Name: induction of hypoxia in living frog and zebrafish embryos
Uploaded: 2017-06-26T14:00:00
Description: Watch this Scientific Journal Video about Induction of Hypoxia in Living Frog and Zebrafish Embryos at JoVE.com

Dette arbejde blev støttet af Support from Wellcome Trust SIA Award 100329 / Z / 12 / Z til WAH og DFG-stipendiet KH 376 / 1-1 tildelt HK

Denne protokol følger dyresundhedsretningslinjerne fra University of Cambridge. 1. Dyrevedligeholdelse <ol><li> Frøembryoer BEMÆRK: Embryoer kan opdrættes og opretholdes i henhold til dyre- og laboratoriefaciliteten. Her er et eksempel på dyrvedligeholdelsen beskrevet. <ol><li> Forbered 0,1x modificeret Barths Solution (MBS) opløsning: 0,88 mM NaCl, 10 μM KCl, 24 μM NaHC03, 100 μM HEPES, 8,2 μM MgS04, 3,3 μM Ca (NO3) 2 og 4,1 μM CaCl2, pH 7,6 . </li><li> Skaff <em...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+genes+encoding+glycolytic+enzymes+by+hypoxia-inducible+factor+1.">Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1.</a> J Biol Chem. 269 (38), 23757-23763 (1994).</li><li>Yuan, Y., Hilliard, G., Ferguson, T., Millhorn, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cobalt+inhibits+the+interaction+between+hypoxia-inducible+factor-alpha+and+von+Hippel-Lindau+protein+by+direct+binding+to+hypoxia-inducible+factor-alpha.">Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-alpha.</a> J Biol Chem. 278 (18), 15911-15916 (2003).</li><li>Elks, P., Renshaw, S. A., Meijer, A. H., Walmsley, S. R., van Eeden, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+HIFs,+buts+and+maybes+of+hypoxia+signalling+in+disease:+lessons+from+zebrafish+models.">Exploring the HIFs, buts and maybes of hypoxia signalling in disease: lessons from zebrafish models.</a> Disease Models &amp; Mechanisms. 8, 1349-1360 (2015).</li><li>Guo, 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=Hypoxia-mimetic+agents+desferrioxamine+and+cobalt+chloride+induce+leukemic+cell+apoptosis+through+different+hypoxia-inducible+factor-1alpha+independent+mechanisms.">Hypoxia-mimetic agents desferrioxamine and cobalt chloride induce leukemic cell apoptosis through different hypoxia-inducible factor-1alpha independent mechanisms.</a> Apoptosis. 11 (1), 67-77 (2006).</li><li>Woods, I. G., Imam, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analysis+of+severe+hypoxic+stress+during+development+in+zebrafish.">Transcriptome analysis of severe hypoxic stress during development in zebrafish.</a> Genom Data. 6, 83-88 (2015).</li><li>Rouhi, P., 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=Hypoxia-induced+metastasis+model+in+embryonic+zebrafish.">Hypoxia-induced metastasis model in embryonic zebrafish.</a> Nat Protoc. 5 (12), 1911-1918 (2010).</li><li>Stevenson, T. J., 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=Hypoxia+disruption+of+vertebrate+CAN+pathfinding+through+EphrinB2+is+rescued+by+magnesium.">Hypoxia disruption of vertebrate CAN pathfinding through EphrinB2 is rescued by magnesium.</a> PLoS Genet. 8 (4), e1002638 (2012).</li><li>Khaliullina, H., Love, N. K., 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=Nutrient-Deprived+Retinal+Progenitors+Proliferate+in+Response+to+Hypoxia:+Interaction+of+the+HIF-1+and+mTOR+Pathway.">Nutrient-Deprived Retinal Progenitors Proliferate in Response to Hypoxia: Interaction of the HIF-1 and mTOR Pathway.</a> J Dev Biol. 4 (2), (2016).</li><li>Nieuwkoop, P. D., Faber, J., Nieuwkoop, D. P., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Table of Xenopus laevis (Daudin). , (1994).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203 (3), 253-310 (1995).</li><li>Kohn, D. F., Wixson, S. K., White, W. J., Benson, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anesthesia and Analgesia in Laboratory Animals. , (1997).</li><li>McDonough, M. 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Her har vi præsenteret en nem, men robust ny metode til at fremkalde hypoxi, der er indstillet til brug med frø- og zebrafiskembryoner, men kan også være egnet til andre vandlevende organismer. Den største fordel ved denne metode ligger i dens enkelhed og omkostningseffektivitet. Ikke desto mindre er de opnåede resultater med denne metode meget robuste. Vi har vist, at hypoxi effektivt kan induceres i kammeret både i hele embryoner såvel som i specifikt væv - her er retina. For at bestemme effektiviteten af ​...

