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

Vi uppskattar stödet från William Archer och Notre Dame Integrated Imaging Facility. Ett särskilt tack riktas till Freimann Life Sciences tekniker för deras kontinuerliga hjälp och deras vård och djurhållning av zebrafisk. Denna studie har finansierats med bidrag från National Eye Institute of NIH till DRH (R01-EY018417, R01-EY024519) och Centrum för Zebrafish Research, University of Notre Dame, Notre Dame, IN.

Obs: Zebrafish höjdes och underhållas i Notre Dame Zebrafish anläggning i Freimann Life Sciences Center. De metoder som beskrivs i detta manuskript är godkända av University of Notre Dame Animal Care och användning kommittén och är i överensstämmelse med uttalandet för användning av djur i en syn forskning av Föreningen för forskning i Vision och Ophthalmology. 1. Lösningar <ol><li> Förbered 70% etanol för att sterilisera vävnadsodling huva och all utrustning / reagenser ...

<ol>
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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. 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Studier som undersöker de mekanismer som styr regenerering av skadade vuxna zebrafisk näthinnan främst används immunocytokemiska metoder 5, 25, 26, 27, 28, 29, 30. Skapa förutsättningar för kultur retinala explants och utföra levande cell imaging på företeelser, såsom INM, ger os...

Till skillnad från människor, zebrafisk (Danio rerio) uppvisar en robust regeneresvar vid celldöd av retinala neuroner 1, 2, 3, 4. Tumörnekrosfaktor α, en signalerande molekyl som frisätts från döende retinala neuroner inducerar Muller Glia bosatt i basal Inre Kärn- Layer (INL) av näthinnan, att proliferera 5 och producera neuronala progenitorceller som fortsätter att föröka sig före differentiera till den neuronala cellen typer som dog 2, 3, 4. Under den proliferativa fasen av förnyelse svar kärnorna av Müller glia och deras härledda neuronala stamceller genomgå en repetitiv migrationsmönster i fas med cellcykeln (Interkinetic Nuclear Migration, INM) 6 </sup<html> &gt;, 7. Kärnor placeras i de basala INL replikera sitt DNA innan den vandrar till yttre kärnlagret (ENDA) där de delar innan de uppstår kärnorna återgår basalt till INL. Denna process beskrevs först under neuroepiteliala utveckling med histologiska metoder, medan levande celler avbildning senare bekräftade tolkningen av Sauer 8, 9, 10, 11, 12. Både histokemiska och live-cell imaging metoder har använts för att bestämma mekanismerna bakom INM och dess funktion i att utveckla neuroepithelia inklusive näthinnan 9, 11, 12, 13. Däremot har de mekanismer som styr INM i vuxen regenere näthinnan inte studerats i stor detaljxref &quot;&gt; 6, 7. Live-cell imaging kommer att vara en ovärderlig metod för att förbättra vår kunskap om de signalvägar som styr INM i vuxen regenere näthinnan. Tills nyligen var levande cell imaging av INM i näthinnan begränsad till antingen levande zebrafisk embryon eller embryonala chick eller postnatal mus näthinnan explants 9, 10, 11, 12, 14, 15, 16. Medan näthinnan explants från vuxna djur av en mängd olika arter, inklusive mus, råtta och zebrafisk har använts för olika cellbiologiska metoder 17, 18, 19, 20, imaging levande cell experiment med användning av retinala explants have varit begränsad till korta tidsperioder och har inte utförts kontinuerligt under flera timmar 21, 22. Här beskriver vi ett detaljerat protokoll för att odla ljus skadade vuxna zebrafisk näthinnor att utföra levande cell imaging experiment övervakning INM med hjälp av multi-foton mikroskopi 6. Live-cell imaging metoder är fördelaktiga över immunhistokemiska metoder när man undersöker de mekanismer som styr INM, eftersom dynamiken i INM, till exempel, hastigheter kan påverkas snarare än placeringen av mitos, som eventuellt inte kan upptäckas med hjälp av immuncytokemi. I framtiden, har denna metod också potential att ändras för att studera andra dynamiska processer under retinal förnyelse, såsom fagocytos att dö fotoreceptorer från Müller glia eller beteende mikroglia.

