Support in Massimo Santoro lab come from HFSP, Marie Curie Action, Telethon and AIRC. We thank Dafne Gays and Emiliano Panieri for critical reading of the manuscript.

1. Preparation of Instruments and Working Solutions
<ol>
	<li>Prepare the fish water solution. Make a stock solution by dissolving 2 g of sea salts &#39;Instant Ocean&#39; in 50 ml of distilled water. Add 1.5 ml of stock fish water to 1 L distilled water to prepare ready to use fish water (60 &#181;g/ml sea salts final concentration). Autoclave the ready to use fish water before usage. This solution is used as zebrafish embryo medium.</li>
	<li>Prepare methylcellulose for embryo mounting. Dissolve 1.5 g of methylcellulose in 50 ml of sterile fish water. Facilitate the dissolution by using a magnet on a stir plate. The complete dissolution of the powder may require several hours. Check the solution for clarity and aliquot into small tubes. Store at -20 &#176;C for months and thaw out aliquots at use. Centrifuge the methylcellulose at 950 x g for 5 min before using. Avoid freeze-thaw cycles of aliquots.</li>
	<li>Prepare 50 ml of tricaine/ethyl 3-aminobenzoate methanesulphonate salt (stock solution) by dissolving 200 mg of tricaine in 100 ml water and adjust pH to 7.0 using Tris-HCl 1 M (pH 9). Store this stock at 4 &#176;C for no more than 30 days. CAUTION: tricaine is toxic. Use in accordance with appropriate handling guidelines.</li>
	<li>Inducing oxidative stress in zebrafish embryos
	<ol>
		<li>Prepare an oxidant solution for generic oxidative stress induction: Make 50 ml of oxidant solution by adding H2O2 stock solution (hydrogen peroxide; 100 mM) to fish water. Use H2O2 of a final concentration between 2 mM and 100 &#956;M. Prepare this solution shortly before usage. The oxidant solution can be applied to both whole mount ROS-detection and single-cell ROS-detection methods. Do not store this solution. CAUTION: H2O2 is dangerous, and harmful by inhalation and if swallowed. Contact with combustible material may cause fire. Handle under a fume hood and wear appropriate personal protective equipment.</li>
		<li>Prepare an oxidant solution for mitochondria-derived oxidative stress induction: Make a oxidant stock solution (5 mM) by dissolving 3.9 mg of rotenone in 2 ml of dimethyl sulfoxide (DMSO). Keep this solution at room temperature in the dark.
		<ol>
			<li>Dissolve rotenone stock solution to 10 ml of fish water to make a ready to use solution. Use rotenone at a concentration between 5-50 &#956;M. Do not use rotenone at concentrations higher than 100 &#956;M. At appropriate concentrations, this oxidant solution can be applied to both whole mount ROS-detection and single-cell ROS-detection methods. CAUTION: Rotenone is toxic and hazardous. Handle according appropriate precautionary statements.</li>
		</ol>
		</li>
		<li>Induce oxidative stress by gene knock-down: Knock-down nrf2a gene expression in zebrafish embryos by morpholino microinjection as previously reported by Timme-Laragy A. et al., 201238.</li>
		<li>Induce oxidative stress after tissue damage: generate a wound margin at the tail fin of zebrafish embryos at 72 hpf as previously described by Niethammer et al., 200939.</li>
	</ol>
	</li>
	<li>Prepare 5 ml of ROS-detection solution for single cell ROS-detection method:
	<ol>
		<li>Solution for general ROS detection: Dissolve the generic ROS-sensitive molecular probe in HBSS (Hanks&#8217;s Balanced Salt Solution) in order to prepare a working solution (concentration range: 2.5-10 &#956;M). This solution must be prepared shortly before usage.</li>
