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, and 4.1 &#181;M CaCl2, pH&#160;7.6.</li>
		<li>Obtain Xenopus laevis embryos by in vitro fertilization.
		<ol>
			<li>Induce ovulation in adult female African clawed frogs (Xenopus laevis) by injection with pregnant mare serum gonadotropin one week (60 IU) and 12 h (500 IU) prior to egg laying.</li>
			<li>Collect oocytes and fertilize them in vitro with homogenized male testis.</li>
			<li>Raise embryos in 0.1x MBS at 16 - 18 &#176;C. Stage the embryos according to the Normal table of Xenopus laevis14. Take care in selecting whole and alive embryos for the experiment.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Zebrafish 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 embryo medium: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 0.00001% methylene blue.</li>
		<li>Maintain and breed WT&#160;AB strain Danio rerio at 26.5 &#176;C.</li>
		<li>Collect the embryos on the morning of fertilization, raise until 4 d post fertilization (dpf) at 28 &#176;C in embryo medium prepared as described above, select and stage as previously described15.</li>
	</ol>
	</li>
</ol>
2. Induction of Hypoxia In Vivo
<ol>
	<li>Construction of the gas incubation chamber
	<ol>
		<li>Use a breeding aquatic tank (Figure 1A) that is normally used in the fish facility for hypoxic chamber construction. This tank should fit a 24-well plate.</li>
		<li>Prepare a mesh-bottom 24-well plate as depicted in Figure 1. Remove the plastic bottom of the 24-well plate (Figure 1B), e.g., by using a milling machine for this purpose.</li>
		<li>Fill in the space between the plastic of the wells and the walls of the plate with epoxy resin. Attach any nylon filter (Figure 1C) with a mesh diameter of 0.1-0.2 mm to the resin-coated plastic and allow the plate to be completely dry.</li>
		<li>Once dry, drill three or four tunnels (Figure 1D) of 10 mm length and 5 mm diameter into the resin- and mesh-coated plate walls. Attach plastic or Teflon rods (Figure 1E) of appropriate diameter and 30 mm length to the tunnels and place the plate into the tank of choice.</li>
		<li>Place the mesh-bottom 24-well plate into the aquatic tank of choice. Using silicone tubing (Figure 1F), attach a gas tank (Figure 1G) with the desired O2/N2 mixture or another gas mixture of choice to a distributor valve (Figure 1H) using an appropriate gas regulator (I) (BOC 200 bar). Here, use gas tanks containing a mixture of 5% oxygen and 95% N2 in the hypoxia experiments presented here.</li>
		<li>Adjust the oxygen flow rate in the chamber medium to 0.0017-0.0019 cubic meters per second. Under this gas flow rate, no disturbance of the medium should be seen in the wells of the plate.
		<ol>
			<li>Use the high purity two-stage gas regulator for this purpose. Set the regulator to reduce the inner pressure of the gas tank (1000 - 2500 PSI) to a delivery pressure of 10 PSI throughout the experiment. Thus, in accordance with the specifications of this gas regulator, a flow rate of 0.00189 cubic meters per second was achieved throughout the experiment.</li>
		</ol>
		</li>
		<li>Connect the silicone tubing and the distributor valve to a ceramic disc diffuser (Figure 1J) inside the aquatic tank. Close the aquatic tank and carefully seal the lid, coating it with silicone grease (Figure 1K) (compare with the Figure 1). If a particular non-room temperature is required, place the hypoxic chamber into a laboratory incubator of required temperature.</li>
		<li>Depending on the type of embryos used in the experiment, fill the hypoxic chamber tank with the appropriate embryo medium and start diffusing the gas mixture through the ceramic disc diffuser. Equilibrate for 10-15 min.</li>
		<li>After equilibration with the desired oxygen mixture, measure oxygen concentration in the water using an oxymeter or oxygen probe. Here, use an oxymeter with the fiber-optic dissolved oxygen and temperature sensor for this purpose.</li>
	</ol>
	</li>
	<li>Induction of hypoxia in embryos
	<ol>
		<li>Carefully select the embryos for the experiment, use whole and alive embryos for the hypoxia incubation. Here, us frog embryos of stage 38 or zebrafish embryos 4 dpf&#160;in the experiments presented here.</li>
		<li>Place the embryo dishes into the incubator (Figure 1K) containing the gas incubation chamber and let them equilibrate to the temperature of the incubator.</li>
		<li>Carefully transfer the embryos into the wells of the meshed 24-well plate (Figure 1B) of the gas incubation chamber, using a plastic pipette. Always use a plastic pipette for embryo transfer throughout the protocol. 
		NOTE: Each well of this plate can contain up to 5 frog embryos of stage 38 or up to 7 zebrafish embryos of 4 dpf. Label the wells carefully if using different genotypes.</li>
		<li>Maintain the gas incubation chamber under a constant infusion with the gas mixture of choice for 5 h or for a longer time that suits the experiment. Use a mixture of 5% O2 and 95% N2 here.</li>
