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

Wij waarderen de steun van William Archer en de Notre Dame Integrated Imaging Facility. Speciale dank zijn gericht op de Freimann Life Sciences technici voor hun voortdurende hulp en hun zorg en verzorging van de zebravis. Deze studie werd ondersteund door subsidies van de National Eye Institute van de NIH om DRH (R01-EY018417, R01-EY024519) en het Centrum voor zebravis Research, Universiteit van Notre Dame, de Notre Dame, IN.

Opmerking: zebravis zijn gerezen en onderhouden in de Notre Dame zebravis faciliteit in de Freimann Life Sciences Center. De in dit manuscript beschreven methoden zijn goedgekeurd door de Universiteit van Notre Dame Animal Care en gebruik Comite en zijn in overeenstemming met de verklaring voor het gebruik van dieren in een visioen onderzoek door de Vereniging voor Onderzoek in Vision en Oogheelkunde. 1. Oplossingen <ol><li> Bereid 70% ethanol aan de weefselkweek kap en alle apparatuur / r...

<ol>
	<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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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. 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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. 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Studies die de mechanismen die de regeneratie van beschadigde volwassen zebravissen netvlies overwegend gebruikte immunocytochemische werkwijzen 5, 25, 26, 27, 28, 29, 30. Voorwaarden worden vastgesteld om cultuur netvlies explantaten en live-cell imaging voeren op verschijnselen, zoals INM...

In tegenstelling tot mensen, de zebravis (Danio rerio) vertonen een robuuste regeneratie reactie op de celdood van retinale neuronen 1, 2, 3, 4. Tumor necrosis factor α, een signaalmolecuul dat vrijkomt uit stervende retinale neuronen induceert Müller glia die in de basale Inner kernlaag (INL) van het netvlies, te prolifereren 5 en produceren neuronale progenitorcellen die blijven prolifereren vóór differentiëren in de neuronale cel soorten die stierven 2, 3, 4. Tijdens de proliferatieve fase van de regeneratie respons, de kernen van Müller glia en daarvan afgeleide neuronale progenitorcellen ondergaan een zich herhalend patroon in trekkende fase van de celcyclus (Interkinetic Nuclear Migration, INM) 6 </sup<html> &gt;, 7. Kernen gepositioneerd in de basale INL repliceren hun DNA vóór de migratie naar de Outer Nuclear Layer (ONL) waar ze verdelen vóór het ontstaan ​​kernen basaal terug te keren naar het INL. Deze werkwijze werd voor het eerst beschreven in neuro-ontwikkeling met behulp van histologische methoden, terwijl levende cellen beeldvormingsbenaderingen later de uitlegging steun Sauer 8, 9, 10, 11, 12. Zowel histochemische en live-cell imaging benaderingen zijn gebruikt om mechanismen verantwoordelijk INM en zijn functie in de ontwikkeling neuroepithelia waaronder het netvlies 9, 11, 12, 13 bepalen. Echter, de mechanismen die INM in de volwassen regenererende netvlies zijn niet onderzocht in veel detailxref &quot;&gt; 6, 7. live-cell imaging zal een waardevolle benadering van onze kennis van de signaalwegen die INM controle in de volwassen regenererende netvlies vooruit zijn. Tot voor kort levende cellen beeldvorming van INM in het netvlies is beperkt tot of levende zebravis embryo&#39;s of embryonale kuiken of postnatale muizen netvlies explantaten 9, 10, 11, 12, 14, 15, 16. Terwijl netvlies explantaten van volwassen dieren van verschillende soorten, waaronder muizen, ratten en zebravis zijn gebruikt voor verschillende celbiologische benaderingen 17, 18, 19, 20, live-cell imaging experimenten met retinale explantaten have beperkt tot periodes kort en hebben niet continu gedurende enkele uren 21, 22 is uitgevoerd. Hier beschrijven we een gedetailleerd protocol om de cultuur-licht beschadigd volwassen zebravissen netvlies live-cell imaging experimenten toezicht INM te voeren met behulp van multi-foton microscopie 6. Live-cell imaging benaderingen zijn gunstig ten opzichte van immunohistochemische methoden bij het onderzoek naar de mechanismen die INM, als de dynamiek van INM, bijvoorbeeld snelheden kunnen worden getroffen in plaats van de locatie van de mitose, dat zou mogelijk niet worden gedetecteerd met behulp van immunocytochemie. In de toekomst, deze werkwijze heeft ook het potentieel om te worden aangepast aan andere dynamische processen tijdens het netvlies regeneratie, zoals fagocytose sterven fotoreceptoren Muller glia of het gedrag van microglia bestuderen.

