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

This work was supported by the Support from Wellcome Trust SIA Award 100329/Z/12/Z to W.A.H. and the DFG fellowship KH 376/1-1 awarded to H.K.

This protocol follows the animal care guidelines of the University of Cambridge.
1. Animal Maintenance
<ol>
	<li>Frog embryos 
	NOTE: Embryos can be raised and maintained according to the animal and laboratory facility. Here, an example of the animal maintenance is described.
	<ol>
		<li>Prepare 0.1x Modified Barth&#39;s Solution (MBS) solution: 0.88 mM NaCl, 10 &#181;M KCl, 24 &#181;M NaHCO3, 100 &#181;M HEPES, 8.2 &#181;M MgSO4, 3.3 &#181;M Ca(NO3)2</...

<ol>
	<li>Grocott, M., Montgomery, H., Vercueil, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-altitude+physiology+and+pathophysiology:+implications+and+relevance+for+intensive+care+medicine.">High-altitude physiology and pathophysiology: implications and relevance for intensive care medicine.</a> Crit Care. 11 (1), 203 (2007).</li><li>Grant, S., 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=Sea+level+and+acute+responses+to+hypoxia:+do+they+predict+physiological+responses+and+acute+mountain+sickness+at+altitude?.">Sea level and acute responses to hypoxia: do they predict physiological responses and acute mountain sickness at altitude?.</a> Brit J Sport Med. 36 (2), 141-146 (2002).</li><li>Kajimura, S., Aida, K., Duan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-like+growth+factor-binding+protein-1+(IGFBP-1)+mediates+hypoxia-induced+embryonic+growth+and+developmental+retardation.">Insulin-like growth factor-binding protein-1 (IGFBP-1) mediates hypoxia-induced embryonic growth and developmental retardation.</a> PNAS. 102 (4), 1240-1245 (2005).</li><li>Ong, K. K., Dunger, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perinatal+growth+failure:+the+road+to+obesity,+insulin+resistance+and+cardiovascular+disease+in+adults.">Perinatal growth failure: the road to obesity, insulin resistance and cardiovascular disease in adults.</a> Best Pact Res Clin Endocrinol Metab. 16, 191-207 (2002).</li><li>Maxwell, P., Salnikow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1:+an+oxygen+and+metal+responsive+transcription+factor.">HIF-1: an oxygen and metal responsive transcription factor.</a> Cancer Bio Ther. 3 (1), 29-35 (2004).</li><li>Semenza, G. L., Roth, P. H., Fang, H. M., Wang, G. 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. 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=Dissection,+Culture,+and+Analysis+of+Xenopus+laevis+Embryonic+Retinal+Tissue.">Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue.</a> JoVE. (70), (2012).</li></ol>

Here we have presented an easy but robust new method to induce hypoxia that is adjusted for use with frog and zebrafish embryos but can also be suited for other aquatic organisms. The major advantage of this method lies in its simplicity and cost efficiency. Nevertheless, the results achieved with this method are very robust. We have shown that hypoxia can be efficiently induced in the chamber both in whole embryos as well as in specific tissue &#8211; here, retinas. To determine the effectiveness of hypoxia induction, w...