Hypoxi forskning har mange anvendelser. Disse omfatter undersøgelse af patogenesen og udvikling af behandlinger for medicinske tilstande præget af hypoxi 1 og akut højhøjde sygdom 2 . Hypoksisk stress forårsager større metaboliske forandringer i alle organismer, der kræver ilt. Hypoksisk stress påvirker også fostervækst og udvikling og patogenesen af ​​adskillige humane sygdomme, herunder intrauterin vækstrestriktion 3 . Hypoksisk stress kan ikke kun føre til nedsat fødselsvægt, føtal og neonatal dødelighed, men kan også resultere i mange komplikationer i voksenlivet, såsom kardiovaskulær sygdom, type 2 diabetes, fedme og hypertension 4 . Hypoksisk stress observeres også ofte under udvikling af fast tumor, når tumorvævet vokser blodforsyningen. Det er derfor afgørende at kunne studere virkningerne af hypoxi in vivo og direkte under embr<html> Yonisk udvikling. Blandt de mest kendte metoder, som har været anvendt til at studere virkninger af hypoxi under udvikling, er brugen af ​​koboltchlorid i vækstmediet eller inkubation af organismen i et hypoxisk kammer. Cobaltchlorid inducerer kunstigt en hypoxisk reaktion under normal oxygenkoncentration på grund af sin rolle i stabiliseringen af ​​hypoxi-inducerbar faktor-1 alpha (HIF-1a) ved at forhindre dens proteosomal nedbrydning 5 , 6 , 7 . Imidlertid er anvendelsen af ​​koboltchlorid såvel som andre lignende kemiske hypoximimetika en uegnet, skadelig virkning på celler og væv, f.eks . Apoptose 9 , som en bekvem metode 8 . Derfor er hypoxiske kamre en bedre metode til induktion af &quot;naturlig hypoxi&quot; i levende organismer gennem normal udvikling. Ntent &quot;&gt; Vi har fokuseret på at udvikle et system til induktion af hypoxi hos akvatiske embryoner. Både frøer og zebrafisk er nu blevet informative hvirveldyrmodellorganismer til studier af mange biologiske processer samt modeller for forskellige menneskelige sygdomme. Frog- og zebrafiskembryoner Udvikle eksternt, eliminere komplikationen af ​​maternisk kompensation. Et hurtigt udviklingsforløb gør det også muligt at manipulere miljøfaktorer og observere de fænotypiske ændringer i organdannelsen i realtid. Desuden er mange komponenter af de store signaltransduktionsveje stærkt bevaret i Disse modelorganismer og er blevet karakteriseret i detaljer af en stor mængde litteratur. Hovedfordelen ved at bruge frøer og zebrafiskembryoner til at undersøge virkningerne af hypoxi på udviklingen af ​​hvirveldyr er, at alle processer kan overvåges direkte, da oxygen hurtigt trænger ind i embryonerne. Således i frøer og zebrafisk, som i modsætning til andre modelorganismer som f.eksMusembryoer, kan indflydelsen af ​​en specifik iltkoncentration undersøges i det væv af interesse uden at tage hensyn til tilstedeværelsen eller manglen på funktionel vaskulatur. De fleste kommercielt tilgængelige opsætninger til hypoxisk inkubation har den ulempe, at de er sammenligneligt store og har tilsvarende høje løbende omkostninger. Bortset fra deres høje indledende omkostninger og gasforbrug kræver ækvilibrering og vedligeholdelse af almindelige hypoxikamre en konstant hypoxisk atmosfære imod gasgradienten, som naturligt forekommer i disse kamre på grund af deres større størrelse og / eller organismernes åndedræt. Dette kræver ansættelse af gasfans og et kølesystem, hvilket øger mængden af ​​yderligere nødvendigt udstyr, forhindrer forskers fingerfærdighed og generelt reducerer enkelheden af ​​forsøgsproceduren. I modsætning hertil er opsætningen vi præsenterer her sammenligneligt robust, men meget omkostningseffektiv, lille, let at etablere og tillader fAst gas-ækvilibrering, stabil hypoxisk atmosfære og simpel udveksling af materialer og opløsninger inde i kammeret. Vores system kan anvendes til brug med enhver vandmiljøorganisme af interesse. Vi har konstrueret et hypoxisk kammer, der er bekvemt lille, og kan derfor placeres inde i en fælles laboratorieinkubator, som let giver eksperimentelle procedurer til enhver bestemt temperatur. Fordelene ved vores system mod de kommercielt tilgængelige hypoxi-inkubatorer ligger bekvemt i kontrol med temperaturen såvel som iltkoncentrationen i mediet, idet den er lille størrelse og omkostningseffektivitet. Således kan vores opsætning etableres ved hjælp af generelle laboratorieforsyninger til rådighed for de fleste forskningslaboratorier og kræver ikke nogen dyre materialer. Derudover genererer vores opsætning ikke varme, i modsætning til de kommercielt tilgængelige hypoxi-inkubatorer, og tillader brug ved temperaturer, der er lavere end stuetemperatur, der anbringes i en inkubator. LaSt er særligt kritisk for arbejdet med koldblodede organismer som frøer og fisk, hvor udviklings- og metaboliske satser er stærkt