Name: semi-automated imaging of tissue-specific fluorescence in zebrafish embryos
Uploaded: 2014-05-17T17:00:00
Description: Watch this Scientific Journal Video about Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos at JoVE.com

Vi takker Susan Farmer og personale UAB zebrafisk forskning facilitet for zebrafisk pleje. Finansiering fra startfonde fra Institut for Farmakologi og Toksikologi.

BEMÆRK: Denne protokol blev godkendt af University of Alabama i Birmingham Institutional Animal Care og brug Udvalg. 1.. Zebrafisk Forædling og Egg Samlinger <ol><li> Tre dage før du begynder kemiske eksponeringer, samle avl tanke med skillevægge til separate hanner og hunner. Fyld hver tank halvvejs med akvakultursystemet vand. Ved hjælp af et net, overføre Tg (5xERE: GFP) c262/c262 fisk til avl tanke, at placere 2 hanner og 3 hunner i hver tank adskilt af en ski...

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	<li>Molina, G., Watkins, S., Tsang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+FGF+reporter+transgenic+zebrafish+and+their+utility+in+chemical+screens.">Generation of FGF reporter transgenic zebrafish and their utility in chemical screens.</a> BMC Developmental Biology. 7, 62 (2007).</li><li>Perz-Edwards, A., Hardison, N. L., Linney, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid-mediated+gene+expression+in+transgenic+reporter+zebrafish.">Retinoic acid-mediated gene expression in transgenic reporter zebrafish.</a> Developmental Biology. , 89-101 (2001).</li><li>Gorelick, D. A., Halpern, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Estrogen+Receptor+Transcriptional+Activation+in+Zebrafish.">Visualization of Estrogen Receptor Transcriptional Activation in Zebrafish.</a> Endocrinology. 152, 2690-2703 (2011).</li><li>Tsang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+A+tool+for+chemical+screens.">Zebrafish: A tool for chemical screens.</a> Birth Defects Research Part C: Embryo Today: Reviews. 90, 185-192 (2010).</li><li>Peravali, 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=Automated+feature+detection+and+imaging+for+high-resolution+screening+of+zebrafish+embryos.">Automated feature detection and imaging for high-resolution screening of zebrafish embryos.</a> Biotechniques. 50, 319-324 (2011).</li><li>Walker, S. 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=Automated+reporter+quantification+in+vivo:+high-throughput+screening+method+for+reporter-based+assays+in+zebrafish.">Automated reporter quantification in vivo: high-throughput screening method for reporter-based assays in zebrafish.</a> PLoS One. 7, (2012).</li><li>Vogt, A., 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=Automated+image-based+phenotypic+analysis+in+zebrafish+embryos.">Automated image-based phenotypic analysis in zebrafish embryos.</a> Developmental Dynamics. 238, 656-663 (2009).</li><li>Vogt, A., Codore, H., Day, B. W., Hukriede, N. A., Tsang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+automated+imaging+and+analysis+for+zebrafish+chemical+screens.">Development of automated imaging and analysis for zebrafish chemical screens.</a> J. Vis. Exp. , (2010).</li><li>Gruber, C. J., Gruber, D. M., Gruber, I. M., Wieser, F., Huber, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+estrogen+response+element.">Anatomy of the estrogen response element.</a> Trends in Endocrinology and Metabolism. 15, 73-78 (2004).</li><li>Rovira, 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=Chemical+screen+identifies+FDA-approved+drugs+and+target+pathways+that+induce+precocious+pancreatic+endocrine+differentiation.">Chemical screen identifies FDA-approved drugs and target pathways that induce precocious pancreatic endocrine differentiation.</a> Proceedings of the National Academy of Sciences USA. 108, 19264-19269 (2011).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zebrafish+Book:+A+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">The Zebrafish Book: A guide for the laboratory use of zebrafish (Danio rerio).</a> , (2000).</li></ol>

Denne protokol beskriver en enkel metode til automatisk billede vævsspecifik fluorescens i zebrafisk embryoner. Protokollen blev udviklet ved hjælp af et Zeiss Axio Observer. Z1 Zen Blå 2011 software, men teknikken kan tilpasses ved hjælp af en inverteret mikroskop med en motoriseret trin og mikroskop control software, der kan udføre flisebelægning at skabe sammensatte billeder. Udstyre et inverteret mikroskop med en motoriseret scene kan give en praktisk, billigere alternativ til at købe specialudstyr til high-c...