Hypoxia research has numerous applications. These include investigating the pathogenesis and developing treatments for medical conditions characterized by hypoxia1 and acute high altitude illness2. Hypoxic stress causes major metabolic changes in all organisms requiring oxygen. Hypoxic stress also influences fetal growth and development and the pathogenesis of several human diseases, including intrauterine growth restriction3. Hypoxic stress can not only lead to reduced birth weight, fetal and neonatal mortality, but can also result in many complications in adult life, such as cardiovascular disease, type-2 diabetes, obesity, and hypertension4. Hypoxic stress is also often observed during solid tumor development, when the tumor tissue outgrows its blood supply. It is therefore crucial to be able to study the effects of hypoxia in vivo and directly during embryonic development.
Among the most well-known methods that have been employed to study effects of hypoxia during development is the use of cobalt chloride in the growth medium or incubation of the organism in a hypoxic chamber. Cobalt chloride artificially induces a hypoxic response under normal oxygen concentration, due to its role in the stabilization of hypoxia-inducible factor-1 alpha (HIF-1&#945;) by preventing its proteosomal degradation5,6,7. However, being a convenient method8, the use of cobalt chloride as well as other similar chemical hypoxia mimetics can have unspecific deleterious effect on cells and tissues, e.g., apoptosis9. Therefore, hypoxic chambers are a better method for induction of &#34;natural hypoxia&#34; in living organisms through the course of normal development.
We have focused on developing a system for induction of hypoxia in aquatic animal embryos. Both frogs and zebrafish have now become informative vertebrate model organisms for studies of numerous biological processes, as well as models for various human diseases. Frog and zebrafish embryos develop externally, eliminating the complication of maternal compensation. Further, a fast course of development makes it possible to manipulate environmental factors and observe the phenotypic changes in organ formation in real time. In addition, many components of major signal transduction pathways are highly conserved in these model organisms and have been characterized in detail by a large body of literature. The main advantage in using frogs and zebrafish embryos to study the effects of hypoxia on vertebrate development is that all processes can be monitored directly, since oxygen quickly penetrates the embryos. Thus, in frogs and zebrafish, as in contrast to other model organisms such as mouse embryos, the influence of a specific oxygen concentration can be studied in the tissue of interest, without taking into consideration the presence or lack of functional vasculature.
Most commercially available setups for hypoxic incubation have the disadvantage of being comparably large and having correspondingly high running costs. Apart from their high initial cost and gas consumption, equilibration and maintenance of common hypoxia chambers requires sustainment of constant hypoxic atmosphere against the gas gradient that naturally occurs in these chambers because of their larger size and/or organism respiration. This requires employment of gas fans and a cooling system, which increases the amount of additional necessary equipment, impedes the dexterity of the researcher and overall decreases simplicity of experimental procedure. In contrast, the setup we present here is comparably robust but very cost-effective, small, easy to establish and allows fast gas equilibration, stable hypoxic atmosphere and simple exchange of materials and solutions within the chamber. Our system can be employed for use with any aquatic model organism of interest.
We have constructed a hypoxic chamber that is conveniently small and therefore can be placed inside a common laboratory incubator, which easily allows experimental procedures at any specific temperature. Providing convenient control of temperature as well as oxygen concentration in the medium, the advantage of our system against the commercially available hypoxia incubators lies in its small size and cost efficiency. Thus, our setup can be established using general laboratory supplies available for the majority of research labs and does not require any expensive materials. In addition, our setup does not generate heat, unlike the commercially available hypoxia incubators, and allows use at temperatures lower than room temperature being placed in an incubator. The last is especially critical for the work with cold-blooded organisms such as frogs and fish where developmental and metabolic rates are strongly temperature dependent.
Being very cost-effective and easily built, our gas incubation chamber is nevertheless very versatile in establishing various hypoxic or hyperoxic conditions, as well as enabling quick and easy administration of different media and solutions for a vast number of experimental conditions. In addition, employing a 24-well plate instead of commonly used dishes or laboratory tanks10,11,12, our system allows observation and experimental treatment of several mutant conditions at once.
To control for correct induction of hypoxia, we have monitored the levels of the HIF-1&#945; protein by Western blot detection. In addition, the number of proliferating cells before and after incubation in the hypoxic chamber can be used to determine if hypoxia has been induced in the tissue. This method is based on our previously published results13, showing that proliferation in embryonic retinal stem cell niche decreases upon induction of hypoxia. Thus, we have monitored the level of retinal stem cell proliferation by adding 5-ethynyl-2&#8242;-deoxyuridine (EdU) to the embryo medium and measuring its incorporation into the DNA of newly proliferating cells.

<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

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 zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

Employing the hypoxic chamber system that we present here allows the study of the effects of hypoxia individually and in vivo in whole living animals. Hypoxia can be induced by placing entire frog or zebrafish embryos in the hypoxic chamber (Figure 1), and be undertaken on different combinations of conditions. An image of our completed gas chamber setup is shown in Figure 2. We have monitored oxygen concentration in the ...

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

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

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 ...

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

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

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

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

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

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

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

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

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

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

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