Name: culture of adult transgenic zebrafish retinal explants for live-cell imaging by multiphoton microscopy
Uploaded: 2017-02-24T15:00:00
Description: Watch this Scientific Journal Video about Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy at JoVE.com

Agradecemos o apoio fornecido por William Archer e do Mecanismo de Notre Dame Integrado Imaging. Agradecimentos especiais são direcionados para os técnicos Freimann Ciências Biológicas por sua ajuda contínua e seus cuidados e criação de peixe-zebra. Este estudo foi apoiado por subsídios da National Eye Institute dos NIH para DRH (R01-EY018417, R01-EY024519) eo Centro de Zebrafish Research, University of Notre Dame, Notre Dame, IN.

Nota: Os peixes-zebra foram criados e mantidos na instalação de Notre Dame Zebrafish na Freimann Life Sciences Center. Os métodos descritos neste manuscrito são aprovados pela Universidade de Notre Dame Animal Care e do Comitê Use e estão em conformidade com a instrução para o uso de animais em pesquisa visão pela Associação de Pesquisa em Visão e Oftalmologia. 1. Soluções <ol><li> Preparar 70% de etanol para esterilizar o capuz de cultura de tecidos e qualquer equipamento / ...

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	<li>Fausett, B. V., Goldman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+alpha1+tubulin-expressing+Muller+glia+in+regeneration+of+the+injured+zebrafish+retina.">A role for alpha1 tubulin-expressing Muller glia in regeneration of the injured zebrafish retina.</a> J.Neurosci. 26 (23), 6303-6313 (2006).</li><li>Kassen, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+analysis+of+gene+expression+during+light-induced+photoreceptor+cell+death+and+regeneration+in+albino+zebrafish.">Time course analysis of gene expression during light-induced photoreceptor cell death and regeneration in albino zebrafish.</a> Dev.Neurobiol. 67 (8), 1009-1031 (2007).</li><li>Vihtelic, T. S., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+rod+and+cone+cell+death+and+regeneration+in+the+adult+albino+zebrafish+(Danio+rerio)+retina.">Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina.</a> J.Neurobiol. 44 (3), 289-307 (2000).</li><li>Bernardos, R. L., Barthel, L. K., Meyers, J. R., Raymond, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late-stage+neuronal+progenitors+in+the+retina+are+radial+Muller+glia+that+function+as+retinal+stem+cells.">Late-stage neuronal progenitors in the retina are radial Muller glia that function as retinal stem cells.</a> J.Neurosci. 27 (26), 7028-7040 (2007).</li><li>Nelson, C. M., Ackerman, K. M., O'Hayer, P., Bailey, T. J., Gorsuch, R. A., Hyde, D. 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K., Raymond, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+self-renewing+division+of+zebrafish+Muller+glial+cells+generates+neuronal+progenitors+that+require+N-cadherin+to+regenerate+retinal+neurons.">A self-renewing division of zebrafish Muller glial cells generates neuronal progenitors that require N-cadherin to regenerate retinal neurons.</a> Development. 140 (22), 4510-4521 (2013).</li><li>Sauer, M. E., Walker, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioautographic+study+of+interkinetic+nuclear+migration+in+the+neural+tube.">Radioautographic study of interkinetic nuclear migration in the neural tube.</a> Proc.Soc.Exp.Biol.Med. 101 (3), 557-560 (1959).</li><li>Pearson, R. A., Luneborg, N. L., Becker, D. L., Mobbs, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctions+modulate+interkinetic+nuclear+movement+in+retinal+progenitor+cells.">Gap junctions modulate interkinetic nuclear movement in retinal progenitor cells.</a> J.Neurosci. 25 (46), 10803-10814 (2005).</li><li>Baye, L. M., Link, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interkinetic+nuclear+migration+and+the+selection+of+neurogenic+cell+divisions+during+vertebrate+retinogenesis.">Interkinetic nuclear migration and the selection of neurogenic cell divisions during vertebrate retinogenesis.</a> J.Neurosci. 27 (38), 10143-10152 (2007).</li><li>Del Bene, F., Wehman, A. M., Link, B. A., Baier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neurogenesis+by+interkinetic+nuclear+migration+through+an+apical-basal+notch+gradient.">Regulation of neurogenesis by interkinetic nuclear migration through an apical-basal notch gradient.