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

Apprezziamo il sostegno fornito da William Archer e il meccanismo di Notre Dame Integrated Imaging. Un ringraziamento particolare sono dirette ai tecnici Freimann Life Sciences per il loro aiuto continuo e la loro cura e l&#39;allevamento del pesce zebra. Questo studio è stato sostenuto da sovvenzioni dal National Eye Institute di NIH per DRH (R01-EY018417, R01-EY024519) e il Centro per la ricerca Zebrafish, Università di Notre Dame, Notre Dame, IN.

Nota: Zebrafish sono state sollevate e mantenuto nella struttura Notre Dame Zebrafish nel Freimann Life Sciences Center. I metodi descritti in questo manoscritto sono approvati dalla University of Notre Dame cura degli animali e del Comitato uso e sono in conformità con la dichiarazione per l&#39;utilizzo degli animali nella ricerca visione dall&#39;Associazione per la Ricerca e la Visione e Oftalmologia. 1. Soluzioni <ol><li> Preparare 70% di etanolo per sterilizzare il cofano coltura di...

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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>

Gli studi che indagano i meccanismi che regolano la rigenerazione dell&#39;adulto danneggiato retina zebrafish metodi utilizzati prevalentemente immunocitochimiche 5, 25, 26, 27, 28, 29, 30. Stabilire condizioni alla cultura espianti di retina e per eseguire live-cell imaging su fenomeni, c...

A differenza di esseri umani, pesce zebra (Danio rerio) mostrano una robusta risposta la rigenerazione delle cellule alla morte dei neuroni della retina 1, 2, 3, 4. Fattore di necrosi tumorale α, una molecola di segnalazione che viene rilasciato dalla morte dei neuroni della retina induce Müller glia residente nel basale strato interno nucleare (INL) della retina, a proliferare 5 e produrre cellule progenitrici neuronali che continuano a proliferare prima differenziazione in cellule neuronali tipi morti 2, 3, 4. Durante la fase proliferativa della risposta rigenerazione, i nuclei di Müller glia e le loro cellule progenitrici neuronali derivate subiscono un modello migratorio ripetitivo in fase con il ciclo cellulare (Interkinetic migrazione nucleare, INM) 6 </sup<html> &gt;, 7. Nuclei posizionato nel basale INL replicare il loro DNA prima di migrare verso lo strato esterno nucleare (ONL) in cui si dividono prima i nuclei derivanti tornano basale alla INL. Questo processo è stato descritto durante lo sviluppo neuroepiteliale utilizzando metodi istologici, mentre approcci di imaging live-cell successivamente confermato l&#39;interpretazione da Sauer 8, 9, 10, 11, 12. Entrambi gli approcci di imaging istochimiche e live-cellulari sono stati utilizzati per determinare i meccanismi sottostanti INM e la sua funzione nello sviluppo neuroepiteli compreso retina 9, 11, 12, 13. Tuttavia, i meccanismi che regolano INM nell&#39;adulto rigenerazione della retina non sono stati studiati in molti dettaglixref &quot;&gt; 6, 7. live-cell imaging sarà un approccio preziosa per far progredire la nostra conoscenza delle vie di segnalazione che controllano INM nella rigenerazione della retina adulta. Fino a poco tempo, live-cell imaging del INM nella retina era limitato a uno zebrafish embrioni vivi o pulcino embrionale o topo postnatale espianti retinici 9, 10, 11, 12, 14, 15, 16. Mentre espianti di retina da animali adulti di una varietà di specie, tra cui topo, ratto e zebrafish sono stati utilizzati per la cella diversa approcci biologici 17, 18, 19, 20, esperimenti di imaging dal vivo di cellule utilizzando espianti di retina have stato limitato a brevi periodi di tempo e non sono stati eseguiti in modo continuativo per diverse ore 21, 22. Qui, descriviamo un protocollo dettagliato alla cultura per adulti retine zebrafish luce danneggiato per eseguire esperimenti di imaging dal vivo di cellule monitoraggio INM utilizzando multi-fotone microscopia a 6. Approcci live-cell imaging sono vantaggiosi rispetto ai metodi di immunoistochimica nell&#39;ambito delle indagini i meccanismi che controllano INM, come le dinamiche di INM, ad esempio, le velocità potrebbero essere interessati, piuttosto che la posizione della mitosi, che non è potenzialmente essere rilevato tramite immunocitochimica. In futuro, questo metodo ha anche il potenziale di essere modificati per studiare altri processi dinamici durante la rigenerazione della retina, come la fagocitosi di morire fotorecettori da Müller glia o il comportamento della 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