temperaturafhængige. At være meget omkostningseffektiv og let opbygget er vores gasinkubationskammer alligevel meget alsidigt ved etablering af forskellige hypoxiske eller hyperoxiske forhold samt mulighed for hurtig og nem administration af forskellige medier og løsninger til et stort antal eksperimentelle forhold. Derudover tillader vores system at anvende en brønd med 24 brønde i stedet for almindeligt anvendte tallerkener eller laboratorietanke 10 , 11 , 12 og observation og eksperimentel behandling af flere mutante forhold på én gang. For at kontrollere for korrekt induktion af hypoxi har vi overvåget niveauerne af HIF-1a-proteinet ved Western blot-detektion. Derudover er antallet af proliferative celler før og efter inkubationN i det hypoksiske kammer kan bruges til at bestemme, om hypoxi er blevet induceret i vævet. Denne metode er baseret på vores tidligere offentliggjorte resultater 13 , der viser, at spredning i embryonal retinal stamcelle niche falder ved induktion af hypoxi. Således har vi overvåget niveauet af retinal stamcelleproliferation ved at tilsætte 5-ethynyl-2&#39;-deoxyuridin (EdU) til embryomediet og måle dets inkorporering i DNA&#39;et af nyudbredende celler.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Sodium chloride</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S7653</td>
			<td style="width:167px;">NaCl / 0.1X MBS, Embryo medium, 10X TBST</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Potassium chloride</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P9333</td>
			<td style="width:167px;">KCl / 0.1X MBS, Embryo mediu,</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Sodium bicarbonate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S5761</td>
			<td style="width:167px;">NaHCO3 / 0.1X MBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">HEPES</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td style="width:167px;">0.1X MBS</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Magnesium sulfate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">M7506</td>
			<td style="width:167px;">MgSO4 / 0.1X MBS, Embryo medium</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Calcium nitrate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">202967</td>
			<td style="width:167px;">Ca (NO3)2 / 0.1X MBS</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Calcium chloride</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C1016</td>
			<td style="width:167px;">CaCl2 / 0.1X MBS, Embryo medium</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Methylene blue</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">M9140</td>
			<td style="width:167px;">Embryo medium</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Pregnant mare serum gonadotropin</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">CG10</td>
			<td style="width:167px;">frog fertilization</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Zebrafish breeding tank</td>
			<td style="width:71px;">Carolina</td>
			<td style="width:119px;">161937</td>
			<td style="width:167px;">gas chamber construction</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">24-well plate</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">142475</td>
			<td style="width:167px;">Nunclon Delta Surface, for gas chamber construction</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Epoxy resin</td>
			<td style="width:71px;">RS Components UK</td>
			<td style="width:119px;">Kit 199-1468</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Gas distributor valve</td>
			<td style="width:71px;">WPI Luer Valves</td>
			<td style="width:119px;">Kit 14011</td>
			<td style="width:167px;">aquatic tank attachment (Schema 1, H)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">High precision gas valve</td>
			<td style="width:71px;">BOC&#160;</td>
			<td style="width:119px;">200 bar HiQ C106X/2B</td>
			<td style="width:167px;">gas tank attachment (Schema 1, I)</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">5% oxygen and 95% N2 gas tank</td>
			<td style="width:71px;">BOC</td>
			<td style="width:119px;">226686-L</td>
			<td style="width:167px;">hypoxic gas mixture</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">ceramic disc diffuser</td>
			<td style="width:71px;">CO2 Art&#160;</td>
			<td style="width:119px;">Glass CO2 Nano Aquarium Diffuser, DG005DG005</td>
			<td style="width:167px;">Schema 1, J</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">silicone grease</td>
			<td style="width:71px;">Scientific Laboratory Supplies</td>
			<td style="width:119px;">VAC1100</td>
			<td style="width:167px;">Schema 1, K</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">oxymeter</td>