Transgen zebrafisk er blevet udviklet, som giver mulighed for direkte visualisering af aktivitet i signalveje, såsom fibroblastvækstfaktorer 1 retinsyre 2 og østrogener 3, i levende embryoer. Sådanne værktøjer muliggør screening for kemikalier, der forstyrrer signalveje (analyseret som ændringer i fluorescensintensitet) eller kemikalier, som modulerer signalering en vævsspecifik måde (ændring i fluorescens lokalisering) 4.. Automatiseret billedoptagelse øger throughput af kemiske skærme dramatisk 5,6. Skærme, der automatisk assay fluorescens i levende zebrafisk ofte afhængige af dyre, specialiseret udstyr. Såkaldte højt indhold screening giver fordelen ved høj opløsning, kvantitativ billeddannelse men på bekostning af hjælp af specialiserede pladelæsere udstyret til konfokal mikroskopi 7,8. Målet med denne metode er at automatisk assay vævsspecifik fluorescens i zebrafisk embryoneri en 96-brønds plade ved anvendelse af en standard epifluorescensmikroskop. Tissue-specifik fluorescens kan skelnes ved hjælp af denne teknik, og kan være en fornuftig tilgang til laboratorier, der mangler adgang til specialiserede pladelæsere eller high-content screening udstyr. I denne protokol, vi bruger automatiseret billeddannelse til at opdage vævsspecifik østrogen receptor (ER)-agonister i levende zebrafisk ved 3 dage efter befrugtning (DPF). De transgene linje Tg (5xERE: GFP) c262/c262 indeholder 5 tandem østrogenresponselementer element DNA-sekvenser (EBS) opstrøms for grønt fluorescerende protein (GFP) 3. I fravær af ligand, ERS normalt inaktive. Ligandbinding udløser en konformationsændring, så receptorer til at binde ERE DNA og således regulere transkription 9. 5xERE: GFP fisk kan anvendes til screening af kemiske biblioteker for ER-modulatorer og kan anvendes til at screene miljømæssige vandprøver til østrogene stoffer.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:100px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:451px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Plastic transfer pipettes; wide bore</td>
			<td style="width:100px;">Fisher</td>
			<td style="width:119px;">13-711-23</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Plastic transfer pipettes; fine tip</td>
			<td style="width:100px;">Fisher</td>
			<td style="width:119px;">13-711-26</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-well, round, flat bottom plates</td>
			<td style="width:100px;">Fisher</td>
			<td style="width:119px;">21-377-203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 x 35mm paltes</td>
			<td style="width:100px;">Fisher</td>
			<td style="width:119px;">08-757-100D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 x 60mm plates</td>
			<td style="width:100px;">Fisher</td>
			<td style="width:119px;">08-757-100B</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dumont #5 fine forceps</td>
			<td style="width:100px;">Fine Science Tools</td>
			<td style="width:119px;">11254-20</td>
			<td>tip dimensions 0.05 x 0.01 mm, for manually removing chorions from embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tricane methanesulfonate</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:119px;">A5040-25G</td>
			<td>see Zebrafish Book for recipe (http://zfin.org/zf_info/zfbook/chapt10.html#wptohtml63)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-phenyl 2-thiourea (PTU)</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:119px;">P7629-10G</td>
			<td>Prepare 20 mM stock (100x) and use at 200 &#956;M in E3B&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss Axio Observer Z1</td>
			<td style="width:100px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:451px;">Protocol requires an inverted fluorescence microscope with a motorized stage</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">20x long working distance objective</td>
			<td style="width:100px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>We use an objective with 0.4 NA and 8.4 mm working distance</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axiocam HRm digital camera</td>
			<td style="width:100px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ZenBlue 2011 microscope control software</td>
			<td style="width:100px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:451px;">Protocol requires microscopy automation and control software to enable capturing of tiled images using a motorized stage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene Blue</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>&#160;MB-1; 25 grams</td>
			<td>make 2% solution in RO water for use in E3B (below)</td>
		</tr>
		<tr height="273">
			<td height="273" style="height:273px;">E3B</td>
			<td style="width:100px;"></td>
			<td style="width:119px;"></td>
			<td style="width:451px;">60X E3 SOLUTION: 
			NaCl&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; - 17.2 grams 
			KCl&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; - 0.76 grams 
			CaCl2-2H2O&#160;&#160;&#160;&#160; - 2.9 grams 
			MgSO4-7H2O&#160; - 4.9 grams or MgSO4 &#8211; 2.39 grams 
			Dissolve in 1 liter Milli-Q water; store in sterile 1 liter bottle. 
			 
			1X E3B SOLUTION FOR ZEBRAFISH: 
			60X E3&#160;&#160;&#160;&#160;&#160; 150mls 
			2% methylene blue&#160;&#160;&#160;&#160; 100&#956;l&#160; 
			Bring to 9 liters with Milli-Q water 
			&#160;</td>
		</tr>
	</tbody>
</table>

semi-automated imaging of tissue-specific fluorescence in zebrafish embryos

Zebrafisk embryoner er et kraftfuldt værktøj til storstilet screening af små molekyler. Transgene zebrafisk, der udtrykker fluorescerende reporter proteiner der ofte bruges til at identificere kemikalier, der modulerer genekspression. Kemiske skærme, assay fluorescens i levende zebrafisk ofte afhængige dyrt, specialudstyr til højt indhold screening. Vi beskriver en procedure ved hjælp af en standard epifluorescensmikroskop med en motoriseret scene til automatisk billed zebrafisk embryoner og opdage vævs-specifik fluorescens. Brug transgene zebrafisk der rapporterer østrogen receptor aktivitet via udtryk af GFP, har vi udviklet en semi-automatiseret procedure for at screene for østrogen-receptor-ligander, der aktiverer reporter i et væv-specifik måde. I denne video beskrives procedurer for gruppering zebrafisk embryoer ved 24-48 timer efter befrugtning (HPF) i en 96-brønds plade og tilsætning af små molekyler, der binder østrogenreceptorer. Ved 72-96 HPF, billeder af hver brønd frahele pladen automatisk opsamles og undersøges manuelt for vævs-specifik fluorescens. Denne protokol demonstrerer evnen til at opdage østrogener, der aktiverer receptorer i hjerteklapper, men ikke i leveren.

Figur 1 viser sammensatte billeder fra individuelle brønde i en 96-brønds plade. Hver sammensatte billede består af 59 individuelle billeder med 5% billede overlap. Bemærk, at de levende zebrafisk er orienteret tilfældigt inden for hver brønd, men vi er i stand til at skelne fluorescens i hjertet fra leveren. Lysfelt billeder er nyttige som henvisninger til at vurdere zebrafisk orientering og visualisere morfologiske abnormiteter (figur 1A og 1C). Automatiseret billedbehandling ve...

Watch this Scientific Journal Video about Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos at JoVE.com

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

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Beskrevet her er en protokol til halvautomatisk billeddannelse af væv-specifik fluorescens i zebrafisk embryoner.

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