		<li>Solution for specific detection ROS induced by mitochondria: Shortly before use, dissolve a mitochondrial ROS-sensitive probe with DMSO making a 5 mM stock solution. Dissolve stock solution in HBSS in order to prepare a working solution (concentration range: 2.5-10 &#956;M). 
		NOTE: Avoid light and oxygen exposure of ROS-detection working solutions and their respective stock solutions. Protect the tube from light by using an aluminum foil. Do not store or re-use probes dissolved in HBSS. Store the stock solutions at -20 &#176;C for a month. 
		CAUTION: Handle molecular probes and DMSO under a fume hood in accordance with appropriate guidelines.</li>
	</ol>
	</li>
	<li>Prepare 5 ml of ROS-detection solution for the whole mount ROS-detection method: Dissolve the generic ROS-sensitive probe stock solution (stabilized in DMSO) in HBSS in order to prepare a working solution (concentration range: 2.5-5 &#956;M). Avoid light and oxygen exposure. Protect the tube from light. Prepare this solution before use. Do not store or re-use this solution. CAUTION: Handle molecular probes under a fume hood in accordance with appropriate guidelines.</li>
	<li>Make 25 ml of Stop Solution by adding 10 ml of fetal bovine serum to 15 ml of PBS 1x. Keep solution sterile. Store this solution at 4 &#176;C. This solution is required only for single cell - ROS detection method.</li>
	<li>Set the zebrafish air incubator at 28 &#176;C.</li>
	<li>Set the centrifuge at 4 &#176;C for the single cell ROS-detection method.</li>
</ol>
2. Mating of Adult Fishes and Selection of Zebrafish Embryos
<ol>
	<li>Set-up adult zebrafish pair crosses according to standard protocols40. Select the appropriate transgenic line in accordance with specific experimental needs.</li>
	<li>Collect eggs and place them into a 90-mm dish with fish water/embryo medium. Keep eggs at 28 &#176;C until embryos will develop and grow to the desired developmental stage (e.g., 48 hpf, 72 hpf; hpf: hours post fertilization).</li>
	<li>Screen for developing embryos. Exclude all unfertilized eggs or underdeveloped embryos.</li>
	<li>Anesthetize embryos by adding 1 ml of tricaine (stock solution) in 50 ml fish water.</li>
	<li>Select Tg fluorescent embryos under a stereomicroscope.</li>
</ol>
3. Treatment of Embryos with Oxidant Agent
<ol>
	<li>Use at least 30 embryos between 48 hpf and 72 hpf per different condition. Wash embryos twice with fresh fish water in order to remove tricaine.</li>
	<li>Split fluorescent embryos into three dishes. Put no more than 30 embryos/dish.</li>
	<li>Remove the washing solution and add 10 ml of the oxidant solution or 10 ml of fish water as control solution.</li>
	<li>Incubate embryos for 10 min to 1 hr at 28 &#176;C. The incubation period depends by the level of oxidative stress to be induced in specific tissues. Short periods are the best as cells affected by oxidative stress progressively undergo cell death.</li>
	<li>Transfer embryos into a new dish containing HBSS and wash embryos by swirling. Pre-warm HBSS at 28 &#176;C.</li>
</ol>
4. Whole Mount ROS Detection Method
<ol>
	<li>Collect embryos after oxidant treatment and put no more than 10 embryos in a small tube. Rinse with HBSS as much as possible. Protect the tube from light using aluminum foil.</li>
	<li>Add 1 ml of ROS-detection solution for each tube.</li>
	<li>Incubate embryos in the dark for 15 min at 28 &#176;C to avoid light exposure.</li>