			<td style="width:71px;">Oxford Optronix&#160;</td>
			<td style="width:119px;">Oxylite, CP/022/001</td>
			<td style="width:167px;">hypoxic chamber setup</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">fibre-optic dissolved oxygen sensor</td>
			<td style="width:71px;">Oxford Optronix</td>
			<td style="width:119px;">HL_BF/OT/E</td>
			<td style="width:167px;">hypoxic chamber setup</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">plastic pasteur pipette</td>
			<td style="width:71px;">Sterilin</td>
			<td>STS3855604D</td>
			<td style="width:167px;">for embryo transfer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">MS222&#160;</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">E10521-50G</td>
			<td style="width:167px;">embryo anesthetic</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">RIPA buffer&#160;</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">R0278-50ML</td>
			<td style="width:167px;">tissue homogenization</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Protease inhibitor</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P8340</td>
			<td style="width:167px;">tissue homogenization</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Tris</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">77-86-1</td>
			<td style="width:167px;">4X Laemmli loading buffer, 10X TBST</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Glycerol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">G5516</td>
			<td style="width:167px;">4X Laemmli loading buffer</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Sodium Dodecyl Sulfate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">L3771</td>
			<td style="width:167px;">SDS, 4X Laemmli loading buffer, 5X Running buffer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">beta-Mercaptoethanol&#160;</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">M6250</td>
			<td style="width:167px;">4X Laemmli loading buffer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Bromophenol Blue</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">B0126</td>
			<td style="width:167px;">4X Laemmli loading buffer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Trizma base&#160;</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">77-86-1</td>
			<td style="width:167px;">5X Running buffer, Transfer buffer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Glycine</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">G8898</td>
			<td style="width:167px;">5X Running buffer, Transfer buffer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Methanol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">34860</td>
			<td style="width:167px;">Transfer buffer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Tween 20</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P2287-500ML</td>
			<td style="width:167px;">10X TBST</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">skim milk powder</td>
			<td style="width:71px;">Sigma</td>
			<td>70166</td>
			<td style="width:167px;">Blocking Solution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Eppendorf microcentrifuge tube</td>
			<td style="width:71px;">Sigma</td>
			<td>T9661</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">tissue homogenizer</td>
			<td style="width:71px;">Pellet Pestle Motor Kontes</td>
			<td style="width:119px;">Z359971</td>
			<td style="width:167px;">tissue homogenization</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">pellet pestles</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">Z359947-100EA</td>
			<td style="width:167px;">tissue homogenization</td>
		</tr>
		<tr height="20">
			<td height="40" style="height:40px;width:216px;">precast 12% gel</td>
			<td rowspan="2" style="width:71px;">Biorad</td>
			<td rowspan="2" style="width:119px;">Mini-ProteinTGX, 456-1043</td>
			<td rowspan="2" style="width:167px;">Western Blot</td>
		</tr>
		<tr height="20">
			<td height="40" style="height:40px;width:216px;">protein ladder</td>
			<td rowspan="2" style="width:71px;">Amersham</td>
			<td rowspan="2" style="width:119px;">Full-Range Rainbow ladder, RPN800E</td>
			<td rowspan="2" style="width:167px;">Western Blot</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">nitrocellulose membrane (0.45 &#181;m)</td>
			<td style="width:71px;">Biorad</td>
			<td style="width:119px;">162-0115</td>
			<td style="width:167px;">Western Blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">anti-HIF-1&#945; antibody</td>
			<td style="width:71px;">Abcam</td>
			<td style="width:119px;">ab2185</td>
			<td style="width:167px;">Western Blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">anti-&#945;-tubulin antibody</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T6074</td>