Un programma di rigenerazione endogena è avviata da Müller glia nel zebrafish adulti (Danio rerio) retina seguente danno neuronale e la morte. Le Müller glia ri-entrare nel ciclo cellulare e producono cellule progenitrici neuronali che sono oggetto di ulteriori cicli di divisioni cellulari e differenziarsi in tipi di cellule neuronali persi. Sia Müller glia e cellule progenitrici neuronali nuclei replicano il loro DNA e sottoposti mitosi in posizioni distinte della retina, cioè migrano tra il basale strato interno nucleare (INL) e lo strato esterno nucleare (ONL) rispettivamente, in un processo descritto come Interkinetic La migrazione nucleare (INM). INM è stata studiata principalmente nella retina sviluppo. Per esaminare le dinamiche di INM nell&#39;adulto rigenerante retina zebrafish in dettaglio, è necessario live-cell imaging di / cellule progenitrici neuronali fluorescenza marcata Müller glia. Qui, forniamo le condizioni per isolare e retine cultura dorsalida Tg [GFAP: nGFP] mi2004 zebrafish che sono stati esposti ad una costante luce intensa per 35 ore. Mostriamo anche che queste culture della retina sono vitali per eseguire esperimenti di imaging live-cell, acquisendo continuamente immagini z-stack per tutto lo spessore della espianto della retina per un massimo di 8 ore usando la microscopia multiphoton per monitorare il comportamento migratorio di GFAP: cellule -positive nGFP . Inoltre, si descrivono i dettagli per effettuare analisi post-imaging per determinare la velocità di INM apicale e basale. Per riassumere, abbiamo stabilito le condizioni per studiare le dinamiche di INM in un modello adulto di rigenerazione neuronale. Questo farà progredire la nostra comprensione di questo processo cellulare cruciale e ci permettono di determinare i meccanismi che controllano INM.

L&#39;isolamento della retina secondo la procedura descritta nello schema di figura 1 consente coltura di una retina dorsale appiattito da adulti light-danneggiato Tg [GFAP: nGFP] mi2004 zebrafish per un periodo di almeno 24 h in un CO 5% 2 / ambiente di aria. Questi espianti di retina piatto montate possono essere utilizzate per immagini piani focali a livello dei tessuti profondi. Un esempio è Müller glia / nuclei neuronali di cellule p...

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

la rigenerazione della retina zebrafish è stato per lo più studiata utilizzando retine fissi. Tuttavia, i processi dinamici come la migrazione nucleare interkinetic si verificano durante la risposta rigenerativa e richiedono live-cell imaging per studiare i meccanismi alla base. Qui, descriviamo condizioni di coltura e di imaging per monitorare Interkinetic migrazione nucleare (INM) in tempo reale utilizzando la microscopia multiphoton.

Cultura di adulti transgenici Zebrafish retina espianti per live-cell imaging per 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

Rene Rigenerazione in Zebrafish adulti da Gentamicina Induced Injury

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

Ricerca

Biologia dello sviluppo

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Cultura della embrionali di topo cocleari espianti e Gene Transfer con elettroporazione

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

Espianti di retina intero da pollo embrioni per trattamenti di elettroporazione e Agenti chimici

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

Monitoraggio celle in Zebrafish GFP-transgenico Utilizzo del sistema fotoconvertibile 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 la coltura di Neurosfere Zebrafish adulti Brain-derived

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

Analisi dello sviluppo del rene Zebrafish con time-lapse imaging Utilizzando una dissezione microscopio dotato di sezionamento ottico

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

Imaging in vivo del transgenico espressione genica nei singoli retinica Progenitori in embrioni di zebrafish Chimerico da Cell studio Influenze non autonomo

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

Imaging Live di Mouse Seconda Palate Fusion

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

Imaging multimodale gerarchica delle sezioni di serie per l'individuazione di specifici obiettivi cellulari all'interno di grandi volumi

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

Live Cell Imaging della segregazione cromosomica durante la mitosi

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