</a> Cell. 134 (6), 1055-1065 (2008).</li><li>Norden, C., Young, S., Link, B. A., Harris, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actomyosin+is+the+main+driver+of+interkinetic+nuclear+migration+in+the+retina.">Actomyosin is the main driver of interkinetic nuclear migration in the retina.</a> Cell. 138 (6), 1195-1208 (2009).</li><li>Becker, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+imaging+of+chick+retinal+development+in+relation+to+gap+junctional+communication.">Multiphoton imaging of chick retinal development in relation to gap junctional communication.</a> J.Physiol. 585 (Pt 3), 711-719 (2007).</li><li>Surzenko, N., Crowl, T., Bachleda, A., Langer, L., Pevny, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX2+maintains+the+quiescent+progenitor+cell+state+of+postnatal+retinal+Muller+glia.">SOX2 maintains the quiescent progenitor cell state of postnatal retinal Muller glia.</a> Development. 140 (7), 1445-1456 (2013).</li><li>Nickerson, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+and+analysis+of+postnatal+mouse+retinal+development.">Live imaging and analysis of postnatal mouse retinal development.</a> BMC Dev.Biol. 13, 24 (2013).</li><li>Suzuki, S. C., Bleckert, A., Williams, P. R., Takechi, M., Kawamura, S., Wong, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone+photoreceptor+types+in+zebrafish+are+generated+by+symmetric+terminal+divisions+of+dedicated+precursors.">Cone photoreceptor types in zebrafish are generated by symmetric terminal divisions of dedicated precursors.</a> Proc.Natl.Acad.Sci.U.S.A. 110 (37), 15109-15114 (2013).</li><li>Johnson, T. V., Martin, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+an+adult+retinal+explant+organotypic+tissue+culture+system+as+an+in+vitro+intraocular+stem+cell+transplantation+model.">Development and characterization of an adult retinal explant organotypic tissue culture system as an in vitro intraocular stem cell transplantation model.</a> Invest.Ophthalmol.Vis.Sci. 49 (8), 3503-3512 (2008).</li><li>Bull, N. D., Johnson, T. V., Welsapar, G., DeKorver, N. W., Tomarev, S. I., Martin, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+an+adult+rat+retinal+explant+model+for+screening+of+potential+retinal+ganglion+cell+neuroprotective+therapies.">Use of an adult rat retinal explant model for screening of potential retinal ganglion cell neuroprotective therapies.</a> Invest.Ophthalmol.Vis.Sci. 52 (6), 3309-3320 (2011).</li><li>Kustermann, S., Schmid, S., Biehlmaier, O., Kohler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival,+excitability,+and+transfection+of+retinal+neurons+in+an+organotypic+culture+of+mature+zebrafish+retina.">Survival, excitability, and transfection of retinal neurons in an organotypic culture of mature zebrafish retina.</a> Cell Tissue Res. 332 (2), 195-209 (2008).</li><li>Williams, P. R., Morgan, J. L., Kerschensteiner, D., Wong, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+imaging+of+retinal+whole+mounts.">In vitro imaging of retinal whole mounts.</a> Cold Spring Harb Protoc. 2013, (2013).</li><li>Johnson, T. V., Oglesby, E. N., Steinhart, M. R., Cone-Kimball, E., Jefferys, J., Quigley, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-Lapse+Retinal+Ganglion+Cell+Dendritic+Field+Degeneration+Imaged+in+Organotypic+Retinal+Explant+Culture.">Time-Lapse Retinal Ganglion Cell Dendritic Field Degeneration Imaged in Organotypic Retinal Explant Culture.</a> Invest.Ophthalmol.Vis.Sci. 57 (1), 253-264 (2016).</li><li>Roehlecke, C., Schumann, U., Ader, M., Knels, L., Funk, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+blue+light+on+photoreceptors+in+a+live+retinal+explant+system.">Influence of blue light on photoreceptors in a live retinal explant system.</a> Mol.Vis. 17, 876-884 (2011).</li><li>Coutu, D. L., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+cellular+processes+by+long-term+live+imaging--historic+problems+and+current+solutions.">Probing cellular processes by long-term live imaging--historic problems and current solutions.</a> J.Cell.Sci. 126 (Pt 17), 3805-3815 (2013).</li><li>Thomas, J. L., Thummel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+light+damage+paradigm+for+use+in+retinal+regeneration+studies+in+adult+zebrafish.">A novel light damage paradigm for use in retinal regeneration studies in adult zebrafish.