A simple fluorescent double staining method for distinguishing neuronal from non-neuronal cells in the insect central nervous system

Being able to discriminate between neurons and non-neuronal cells such as glia and tracheal cells has been a major problem in insect neuroscience, because glia-specific antisera are available for only a small number of species such as Drosophila melanogaster and Manduca sexta. Especially development...

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Published in:	Journal of neuroscience methods Vol. 155; no. 2; pp. 202 - 206
Main Authors:	Loesel, Rudi, Weigel, Stefan, Bräunig, Peter
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 15-09-2006
Subjects:	Animals Cell culture Cell Nucleus - metabolism Cells, Cultured Central Nervous System - cytology Confocal laser-scanning microscopy DAPI DNA - metabolism Double fluorescent staining Fluoresceins - metabolism Glia Horseradish peroxidase Horseradish Peroxidase - immunology Horseradish Peroxidase - metabolism Immune Sera - metabolism Immunocytochemistry Indoles Insect brain Insecta Neuroglia - cytology Neuroglia - metabolism Neurons - cytology Neurons - metabolism Cell culture Confocal laser-scanning microscopy Immunocytochemistry Horseradish peroxidase DAPI Insect brain Glia Double fluorescent staining
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Abstract	Being able to discriminate between neurons and non-neuronal cells such as glia and tracheal cells has been a major problem in insect neuroscience, because glia-specific antisera are available for only a small number of species such as Drosophila melanogaster and Manduca sexta. Especially developmental or comparative studies often require an estimate of neuron numbers. Since neuronal and glial cell bodies are in many cases indiscernible in situ, a method to distinguish neurons from non-neuronal cells that works in any given species is wanting. Another application is cell culturing. Cultured cells usually change their outward shape dramatically after being isolated so that it is frequently impossible to tell neurons and glia apart. Here, we present a simple method that uses a commercially available antiserum directed against horseradish peroxidase, which specifically stains neurons but no other cell type in every insect species investigated. Counterstaining with DAPI, a fluorescent chromophore that binds to double-stranded DNA in the nuclei of all cells, yields the total number of cells in a given sample. Thus, double labeled cells can be identified as neurons, cells that carry only DAPI staining are non-neuronal.
AbstractList	Being able to discriminate between neurons and non-neuronal cells such as glia and tracheal cells has been a major problem in insect neuroscience, because glia-specific antisera are available for only a small number of species such as Drosophila melanogaster and Manduca sexta. Especially developmental or comparative studies often require an estimate of neuron numbers. Since neuronal and glial cell bodies are in many cases indiscernible in situ, a method to distinguish neurons from non-neuronal cells that works in any given species is wanting. Another application is cell culturing. Cultured cells usually change their outward shape dramatically after being isolated so that it is frequently impossible to tell neurons and glia apart. Here, we present a simple method that uses a commercially available antiserum directed against horseradish peroxidase, which specifically stains neurons but no other cell type in every insect species investigated. Counterstaining with DAPI, a fluorescent chromophore that binds to double-stranded DNA in the nuclei of all cells, yields the total number of cells in a given sample. Thus, double labeled cells can be identified as neurons, cells that carry only DAPI staining are non-neuronal. Being able to discriminate between neurons and non-neuronal cells such as glia and tracheal cells has been a major problem in insect neuroscience, because glia-specific antisera are available for only a small number of species such as Drosophila melanogaster and Manduca sexta. Especially developmental or comparative studies often require an estimate of neuron numbers. Since neuronal and glial cell bodies are in many cases indiscernible in situ, a method to distinguish neurons from non-neuronal cells that works in any given species is wanting. Another application is cell culturing. Cultured cells usually change their outward shape dramatically after being isolated so that it is frequently impossible to tell neurons and glia apart. Here, we present a simple method that uses a commercially available antiserum directed against horseradish peroxidase, which specifically stains neurons but no other cell type in every insect species investigated. Counterstaining with DAPI, a fluorescent chromophore that binds to double-stranded DNA in the nuclei of all cells, yields the total number of cells in a given sample. Thus, double labeled cells can be identified as neurons, cells that carry only DAPI staining are non-neuronal.
Author	Bräunig, Peter Loesel, Rudi Weigel, Stefan
Author_xml	– sequence: 1 givenname: Rudi surname: Loesel fullname: Loesel, Rudi email: loesel@bio2.rwth-aachen.de organization: Institute of Biology II (Zoology), Unit of Developmental Biology and Morphology of Animals, Kopernikusstraße 16, RWTH Aachen University, D-52074 Aachen, Germany – sequence: 2 givenname: Stefan surname: Weigel fullname: Weigel, Stefan organization: Institute of Thin Films and Interfaces (ISG2), Research Center Jülich, D-52425 Jülich, Germany – sequence: 3 givenname: Peter surname: Bräunig fullname: Bräunig, Peter organization: Institute of Biology II (Zoology), Unit of Developmental Biology and Morphology of Animals, Kopernikusstraße 16, RWTH Aachen University, D-52074 Aachen, Germany
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Cites_doi	10.1016/j.ydbio.2004.11.022 10.1016/j.tins.2004.09.001 10.1073/pnas.92.12.5396 10.1007/BF02631275 10.1139/o04-119 10.1016/0042-6822(60)90205-1 10.1046/j.1471-4159.1995.65010434.x 10.1242/jeb.95.1.61 10.1002/(SICI)1096-9861(19960401)367:2<235::AID-CNE6>3.0.CO;2-6 10.1016/j.pneurobio.2004.04.004 10.1016/0165-3806(85)90049-5 10.1007/s004270100173 10.1007/s00441-004-0905-7 10.1073/pnas.79.8.2700 10.1242/dev.121.2.317 10.1242/dev.117.4.1251 10.1016/j.conb.2005.01.007 10.1007/BF00744876 10.1073/pnas.96.18.10438 10.1146/annurev.ento.48.091801.112654
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Snippet	Being able to discriminate between neurons and non-neuronal cells such as glia and tracheal cells has been a major problem in insect neuroscience, because...
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SubjectTerms	Animals Cell culture Cell Nucleus - metabolism Cells, Cultured Central Nervous System - cytology Confocal laser-scanning microscopy DAPI DNA - metabolism Double fluorescent staining Fluoresceins - metabolism Glia Horseradish peroxidase Horseradish Peroxidase - immunology Horseradish Peroxidase - metabolism Immune Sera - metabolism Immunocytochemistry Indoles Insect brain Insecta Neuroglia - cytology Neuroglia - metabolism Neurons - cytology Neurons - metabolism
Title	A simple fluorescent double staining method for distinguishing neuronal from non-neuronal cells in the insect central nervous system
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