol is suitable for various embryonic stages (Calderon de Anda et al., 2010; ...
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