</a> J.Vis.Exp. 80, e51017 (2013).</li><li>Conner, C., Ackerman, K. M., Lahne, M., Hobgood, J. S., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repressing+notch+signaling+and+expressing+TNFalpha+are+sufficient+to+mimic+retinal+regeneration+by+inducing+Muller+glial+proliferation+to+generate+committed+progenitor+cells.">Repressing notch signaling and expressing TNFalpha are sufficient to mimic retinal regeneration by inducing Muller glial proliferation to generate committed progenitor cells.</a> J.Neurosci. 34 (43), 14403-14419 (2014).</li><li>Nelson, C. M., Gorsuch, R. A., Bailey, T. J., Ackerman, K. M., Kassen, S. C., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stat3+defines+three+populations+of+Muller+glia+and+is+required+for+initiating+maximal+muller+glia+proliferation+in+the+regenerating+zebrafish+retina.">Stat3 defines three populations of Muller glia and is required for initiating maximal muller glia proliferation in the regenerating zebrafish retina.</a> J.Comp.Neurol. 520 (18), 4294-4311 (2012).</li><li>Gorsuch, R. A., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Muller+glial+dependent+neuronal+regeneration+in+the+damaged+adult+zebrafish+retina.">Regulation of Muller glial dependent neuronal regeneration in the damaged adult zebrafish retina.</a> Exp.Eye Res. 123, 131-140 (2014).</li><li>Zhao, X. F., Wan, J., Powell, C., Ramachandran, R., Myers, M. G., Goldman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leptin+and+IL-6+family+cytokines+synergize+to+stimulate+Muller+glia+reprogramming+and+retina+regeneration.">Leptin and IL-6 family cytokines synergize to stimulate Muller glia reprogramming and retina regeneration.</a> Cell.Rep. 9 (1), 272-284 (2014).</li><li>Wan, J., Ramachandran, R., Goldman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HB-EGF+is+necessary+and+sufficient+for+Muller+glia+dedifferentiation+and+retina+regeneration.">HB-EGF is necessary and sufficient for Muller glia dedifferentiation and retina regeneration.</a> Dev.Cell. 22 (2), 334-347 (2012).</li><li>Lenkowski, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+regeneration+in+adult+zebrafish+requires+regulation+of+TGFbeta+signaling.">Retinal regeneration in adult zebrafish requires regulation of TGFbeta signaling.</a> Glia. 61 (10), 1687-1697 (2013).</li><li>Weber, I. P., Ramos, A. P., Strzyz, P. J., Leung, L. C., Young, S., Norden, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+position+and+morphology+of+committed+precursor+cells+in+the+zebrafish+retina+adapt+to+architectural+changes+upon+tissue+maturation.">Mitotic position and morphology of committed precursor cells in the zebrafish retina adapt to architectural changes upon tissue maturation.</a> Cell.Rep. 7 (2), 386-397 (2014).</li><li>Magidson, V., Khodjakov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circumventing+photodamage+in+live-cell+microscopy.">Circumventing photodamage in live-cell microscopy.</a> Methods Cell Biol. 114, 545-560 (2013).</li><li>Bailey, T. J., Fossum, S. L., Fimbel, S. M., Montgomery, J. E., Hyde, D. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+dynamic+imaging+of+retinal+microglia+using+time-lapse+confocal+microscopy.">Ex vivo dynamic imaging of retinal microglia using time-lapse confocal microscopy.</a> Invest.Ophthalmol.Vis.Sci. 49 (9), 4169-4176 (2008).</li><li>Zhao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+phagocytosis+of+living+photoreceptors+contributes+to+inherited+retinal+degeneration.">Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration.</a> EMBO Mol.Med. 7 (9), 1179-1197 (2015).</li><li>Peri, F., Nusslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+neuronal+degradation+by+microglia+reveals+a+role+for+v0-ATPase+a1+in+phagosomal+fusion+in+vivo.">Live imaging of neuronal degradation by microglia reveals a role for v0-ATPase a1 in phagosomal fusion in vivo.</a> Cell. 133 (5), 916-927 (2008).</li><li>Morsch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+characterization+of+microglial+engulfment+of+dying+neurons+in+the+zebrafish+spinal+cord.">In vivo characterization of microglial engulfment of dying neurons in the zebrafish spinal cord.</a> Front.Cell.Neurosci. 9, 321 (2015).</li></ol>