	<li>During the incubation time prepare a glass slide for whole embryo analysis. Put 300 &#956;l of methylcellulose on a &#8220;depression&#8221; glass slide and spread it on the glass surface with a pipette tip. Avoid bubbles while releasing the methylcellulose.</li>
	<li>At the end of the incubation time, immediately remove the ROS-detection solution and wash twice with 2 ml of HBSS. Wash embryos by inverting the tube several times. Do not pipette or vortex.</li>
	<li>Aspirate embryos up into a glass Pasteur pipette. Position embryos near the opening of the pipette and gently eject them into the methylcellulose. Orientate the embryos appropriately with a fine nylon line.</li>
	<li>Compare the fluorescence of control embryos with treated embryos. Alternatively, fix parameters of the fluorescence stereomicroscope or confocal microscope so that all embryos are imaged using the same imaging settings.</li>
</ol>
5. Single Cell ROS-detection Method
<ol>
	<li>Start from step: 3.5. Make sure to have at least 35 embryos for each condition.</li>
	<li>Transfer embryos into a new dish. Remove fish water as much as possible. Add 10 ml of ice-cold PBS 1x and 400 &#956;l of Protease Inhibitors Cocktail. Manually dechorionate embryos with forceps and remove the yolk sack with a fine needle. Note: Removing yolk sack ensures clean samples for the following FACS analysis. This step can be avoided according to FACS instrument.</li>
	<li>Transfer de-yolked embryos into a 24-well multiwell plate (15 embryos/well) and remove all fish water.</li>
	<li>Add 300 &#956;l of HBSS, 30 &#956;l collagenase P, 50 &#956;l Trypsin-EDTA in each well.</li>
	<li>Homogenize embryos by gently pipetting up and down with a tight pipette tip (1,000 &#956;l).</li>
	<li>Incubate at 28 &#176;C for 20 min and homogenize samples by pipetting every 5 min.</li>
	<li>Check tissue disruption by observing an aliquot of homogenate at the compound microscope. Facilitate single cell suspension by pipetting. Do not incubate embryos for more than 30 min.</li>
	<li>Stop the reaction by adding 200 &#956;l of Stop Solution. Mix by pipetting gently.</li>
	<li>Transfer cell suspension into a pre-chilled tube with tight bottom. Keep tube on ice and avoid light exposure.</li>
	<li>Centrifuge for 5 min at 250 x g at 4 &#176;C.</li>
	<li>Remove the upper phase and re-suspend cells with ice cold HBSS by gently pipetting.</li>
	<li>Count cells and make sure you have the same number of cells in all of your samples. Do not use less than 2 x 106 cells.</li>
	<li>Centrifuge cells for 5 min at 250 x g at 4 &#176;C.</li>
	<li>Remove supernatant and suspend the pellet with 1 ml of ice chilled HBSS containing the ROS-sensitive probe.</li>
	<li>Incubate the tube at room temperature for 3 min&#160;in the dark. Vary incubation time according to the level of oxidative stress and to the sensitivity of the probe.</li>
	<li>Transfer cells to a FACS tube. Keep tubes on ice and avoid light exposure before FACS analysis.</li>
	<li>Sort cells with a fluorescence-activated cell sorter (FACS). Set wavelengths in accordance with Tg tissue fluorescence (e.g., GFP) and the excitation/emission spectra of the molecular probe used for ROS detection (e.g., specific mitochondrial ROS-sensitive probes, generic ROS-sensitive probes).</li>
	<li>For each condition, measure all samples within the same experimental session by applying the same FACS settings. Calculate the percentage of fluorescent cells as the mean of different biological replicates. Analyze at least two replicates for each condition.</li>
</ol>