		<li>Carefully transfer the embryos from the gas incubation chamber back to the normoxic medium corresponding to the embryo type and immediately proceed with the Steps 3 or 4.</li>
		<li>If a further normoxic treatment is required, carefully transfer the embryos back to the normoxic medium corresponding to the embryo type.</li>
	</ol>
	</li>
</ol>
3. Control of Successful Hypoxia Induction by Monitoring HIF-1&#945; Levels
<ol>
	<li>Preparations
	<ol>
		<li>Prepare 0.4 mg/mL MS222 solution in 0.1x MBS.</li>
		<li>Prepare homogenization buffer: Purchase Radioimmunoprecipitation Assay buffer (RIPA buffer) for homogenization of tissue and supplement the amount of buffer necessary for your experiment with Protease Inhibitor to a final concentration 1:100 v/v.</li>
		<li>Prepare solutions for Western blot.
		<ol>
			<li>Prepare 4x Laemmli gel loading buffer: 425 mM Tris pH 8.0, 40% v/v Glycerol, 8% v/v SDS, 4% v/v beta-mercaptoethanol, 0.5% w/v Bromophenol Blue, 10 mL total volume ddH2O.</li>
			<li>Prepare 5x running buffer: 15 g Trizma base, 72 g Glycine, 5 g SDS, 1 L total volume ddH2O.</li>
			<li>Prepare Transfer buffer: 2.66 g Trizma base, 14.4 g glycine, 200 mL 100% methanol, 1 L total volume ddH2O.</li>
			<li>Prepare 10x&#160;TBST: 5 mL 1 M Tris pH 8.0, 30 mL 5M NaCl, 2.5 mL Tween 20, 1 L total volume H2O.</li>
			<li>Prepare blocking solution: 4% w/v skim milk powder in 1x TBST.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Collection of tissue
	<ol>
		<li>Anaesthetize the embryos by bathing in 0.4 mg/mL MS222 solution16,17 and transfer them into a plastic reaction tube filled with homogenization buffer using a plastic pipette. Use 20 &#181;L buffer per embryo. Note that at least 5 frog embryos of stage 38 or 7 zebrafish embryos of 4 dpf are required to determine a significant change in HIF1&#945; protein levels.</li>
		<li>Homogenize the tissue using an appropriate tissue homogenizer or a sonicator. Remove the debris by centrifugation (15 min; 13,000 x g; 4 &#176;C).
		<ol>
			<li>Alternatively, dissect the retinas from the anaesthetized frog embryos17 and homogenize as above. Use 20 &#181;L of homogenization buffer per 10 retinas.</li>
		</ol>
		</li>
		<li>Take the supernatant using a pipette, denature at 85 &#176;C for 5 min and supplement with Laemmli gel loading buffer to a final concentration of 1x v/v (e.g., add 5 &#181;L of 4x Laemmli buffer to 15 &#181;L homogenate). Use this supernatant for the Western blot or freeze the samples at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Western Blot
	<ol>
		<li>Load the samples prepared as described in Step 3.2 on a precast 12% gel in an electrophoresis system using 1x v/v Running buffer. Use a protein ladder for protein size determination. Transfer the proteins onto a nitrocellulose membrane (0.45 &#181;m membrane) in Transfer buffer for 1 h packed on ice at 4 &#176;C, as per manufacturer&#39;s protocol.</li>
		<li>Block unspecific binding sites incubating the membrane in Blocking buffer for 60 min. Follow with 3 x 10 min washes in 1x TBST.</li>
		<li>Incubate the membrane in solutions of rabbit anti-HIF-1&#945; antibody and mouse anti-&#945;-tubulin diluted 1:100 v/v and 1:5000 v/v, respectively, in Blocking solution, for 2.5 h&#160;at RT. Follow with 3 x 10 min washes in 1x TBST.</li>
		<li>For detection, incubate in goat anti-rabbit and goat anti-mouse HRP-conjugated antibodies diluted 1:1000 v/v in Blocking solution for 1 h. Follow with 3 x 10 min washes in 1x TBST and visualize the HRP staining using a commercial kit and a developer machine of choice.</li>
		<li>Quantify the amount of HIF1&#945; protein using digital quantification.</li>
	</ol>
	</li>
</ol>
4. Monitoring Cell Proliferation in Hypoxia
<ol>
	<li>Preparations
	<ol>
		<li>Prepare 5 mM EdU solution in the embryo medium of choice. The solution can be used immediately or aliquoted and stored at -20 &#176;C for up to 6 months.</li>
		<li>Prepare Phosphate-Buffered Saline (PBS) or purchase PBS from commercial resources. Autoclave for 10 min at 115 &#176;C.</li>
		<li>Prepare solutions for the detection of EdU incorporation.
		<ol>
			<li>Prepare fixation solution: 4% v/v of 16% Formaldehyde stock solution in PBS.</li>
			<li>Prepare sucrose solution: 30% w/v sucrose in PBS.</li>
			<li>Prepare PBST: 0.1% v/v Triton X-100 in PBS.</li>
			<li>Prepare Blocking solution: 10% v/v Heat-inactivated Goat Serum (HIGS) in PBST.</li>
			<li>Prepare DAPI solution: 1:1000 v/v 4&#39;,6-diamidino-2-phenylindole (DAPI) in PBST.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Incubation of embryos in hypoxia for EdU incorporation
	<ol>
		<li>Fill the gas incubation chamber with 400-500 mL of 5 mM EdU solution supplemented with 1% v/v DMSO. Pre-equilibrate the solution with the same gas mixture used in the experiment for 15 min prior to the experiment. Note that the volume of the incubation solution can be minimized by attaching shorter rods (Figure 1E) to the mesh-bottom plate.</li>