Estudos investigando os mecanismos que regem a regeneração do adulto danificado retina zebrafish métodos utilizados predominantemente imunocitoquímicos 5, 25, 26, 27, 28, 29, 30. Estabelecer condições para a cultura de explantes de retina e para realizar processamento de imagem de cé...

Ao contrário dos humanos, peixe-zebra (Danio rerio) apresentam uma resposta regeneração robusta sobre a morte celular dos neurônios da retina 1, 2, 3, 4. Factor de necrose tumoral α, uma molécula de sinalização que é libertado a partir de morrer neurónios retinais induz Müller glia residente no basal camada interna Nuclear (INL) da retina, a proliferar 5 e produzir células progenitoras neuronais que continuam a proliferar antes de se diferenciar em a célula neuronal tipos que morreram 2, 3, 4. Durante a fase proliferativa da resposta de regeneração, os núcleos de células da glia Müller e suas células progenitoras neuronais provenientes passam por um padrão repetitivo migratório em fase com o ciclo celular (migração nuclear Interkinetic, INM) 6 </sup<html> &gt;, 7. Núcleos posicionado no INL basal replicar o seu DNA antes de migrar para a camada exterior Nuclear (ONL), onde se dividem antes de retornar os núcleos resultantes basalmente para o INL. Este processo foi descrito pela primeira vez durante o desenvolvimento neuroepithelial usando métodos histológicos, enquanto abordagens de imagens ao vivo de células mais tarde confirmado a interpretação por Sauer 8, 9, 10, 11, 12. Ambas as abordagens de imagem histoquímicos e em células vivas, foram usadas para determinar os mecanismos subjacentes INM e a sua função no desenvolvimento neuroepithelia incluindo a retina 9, 11, 12, 13. No entanto, os mecanismos que regem INM no adulto regenerar retina não foram estudadas em grande detalhexref &quot;&gt; 6, 7. imagens ao vivo de células será uma abordagem de valor inestimável para avançar o nosso conhecimento sobre as vias de sinalização que controlam o INM na retina regenerar adulto. Até recentemente, a criação de imagens de células vivas de INM na retina foi limitada a qualquer embriões de peixe-zebra ao vivo ou para pinto embrionário ou o rato pós-natal explantes de retina 9, 10, 11, 12, 14, 15, 16. Enquanto explantes de retina de animais adultos de uma variedade de espécies, incluindo ratinhos, ratos e peixes-zebra foram utilizadas para diferentes células abordagens biológicas 17, 18, 19, 20, experimentos com imagens em células vivas a partir de explantes de retina have sido restrita a breves períodos de tempo e não foram executadas de forma contínua ao longo de várias horas 21, 22. Aqui, descrevemos um protocolo detalhado para a cultura adultos retinas de peixe-zebra danificado de luz para realizar experimentos com imagens em células vivas, monitoramento INM usando microscopia multi-fotão 6. Abordagens de imagem de células vivas são vantajosos em relação aos métodos de imuno-histoquímica ao investigar os mecanismos que controlam INM, como a dinâmica do INM, por exemplo, as velocidades podem ser afetadas em vez de a localização da mitose, o que não seria potencialmente ser detectada usando imunocitoquímica. No futuro, este método tem também o potencial de ser modificado para estudar outros processos dinâmicos durante a regeneração da retina, tais como fagocitose de morrer fotorreceptores por Muller glia ou o comportamento da microglia.