The authors have nothing to disclose.

Critical Steps
The procedure for oxidative stress detection in zebrafish embryos herein described comprises two different methods. The whole mount ROS-detection method is mainly a qualitative assay for ROS-detection, while the single cell ROS-detection method allows more specific quantitative measurements (Figure 1). Both methods offer a quick and easy way to assess in vivo ROS-detection on zebrafish embryos. However, they both present some critical steps.
<p class=...

Oxidative stress is specifically defined as a condition that results from an unbalanced cellular redox state. The complex redox reactions that routinely occur inside cells determine the cellular redox-state. Redox reactions consist of all chemical reactions that consist in the transfer of electrons between atoms of biological molecules producing reduction and oxidation of molecules (i.e. redox reactions). These reactions are catalyzed by electronically activated species (i.e. pro-oxidative species), which are characterized by an extreme structural instability and spontaneous activation of unbalanced electrons that exchange with neighboring biomolecules. These irregular reactions result into DNA damage, protein carboxylation, and lipid oxidation, and eventually lead to cell death1. Increased levels of oxidative stress have been associated with aging and the progression of different pathological states2. Oxidative stress has been reported to be responsible for vascular alterations in diabetes and cardiovascular diseases3,4. It also plays a critical role in neuronal degeneration in Alzheimer&#39;s disease and Parkinson&#39;s disease5. Moreover, oxidative stress has been demonstrated as a critical factor in governing cancer progression and metastatic events6,7. In addition, inflammation and immune responses may elicit and further support oxidative stress8.
In living cells, pro-oxidative species are derived from oxygen (ROS; reactive oxygen species) or nitrogen (RNS; reactive nitrogen species). ROS include the hydroxyl radical (.OH), the superoxide anion (O2-), and the hydrogen peroxide (H2O2). The primary RNS is nitrous oxide (NO.). A series of secondary reactive species can be generated by spontaneous interactions between ROS and RNS or free metals ions9. For example, the superoxide anion reacts with nitrous oxide to form peroxynitrate (ONOO-), while H2O2 reacting with Fe2+ generates hydroxyl radicals. ROS and RNS, due to their ability to react with several biomolecules, are considered a dangerous threat for the maintenance of the physiological redox state10. To maintain the redox state cells are equipped with a series of detoxifying anti-oxidant molecules and enzymes. The superoxide dismutase (SOD), Catalase, Glutathione peroxidase and Peroxiredoxins essentially constitute the anti-oxidant enzymatic-arsenal that provides cellular protection from pro-oxidative species including H2O2 , .OH and OONO- 11. Also anti-oxidant molecules like vitamin C and E, polyphenols and CoenzymeQ10 (CoQ10) are of critical importance to quench ROS and their dangerous derivatives12,13. However, an excessive production of ROS and RNS, or a dysfunction in the anti-oxidant system, shifts the cellular redox-state toward oxidative stress14.
Besides their negative connotation, ROS can play various physiological roles in cells of different origin. Cells normally produce ROS as signaling molecules to mediate normal biological events such as host defense and wound repair15-17. Reactive species are normally produced in cells by intracellular enzymes such as NOX (NADPH Oxidase) and XO (Xantine Oxidase) in response to signaling factors, growth factors, and intracellular fluctuations of calcium levels18,19. It has been reported that ROS may differentially modulate the activity of important nuclear factors such as p53 or cellular components such as the ATM-kinase, a master regulator of the response to DNA damage20. Analogously ROS strongly influence cellular signaling by mediating the oxidation and inactivation of protein tyrosine phosphatases (PTPs), which are established as critical regulators of signal transduction21. Moreover, proteomic based methodologies demonstrate that RNS are also responsible for specific protein modifications and alterations of molecular signaling. RNS react with the cysteine thiol groups modifying them into S-nitrothiols (SNO) and triggering molecular pathways concomitant with pathological states such as inflammatory and autoimmune diseases22,23.
Since cell culture experiments only partially reproduce the multitude of factors acting in vivo, it is of great interest to perform redox studies in animal models24,25. To achieve this, the zebrafish has been considered a suitable vertebrate animal model to study oxidative stress dynamics26. The zebrafish is a new model system that grants several advantages to study cellular and genetic events during vertebrate development and disease. Large clusters of embryos can be generated and available weekly for experimental needs. Moreover the extraordinary optical clarity of zebrafish embryos, as well their small size, enables single cell imaging and dynamic tracking in a whole organisms27. In the last decade, a considerable number of zebrafish mutants have been generated to model human pathological conditions such as cancer and genetic diseases28-31. Most importantly, a multitude of transgenic lines has been produced to allow extensive opportunities of genetic and biological manipulations32. For example, transgenic tissue-specific zebrafish lines are regularly utilized for in vivo studies. These lines express a fluorescent protein under the control of a selected promoter, offering the ability to identify single cells in vivo, as well as the anatomical structure they comprise.
Several toxicological studies have already used the zebrafish to evaluate the in vivo effect of chemicals on redox homeostasis, suggesting the suitability of this vertebrate as an animal model for the field of drug discovery and oxidative stress33-35. Even though some fluorescent probes have been tested to monitor oxidative stress in zebrafish larvae36,37, there are no established assays to detect and measure the levels of oxidative stress in zebrafish tissues and living cells. Here we describe a procedure for in vivo quantification of oxidative stress in living cells of zebrafish embryos. Imaging tools, FACS sorting, fluorescent probes and pro-oxidative conditions are all combined to generate a simple assay for the detection and quantification of oxidative species in zebrafish embryos and tissues.