<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

En endogen förnyelseprogram initieras av Müller glia i den vuxna zebrafisk (Danio rerio) näthinnan efter nervskada och dödsfall. Muller Glia tillbaka in i cellcykeln och producera neuronala progenitorceller som genomgår efterföljande omgångar av celldelning och differentierar till de förlorade neuronala celltyper. Både Müller Glia och neuronal progenitor cellkärnor replikera sitt DNA och genomgår mitos i distinkta platser i näthinnan, dvs de migrerar mellan de basala Inre Kärn- Layer (INL) och yttre kärnlagret (ONL), respektive, i en process som beskrivs som Interkinetic Nuclear Migration (INM). INM har främst studerats i utvecklings näthinnan. För att undersöka dynamiken i INM i vuxen regenererande zebrafisk näthinnan i detalj krävs levande cell imaging av fluorescensmärkta Müller glia / neuronala stamceller. Här ger vi förutsättningar för att isolera och kultur rygg näthinnorfrån Tg [GFAP: nGFP] mi2004 zebrafisk som utsattes för konstant intensivt ljus för 35 h. Vi visar också att dessa retinala kulturer är lönsamt att utföra levande cell imaging experiment kontinuerligt förvärva z-stack bilder hela tjockleken av näthinnans Explantation för upp till 8 h med multifotonmikroskop att övervaka migrations beteende GFAP: nGFP -positiva celler . Dessutom beskriver vi detaljerna att utföra efter bildanalys för att bestämma hastigheten för apikala och basala INM. Sammanfattningsvis har vi etablerat förutsättningar för att studera dynamiken i INM i en vuxen modell av neuronal regeneration. Detta kommer att öka vår förståelse av denna viktiga cellulär process och ger oss möjlighet att bestämma de mekanismer som styr INM.

Isoleringen av näthinnan i enlighet med förfarandet som skisseras i den schematiska i fig 1 medger odling av en tillplattad dorsal näthinna från ljus-skadade vuxna Tg [GFAP: nGFP] mi2004 zebrafisk under en period av minst 24 timmar i en 5% CO 2 / luftmiljö. Dessa platt-monterade retinala explants kan användas för att avbilda fokalplan på djupa vävnadsnivåer. Ett exempel är Müller Glia / neuronala stamfadercellkärnor märkta med...

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

Zebrafisk retinal förnyelse har mestadels studerats med hjälp av fasta näthinnor. Men dynamiska processer såsom interkinetic kärn migration inträffar under den regenerativa svar och kräver levande cell imaging att undersöka de bakomliggande mekanismerna. Här beskriver vi kultur och avbildningsbetingelser för att övervaka Interkinetic Nuclear Migration (INM) i realtid med hjälp av multifotonmikroskop.

Kultur av vuxna Transgena Zebrafish retinal explants för Live-cell imaging av multifotonMicrosCopy

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

Njure Regeneration i vuxen Zebrafish av Gentamicin inducerad skada

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

Kultur av embryonala Mouse Cochlear Explantat och Gene Transfer med elektroporering

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

Hela Retinala Explantat från kycklingembryon för elektroporering och kemiskt reagens Behandlingar

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

Spåra celler i GFP-transgen zebrafisk Använda 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

Isolering och kultur av vuxna Zebrafish Brain-härledda Neuro

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

Analys av zebrafisk njure utveckling med Time-lapse avbildning med ett dissektionsmikroskop Utrustad för optisk Sektione

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 avbildning av Transgena genuttryck i Individuell Retinal stamceller i chimär Zebrafish embryon att studera Cell Nonautonomous Influenser

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

Multimodal hierarkiska avbildning av seriell avsnitt för att hitta specifika cellulära mål inom stora volymer

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

Levande cellen avbildning av kromosomsegregering under Mitos

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