			<td style="width:167px;">Western Blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">goat anti-rabbit antibody</td>
			<td style="width:71px;">Abcam</td>
			<td style="width:119px;">ab6789</td>
			<td style="width:167px;">Western Blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">goat anti-mouse antibody</td>
			<td style="width:71px;">Abcam</td>
			<td style="width:119px;">ab97080</td>
			<td style="width:167px;">Western Blot</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Pierce ECL 2 reagent&#160;</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">80196</td>
			<td style="width:167px;">Western Blot</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:216px;">ECL films Hyperfilm</td>
			<td style="width:71px;">GE Healthcare Amersham</td>
			<td style="width:119px;">28906837</td>
			<td style="width:167px;">Western Blot</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">5-Ethynyl-2&#8242;-deoxyuridine &#160;</td>
			<td style="width:71px;">santa cruz</td>
			<td style="width:119px;">CAS&#160;61135-33-9</td>
			<td style="width:167px;">EdU, EdU incorporation</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Phosphate-buffered Saline</td>
			<td style="width:71px;">Oxoid</td>
			<td style="width:119px;">BR0014G</td>
			<td style="width:167px;">1X PBS</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Formaldehyde</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">28908</td>
			<td style="width:167px;">Fixation solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sucrose</td>
			<td style="width:71px;">Fluka</td>
			<td style="width:119px;">S/8600/60</td>
			<td style="width:167px;">Solution solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Triton X-100</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T9284-500ML</td>
			<td style="width:167px;">PBST</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Heat-inactivated Goat Serum</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">G6767-100ml</td>
			<td style="width:167px;">HIGS, Blocking solution (EdU incorporation)</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">4&#39;,6-diamidino-2-phenylindole&#160;</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td style="width:119px;">D1306</td>
			<td style="width:167px;">DAPI, EdU incorporation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Dimethyl sulfoxide</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">C10338</td>
			<td style="width:167px;">DMSO, EdU incorporation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">glass vial</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">98178853</td>
			<td style="width:167px;">EdU incorporation analysis</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Tissue-Plus optimal cutting temperature compound&#160;</td>
			<td style="width:71px;">Scigen</td>
			<td style="width:119px;">4563</td>
			<td style="width:167px;">embedding medium, EdU incorporation analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">cryostat Jung Fridgocut 2800E</td>
			<td style="width:71px;">Leica&#160;</td>
			<td style="width:119px;">CM3035S</td>
			<td style="width:167px;">EdU incorporation analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">microscope slides Super-Frost plus Menzel glass</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">J1800AMNZ</td>
			<td style="width:167px;">EdU incorporation analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">EdU Click-iT chemistry kit</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">C10338</td>
			<td style="width:167px;">EdU incorporation analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">FluorSave</td>
			<td style="width:71px;">Calbiochem</td>
			<td style="width:119px;">D00170200</td>
			<td style="width:167px;">mounting medium, EdU incorporation analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">coverslips</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">ECN631-1575</td>
			<td style="width:167px;">EdU incorporation analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">fluorescent microscope</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">Eclipse 80i</td>
			<td style="width:167px;">EdU incorporation analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">confocal scanning microscope</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">Fluoview FV1000</td>
			<td style="width:167px;">EdU incorporation analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Volocity software</td>
			<td style="width:71px;">PerkinElmer</td>
			<td style="width:119px;">Volocity 6.3</td>
			<td style="width:167px;">EdU incorporation analysis</td>
		</tr>
	</tbody>
</table>