<table border="0" cellpadding="0" cellspacing="0" width="876">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:491px;">Dumont forceps size #5</td>
			<td style="width:201px;">World precision instruments</td>
			<td align="right" style="width:113px;">14098</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont forceps size #5, 45&#176; angle</td>
			<td>World precision instruments</td>
			<td align="right">14101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">McPherson-Vannas scissors</td>
			<td>World precision instruments</td>
			<td align="right">501233</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluordishes</td>
			<td>World precision instruments</td>
			<td>FD35-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereomicroscope</td>
			<td>Nikon&#160;</td>
			<td>SMZ-1B</td>
			<td>similar type of dissection stereomicroscope will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biological Safety Cabinet class type A2</td>
			<td>Labconco</td>
			<td></td>
			<td>equivalent type will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tissue culture incubator</td>
			<td>Thermoscientific</td>
			<td>HEPA-class 100</td>
			<td>equivalent type will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sylvania fluorescent lamps OSFP5835HOECO</td>
			<td>Bulbtronics</td>
			<td align="right">31850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 &#181;m pore-size Acrodisc syringe filter</td>
			<td>VWR</td>
			<td align="right">4192</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 ml Luer-lok syringe</td>
			<td>VWR</td>
			<td>BD309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 ml Luer-lok syringe</td>
			<td>VWR</td>
			<td>BD309653</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaHCO3</td>
			<td>FischerScientific</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CaCl2</td>
			<td>ThermoScientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgCl2</td>
			<td>EMD Millipore</td>
			<td align="right">5980</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">HBSS w/o Ca2+/Mg2+, w/o phenol red, Gibco</td>
			<td>ThermoScientific</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM w/o phenol red, Gibco</td>
			<td>ThermoScientific</td>
			<td>5100-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Horse serum, heat-inactivated</td>
			<td>ThermoScientific</td>
			<td>26050-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">penicillin/streptomycin</td>
			<td>VWR</td>
			<td>16777-164</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrapure low melting point agarose</td>
			<td>ThermoScientific</td>
			<td>16520-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol, absolute</td>
			<td>ThermoScientific</td>
			<td>BP2818-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-phenoxyethanol</td>
			<td>Sigma</td>
			<td align="right">77699</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning Cell-Tak cell and tissue adhesive&#160;</td>
			<td>VWR</td>
			<td align="right">354240</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">refractive index liquid&#160;</td>
			<td>Cargille Lab</td>
			<td>1803Y</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon A1 multiphoton microscope equipped with a MaiTai infrared laser</td>
			<td>Nikon</td>
			<td></td>
			<td>equivalent system will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40x Apo long-distance water immersion objective (N.A. 1.15)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">environmental chamber equipped with insert for 35 mm petridishes</td>
			<td>Okolab</td>
			<td></td>
			<td>equivalent system will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NIS analysis software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

culture of adult transgenic zebrafish retinal explants for live-cell imaging by multiphoton microscopy