<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

Een endogeen regeneratie programma is geïnitieerd door Müller glia in de volwassen zebravissen (Danio rerio) retina volgende neuronale schade en de dood. De Müller glia opnieuw in te voeren de celcyclus en produceren van neuronale voorlopercellen die volgende rondes van celdelingen ondergaan en differentiëren in de verloren neuronale celtypen. Zowel Müller glia en neuronale voorlopercellen celkernen repliceren hun DNA en ondergaat mitose in verschillende locaties van het netvlies, dat wil zeggen ze migreren tussen de basale Inner kernlaag (INL) en de buitenste nucleaire laag (ONL), respectievelijk in een werkwijze genaamd Interkinetic Nuclear Migratie (INM). INM is voornamelijk bestudeerd in de ontwikkelende retina. Om de dynamiek van INM in de volwassen zebravissen netvlies regenereren in detail te onderzoeken, is voor live-cell imaging van fluorescent-gelabelde Müller glia / neuronale voorlopercellen nodig. Hier bieden we de voorwaarden te isoleren en cultuur dorsale netvliezenvan Tg [GFAP: nGFP] mi2004 zebravis die werden blootgesteld aan een constante intens licht voor 35 uur. We tonen ook dat deze retinale culturen levensvatbaar live-cell imaging experimenten, continu verwerven z-stack beelden over de dikte van het netvlies explantaat tot 8 h en multifoton microscopie het migrerende gedrag van GFAP controleren: nGFP -positieve cellen . Daarnaast beschrijven we de gegevens om na de beeldvorming uit te voeren om de snelheid van apicale en basale INM bepalen. Om samen te vatten, hebben we voorwaarden aan de dynamiek van INM in een naaktmodel van neuronale regeneratie te bestuderen. Dit zal ons begrip van deze cruciale cellulaire proces te bevorderen en ons toelaten om de mechanismen die INM controle te bepalen.

De isolatie van het netvlies volgens de procedure in het schema weergegeven in figuur 1 maakt het kweken van een afgeplatte dorsale netvlies tegen licht beschadigde adult Tg [GFAP: nGFP] mi2004 zebravis gedurende ten minste 24 uur in een 5% CO 2 / lucht-omgeving. Deze flat-gemonteerde netvlies explantaten kan worden gebruikt om het focale vliegtuigen bij diepe weefsel niveaus. Een voorbeeld is Müller glia / neuronale voorlopercellen celker...

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

Zebravis netvlies regeneratie is meestal onderzocht met behulp van vaste netvliezen. Echter, dynamische processen zoals interkinetic nucleaire migratie optreden tijdens de regeneratieve respons en vereisen voor live-cell imaging om de onderliggende mechanismen te onderzoeken. We beschrijven hier de cultuur en beeldvorming voorwaarden te controleren Interkinetic Nuclear Migratie (INM) in real-time met behulp van multiphoton microscopie.

Cultuur van volwassen transgene zebravis Retina Explantaten voor Live-cell imaging van Multiphoton Microscopy

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

Nier Regeneratie in Adult zebravis door Gentamicin geïnduceerd letsel

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

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Cultuur van embryonale Mouse Cochlear Explantaten en Gene Transfer door elektroporatie

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

Hele netvlies Explantaten van kippenembryo&#39;s voor Elektroporatie en chemische reagens Behandelingen

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

Het bijhouden van cellen in GFP-transgene zebravis Met behulp van de photoconvertible PSmOrange System

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

Isolatie en cultuur van de Adult zebravis Brain-afgeleide Neurosferen

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

Analyse van de zebravis Kidney Development met Time-lapse Imaging Met behulp van een dissectie microscoop Uitgerust voor Optische-Sectie

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 beeldvorming van transgene Gene Expression Individuele retinale voorouders in chimère zebravis embryo&#39;s to Cell Nonautonomous Invloeden Studie

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

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

Multimodale hiërarchische beeldvorming van seriële secties voor het vinden van specifieke cellulaire doelen binnen grote 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

Levende cel beeldvorming van chromosoom segregatie tijdens de mitose

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