induction of hypoxia in living frog and zebrafish embryos

Her introducerer vi et nyt system til hypoxi induktion, som vi udviklede til at studere hypoxiets virkninger i vandlevende organismer som frog og zebrafish embryoner. Vores system består af et kammer med en simpel opsætning, som alligevel er robust til at fremkalde og opretholde en specifik iltkoncentration og -temperatur i en hvilken som helst forsøgsopløsning. Det præsenterede system er meget omkostningseffektivt men meget funktionelt, det tillader induktion og vedligeholdelse af hypoxi til direkte forsøg in vivo og i forskellige tidsperioder op til 48 timer. For at overvåge og undersøge virkningerne af hypoxi har vi brugt to metoder - måling af niveauer af hypoxi-inducerbar faktor 1alpha (HIF-1α) i hele embryoner eller specifikke væv og bestemmelse af retinalt stamcelleproliferation med 5-ethynyl-2&#39;- Deoxyuridin (EdU) inkorporering i DNA&#39;et. HIF-1a niveauer kan tjene som en generel hypoxi markør i hele embryoet eller vævetEfter eget valg, her embryonalt nethinden. EdU inkorporering i de proliferative celler af embryonalt retina er en specifik udgang af hypoxi induktion. Således har vi vist, at hypoxiske embryonale retinale forfædre reducerer proliferation inden for 1 time inkubation under 5% oxygen af ​​både frø og zebrafiskembryoner. Når vi har mestret, kan vores opsætning anvendes til brug med små vandmiljøorganismer, til direkte in vivo forsøg, en given tidsperiode og under normal, hypoxisk eller hyperoxisk iltkoncentration eller under en anden given gasblanding.

Anvendelse af det hypoxiske kammersystem, som vi præsenterer her, gør det muligt at undersøge virkningerne af hypoxi individuelt og in vivo hos hele levende dyr. Hypoxi kan induceres ved at placere hele frø eller zebrafiskembryoner i det hypoksiske kammer ( Figur 1 ) og udføres på forskellige kombinationer af tilstande. Et billede af vores komplette gaskammeropsætning er vist i figur 2 . Vi har overvåget iltkonc...

Watch this Scientific Journal Video about Induction of Hypoxia in Living Frog and Zebrafish Embryos at JoVE.com

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Vi introducerer et nyt hypoxisk kammersystem til brug sammen med vandorganismer som frog og zebrafish embryoner. Vores system er enkelt, robust, omkostningseffektivt og muliggør induktion og vedligeholdelse af hypoxi in vivo og i op til 48 timer. Vi præsenterer 2 reproducerbare metoder til overvågning af effektiviteten af ​​hypoxi.