Um programa de regeneração endógena é iniciada por Müller glia no peixe-zebra adulto (Danio rerio) retina seguintes danos e morte neuronal. As Müller glia re-entrar no ciclo celular e produzir células progenitoras neuronais que são submetidos a ciclos subsequentes de divisões celulares e diferenciar em tipos de células neuronais perdidas. Ambos Müller glia e células progenitoras neuronais núcleos replicar o seu ADN e sofrem mitose em locais distintos da retina, ou seja, elas migram entre o basal camada interna Nuclear (INL) e a camada exterior Nuclear (ONL), respectivamente, em um processo descrito como Interkinetic Migração nuclear (INM). INM foi predominantemente estudados na retina em desenvolvimento. Para examinar a dinâmica do INM no adulto regenerar retina zebrafish em detalhe, processamento de imagem de células vivas de células fluorescentes marcado Müller glia / neuronais progenitoras é necessária. Aqui, nós fornecemos as condições para isolar e retinas cultura dorsaisde Tg [GFAP: nGFP] mi2004 peixe-zebra que foram expostos à luz intensa constante por 35 h. Também mostram que estas culturas de retina são viáveis para efectuar experiências de imagiologia ao vivo de células, a aquisição de imagens continuamente Z-pilha, através da espessura do explante da retina por até 8 h, usando microscopia multifotônica para monitorar o comportamento migratório das GFAP: células -positivas nGFP . Além disso, descrevemos os detalhes para realizar a análise pós-imagem para determinar a velocidade do INM apical e basal. Para resumir, nós estabelecemos condições para o estudo da dinâmica do INM em um modelo adulto da regeneração neuronal. Isto irá avançar nossa compreensão deste processo celular crucial e nos permitem determinar os mecanismos que controlam INM.

O isolamento da retina de acordo com o procedimento delineado no esquema na Figura 1 permite a cultura de uma retina dorsal achatada de luz-danificado adulto Tg [GFAP: nGFP] mi2004 peixe-zebra ao longo de um período de pelo menos 24 horas numa atmosfera de 5% de CO 2 ambiente / ar. Estes explantes de retina montados plana pode ser usado para planos focais imagem em níveis tecidos profundos. Um exemplo é Müller glia / núcleos neuronais ...

Watch this Scientific Journal Video about Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy at JoVE.com

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

regeneração de retina de peixe-zebra foi principalmente estudada usando retinas fixos. No entanto, os processos dinâmicos, como a migração nuclear interkinetic ocorrer durante a resposta regenerativa e exigem processamento de imagem de células vivas para investigar os mecanismos subjacentes. Aqui, descrevemos as condições de cultura e de imagem para monitorar Migração Nuclear Interkinetic (INM) em tempo real através de microscopia multiphoton.

Cultura do Adulto Transgênicos Zebrafish Retinal explantes for Imaging Em células vivas, por Multiphoton Microscopia

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Research

JoVE Journal

Developmental Biology

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

A regeneração renal em Zebrafish Adulto por lesão induzida pela gentamicina

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

Biologia do Desenvolvimento

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Cultura da embrionárias mouse cocleares explantes e Gene Transferência de Eletroporação

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

Os explantes de retina inteiras de frango embriões para tratamentos de reagentes Eletroporação e Chemical

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

Acompanhando as células GFP em peixes-zebra transgénicos Usando o Sistema Photoconvertible PSmOrange

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

Isolamento e Cultura de neuroesferas Zebrafish Adulto derivado do cérebro

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

Análise de Desenvolvimento de rim Zebrafish com imagem Time-lapse Usando um microscópio de dissecação Equipado para Seccionamento Optical

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

In vivo de imagens de Gene Transgenic Expressão em Individual Retinal Progenitores em embriões Zebrafish Quimérico para estudar celular Influências não-autônomos

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Live Imaging of Mouse Secondary Palate Fusion

Imagem ao vivo do mouse Fusão do palato secundário

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Multimodal Imaging hierárquica das seções Serial para encontrar alvos celulares específicos dentro de grandes Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Imagem de célula viva de segregação do cromossomo durante a mitose

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...