		<li>Connect the gas tubing to the gas cylinder containing a gas mixture of 5% O2 and 95% N2. Incubate the solution for 15 min.</li>
		<li>Transfer the embryos to the hypoxic chamber, distributing them evenly between the wells and incubate for 2 h. Each well can fit up to 5 frog embryos of stage 38 or up to 7 zebrafish embryos 4 dpf.</li>
		<li>Analyze the embryos for EdU incorporation.</li>
	</ol>
	</li>
	<li>Time-course of embryo incubation in hypoxia and EdU incorporation
	<ol>
		<li>Fill the gas incubation chamber with 500 mL of embryo medium of choice. Connect the gas tubing to the gas cylinder containing a gas mixture of 5% O2 and 95% N2 and equilibrate the medium with the gas mixture for 15 min.</li>
		<li>Transfer the embryos to the hypoxic chamber, distributing them evenly between the wells and incubate for desired time period up to 48 h. For the last 1 h, substitute the embryo medium with 5 mM EdU solution supplemented with 1% v/v DMSO.</li>
		<li>Transfer the embryos back to the normoxic dish and analyze for EdU incorporation.</li>
	</ol>
	</li>
	<li>EdU incorporation detection and analysis
	<ol>
		<li>Anaesthetize the embryos by bathing in 0.4 mg/mL MS222 solution.</li>
		<li>Transfer the embryos to an appropriate glass vial and fix by incubating in Fixation solution for 2 h at RT or O/N at 4 &#176;C. Follow with 3 x 10 min washes in PBS. Carefully transfer the embryos to the sucrose solution for tissue cryoprotection. Incubate for 4 h or until the embryos sink to the bottom of the glass vial.</li>
		<li>Embed the embryos in embedding medium (optimal cutting temperature compound). Cryosection blocks with embryos immediately or store for up to 14 d&#160;at -80 &#176;C.</li>
		<li>Cryosection the blocks containing embryos using a cryostat. Collect sections of 12 &#181;m (zebrafish embryos) or 16 &#181;m (frog embryos) thickness on microscope slides and let dry. Process cryosections immediately or store at -20 &#176;C for up to 3 months.</li>
		<li>Detect EdU incorporation by immunofluorescent staining using a commercial chemistry kit. Prepare the amount of EdU Reaction Mix necessary for the number of cryosections in the experiment, as described by the manufacturer.
		<ol>
			<li>For 500 &#181;L of EdU Reaction Mix, use 430 &#181;L of 1X EdU reaction buffer, 20 &#181;L of CuSO4, 1.2 &#181;L of Alexa Fluor azide, 50 &#181;L of 1X EdU buffer additive.</li>
		</ol>
		</li>
		<li>Wash the cryosections once with PBS to remove the embedding medium and 3 x 5 min with PBST to permeabilize the tissue. Incubate the cryosections in Blocking solution for 30 min.</li>
		<li>Incubate the cryosections with the EdU Reaction Mix for 1 h and followed by 3 x 10 min washes in PBST. Costain the cryosections with DAPI solution for 15 min, followed by 3 x 10 min washes in PBST. Mount the slides with stained cryosections in mounting medium, cover with coverslips and leave to dry before analyzing under a fluorescent microscope or storing at 4 &#176;C for up to 1 year.</li>
		<li>Analyze the cryosections with anti-EdU staining under an inverted confocal scanning microscope and determine the number of EdU-positive cells using digital quantification.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

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 ...

induction-of-hypoxia-in-living-frog-and-zebrafish-embryos

Research

JoVE Journal

Developmental Biology

9.7K Views.  University of Cambridge. 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.

Video: Induction of Hypoxia in Living Frog and Zebrafish Embryos

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 systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

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

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

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 and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

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 imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

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 described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

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 embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

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 cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

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

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

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

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

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 disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

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 processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

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. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

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