<table border="0" cellpadding="0" cellspacing="0" width="1261">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:285px;">Catalog Number</td>
			<td style="width:528px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Hydrogen peroxide solution</td>
			<td style="width:147px;">SIGMA</td>
			<td style="width:285px;">516813</td>
			<td>DO NOT STORE DILUITIONS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Hank&#39;s Balanced Salt Solution 1X</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:285px;">14025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Methyl cellulose</td>
			<td style="width:147px;">SIGMA</td>
			<td style="width:285px;">M0387</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Instant Ocean Aquarium Sea Salt Mixture</td>
			<td style="width:147px;">INSTANT OCEAN</td>
			<td style="width:285px;">SS15-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tricaine</td>
			<td style="width:147px;">SIGMA</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:301px;">Cgeneric ROS-sensitive probe:&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; CellROX Deep Red Reagent</td>
			<td style="width:147px;">INVITROGEN</td>
			<td style="width:285px;">C10422</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:301px;">Mitochondria specific ROS-sensitive probe: MitoSOX&#160;</td>
			<td style="width:147px;">INVITROGEN</td>
			<td style="width:285px;">M36008</td>
			<td>dissolve one vial with 13&#956;l of DMSO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Hydroethidine</td>
			<td style="width:147px;">INVITROGEN</td>
			<td style="width:285px;">D23107</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Rotenone</td>
			<td style="width:147px;">SIGMA</td>
			<td style="width:285px;">R8875</td>
			<td>Prepare 5mM stock solution in DMSO.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Dimethyl sulfoxide</td>
			<td style="width:147px;">SIGMA</td>
			<td style="width:285px;">D2650</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:301px;">VAS2870; 3-Benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo(4,5-d)pyrimidine</td>
			<td style="width:147px;">EnzoLifeScience</td>
			<td>BML-EI395</td>
			<td>dissolve the powder in DMSO; diluite in fish water</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:301px;">Propidium Iodide&#160;</td>
			<td style="width:147px;">Molecular probes&#160;&#160;&#160;&#160;&#160;&#160; (Life Technologies)&#160;</td>
			<td>P3566</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:301px;">7-aminoactinomycin D (7-AAD)&#160;</td>
			<td style="width:147px;">Molecular probes&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (Life Technologies)&#160;</td>
			<td>A1310</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Nrf2a Morpholino</td>
			<td style="width:147px;">GeneTools</td>
			<td style="width:285px;">5&#39;-CATTTCAATCTCCATCATGTCTCAG-3&#39;</td>
			<td>Ref: Timme-LaLaragy et al; 2012 (PMID: 22174413); Kobayashi et al; 2002(PMID:12167159 )</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase P</td>
			<td style="width:147px;">ROCHE</td>
			<td>11213857001</td>
			<td>Dissolve the powder at 100mg/ml in sterile HBSS. Store aliquots at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Phosphate-Buffered Saline (PBS)</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:285px;">10010-056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum&#160;</td>
			<td style="width:147px;">GIBCO</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Complete Protease Inhibitor Cocktail Tablets</td>
			<td style="width:147px;">ROCHE</td>
			<td style="width:285px;"></td>
			<td>Dissolve one tablet in 1ml of water</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% Trypsin-EDTA (10x), no phenol red</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:285px;">15400-054</td>
			<td>Prepare 1X working solution before usage</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Compound microscope&#160;</td>
			<td>ZEISS</td>
			<td style="width:285px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereo microscope with fluorescent illumination</td>
			<td>Nikon</td>
			<td style="width:285px;">AZ100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">camera&#160;</td>
			<td>ZEISS</td>
			<td style="width:285px;">AxioCamMRm</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">software for fluorescence image acquisition</td>
			<td>ZEISS</td>
			<td style="width:285px;">ZEN 2011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence-activated cell sorter</td>
			<td style="width:147px;">BD FACSCalibur</td>
			<td style="width:285px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge&#160;</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:285px;">5417R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACS tubes&#160;</td>
			<td style="width:147px;">BD</td>
			<td>342065</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multiwell Plate&#160;</td>
			<td>BD Falcon</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Sterilized, non treated Petri dishes 90mm</td>
			<td style="width:147px;">VWR</td>
			<td style="width:285px;">391-1915</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:301px;">Confocal microscope</td>
			<td style="width:147px;">Leica</td>
			<td style="width:285px;">Leica SP5</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of oxidative stress in zebrafish embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

By applying the method here described, we can easily measure and detect oxidative stress (and ROS levels) in zebrafish embryonic tissues. After crossing adult zebrafish, eggs are collected and allowed to develop at 28 &#176;C to 72 hr&#160;post fertilization (hpf). In order to induce oxidative stress, we propose two different approaches: 1) the treatment of embryos with strong pro-oxidant reagents or 2) promoting ROS formation after tissue injury.
In the first approach, we employed two differe...

Watch this Scientific Journal Video about Analysis of Oxidative Stress in Zebrafish Embryos at JoVE.com

Analysis of Oxidative Stress in Zebrafish Embryos

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

analysis-of-oxidative-stress-in-zebrafish-embryos

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36.9K Views.  University of Torino. Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses. 

Video: Analysis of Oxidative Stress in Zebrafish Embryos

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

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

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...