Induktion af hypoxi i levende frø og zebrafiskembryoner

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

The overall goal of this invention is to study the effects of hypoxia or hyperoxia on the embryonic tissues of any aquatic organism of interest. This method can help answer key questions in the field of developmental and cell biology, such as embryonic growth restriction, fetal hypoxia, high-altitude adaptation, obesity and solid tumor development. The main advantages of this technique are simplicity, cost-effectiveness and robustness.It allows induction and sustainment of hypoxi in vivo for long time periods. After collecting and raising xenopus and zebrafish embryos, prepare a mesh-bottom 24-well plate according to the text protocol. Attach plastic rods to the mesh-bottom 24-well plate and place it into the aquatic tank of choice, then using silicone tubing, attach a gas tank with the desired oxygen nitrogen mixture, or another gas mixture of choice, to a distributor valve using an appropriate gas regulator.Next connect the silicone tubing and the distributor valve to a ceramic disk diffuser inside the aquatic tank. Depending on the type of embryos used in the experiment, fill the hypoxic chamber tank with the appropriate embryo medium, then close the aquatic tank and use silicone grease to coat the lid and carefully seal it. Start diffusing the gas mixture through the ceramic disk diffuser.Allow the system to equilibrate for 10 to 15 minutes. Adjust the oxygen flow rate in the chamber medium to 0.0017 to 0.0019 cubic meters per second. Under this gas flow rate, no disturbance of the medium should be seen in the wells of the plate.The oxygen flow rate depends on the internal pressure of the gas tank and the outlet pressure of the gas regulator. Both these parameters should be adjusted according to the monitor of the gas regulator. After equilibration, use an oximeter or oxygen probe to measure the oxygen concentration in the water.If a particular non-room temperature is required, place the hypoxic chamber into a laboratory incubator of the required temperature. Carefully select the embryos for the experiment using whole and live embryos for hypoxia incubation. Place the embryo dishes into the incubator containing the gas incubation chamber, and let them equilibrate to the temperature of the incubator.Then use a plastic pipette to carefully transfer the embryos into the wells of the meshed 24-well plate of the gas incubation chamber. Carefully label the wells with different genotypes. Ensure minimal reoxygenation of the chamber while placing the embryos into the wells.Transfer the sample swiftly, and quickly reseal the chamber. Maintain the gas incubation chamber under a constant infusion with the gas mixture of choice for five hours, or for a longer time that suits the experiment. A mixture of 5%oxygen and 95%nitrogen is used here.Carefully transfer the embryos from the gas incubation chamber back to the normoxic medium corresponding to the embryo type. After preparing buffers and media, and anesthetizing the embryos according to the text protocol, use a tissue homogenizer or a sonicator to homogenize the embryo tissue. Then centrifuge the homogenates to remove debris.Collect the supernatant and denature the sample at 85 degrees Celsius for five minutes, then add Laemmli gel loading buffer to one X volume by volume. Use the supernatant for western blot analysis, or freeze the samples at negative 20 degrees Celsius. To incorporate EdU into the embryos, fill the gas incubation chamber with 400 to 500 milliliters of five millimolar EuD solution supplemented with 1%volume by volume DMSO.Connect the gas tubing to the gas cylinder containing a gas mixture of 5%oxygen and 95%nitrogen. Incubate the solution for 15 minutes. Pre-equilibrate the solution with the same gas mixture used in the experiment for 15 minutes prior to the experiment.Transfer the embryos to the hypoxic chamber, distributing them evenly between the wells, and incubate the embryos for two hours. Finally, analyze the embryos for EdU incorporation according to the text protocol. As shown in this table, desired oxygen concentrations in the incubation medium were induced at different time points throughout the experiments, as monitored by a fiber optic oxygen sensor.In this experiment HIF-1 alpha which is stabilized under low oxygen concentrations, was measured in whole frog embryo lysates kept under normal oxygen concentrations, or subjected to 5%hypoxia and in lysates of their isolated retinas. In both samples HIF-1 alpha was stabilized under hypoxia. Here frog and zebrafish embryos were incubated in a hypoxic chamber maintained at 5%oxygen, and retinal proliferation was assessed by monitoring EdU incorporation.As expected normoxic control retinas of frog and zebrafish embryos showed intensive staining in the CMZ, where proliferating progenitors reside. Following the oxygen incubation in the hypoxic chamber a strong decrease in the CMZ progenitor proliferation was observed in both frog and zebrafish embryos. By carrying out a time course it could be shown that the decrease in retinal progenitor proliferation was acute, and greatest within two hours, and persisted for many hours while embryos developed normally according to their developmental stage.Once mastered, this technique can be done in 30 minutes if it is performed properly. While attempting this procedure it's important to remember to ensure correct gas flow rate, and to avoid unnecessary and excessively long reopening of the chamber. Following this procedure other methods like QPCR can be performed in order to answer additional questions like activation, or down-regulation of certain target genes of HIF-1 alpha and hypoxia, or hyperoxia respectively.After its development this technique paved the way for the researchers in the field of developmental biology to explore retinal cell proliferation and hypoxia and restricted nutrition in frog and zebrafish embryos. After watching this video you should have a good understanding of how to induce hypoxia or hyperoxia in any aquatic organism, and how to study its effects on the tissue and phenomenon of interest, such as retinal cell proliferation. Don't forget that working with oxygen gas mixtures can be extremely dangerous, and precautions such as ensuring correct use and attachment of gas regulators should always be taken while performing this procedure.

Research

JoVE Journal

Developmental Biology

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Måling Protein Stabilitet i Living zebrafisk embryoner anvendelse af fluorescens Decay Efter fotoomdannelse (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Mechanical Vessel Injury in Zebrafish Embryos

Mekanisk karskade i zebrafisk embryoner

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

Imaging Subcellulære Strukturer i Living Zebrafisk Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

Isolering og karakterisering af enkeltceller fra zebrafisk embryoer

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

Affinity Labeling Påvisning af endogene receptorer fra zebrafisk embryoer

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

Biosensing Motor Neuron Membran Potential i Live Zebrafish Embryoer

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Lys Sheet-baserede Fluorescence Microscopy af Leve- eller fikseres og farves<em&gt; Tribolium castaneum</em&gt; Embryoner

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Single-cell Photoconversion in Living Intact Zebrafish

Encellede Photoconversion i levende intakt zebrafisk

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Visualisering af cellulære elektriske aktivitet i zebrafisk tidlige embryoner og tumorer

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Hele Mount Immunhistokemi i Zebrafish Embryoner og Larver

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...