The authors thank Meng-Chieh Shen for images, Jennifer Anderson for providing helpful comments on the manuscript, and members of the Farber laboratory for their contributions in developing these techniques. This study was funded by NIDDK-NIH award RO1DK093399 (S.A.F.), RO1GM63904 (The Zebrafish Functional Genomics Consortium: PI Stephen Ekker and Co-PI S.A.F), and F32DK096786 (J.P.O.). This content is solely the responsibility of the authors and does not necessarily represent the official views of NIH. Additional support was provided by the G. Harold and Leila Y. Mathers Charitable Foundation to the laboratory of S.A.F and the Carnegie Institution for Science endowment.

These protocols have been approved by the Carnegie Institution for Science Institutional Animal Care and Use Committee (protocol no. 139).
1. Animal Preparation
<ol>
	<li>Maintain adults and larvae at 28 &#176;C on a 14 hr:10 hr light:dark cycle. Feed adults twice daily with shell free Artemia (decapsulated, non-hatching, starting at 14 dpf) and commercial micropellets.</li>
	<li>These protocols are optimized for the use of 6-7 dpf larvae collected by natural spawning of the AB background. Protocols can be modified for larvae of other ages and backgrounds. Do not provide exogenous food prior to 6 dpf.</li>
	<li>Anesthetize larvae with Tricaine in embryo media (EM) (4.2 ml of 4 mg/ml Tricaine per 100 ml EM) at room temperature (RT).</li>
</ol>
2. Preparation of Lipid-rich Egg Yolk Feed
<ol>
	<li>Prepare and store the chicken egg yolks. Separate the yolk and white of 12 chicken eggs. Pool the yolks, aliquot 1 ml into 1.5 ml tubes, and store at -80 &#176;C up to 1 yr (12 yolks will make ~80 aliquots). Pool the whites, store, and use as a lipid-poor, protein-rich feed.</li>
	<li>Prepare fluorescent lipid analog(s) to be added to the egg yolk feed if desired.
	<ol>
		<li>Suspend the purchased, powdered fluorescent lipid analog in 100% ethanol or 100% chloroform at 0.1 &#181;g/&#181;l (see following note discussing solvents), aliquot in opaque glass tubes, seal with parafilm, and store according to manufacturer&#39;s instructions. Due to rapid evaporation of organic solvents do not use aliquot volumes less than ~400 &#181;l for long-term storage. 
		Note: If the lipid analog is suspended in ethanol, large volumes of the lipid analog will be used because it dries faster under N2 than chloroform. If the lipid analog is suspended in ethanol it does not have to be dried and resuspended before adding it to the liposome feed in step 2.2.4, but the total ethanol concentration should not exceed 0.1% in the liposome feed to avoid potentially confounding physiological effects of ethanol.</li>
		<li>For fluorescent fatty acid analogs: Prepare a total volume of 20 ml 5% egg yolk emulsion in EM. From that prepare 5 ml aliquots with a final concentration of 6.4 &#181;M 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY C16) for live confocal imaging and food intake assays or 1 &#181;g/ml BODIPY C12 for stereomicroscope imaging. Transfer the desired amount of the fluorescent lipid analog into a 1.5 ml plastic tube. Dry the lipid under a stream of N2, taking care not to blow the lipid out of the tube.</li>
		<li>Immediately, resuspend in 5 &#181;l 100% ethanol by pipetting a stream down the sides of the tube, add 95 &#181;l EM, and mix well. 
		Note: The mixture should appear homogenous; if colored lipid remains on the sides of the tube or the liquid appears clumpy, the lipid has not fully solubilized and requires more ethanol. Protect the tube from light and store on ice.</li>
		<li>For fluorescent cholesterol analog: prepare a final concentration of 2.4 &#181;g/ml cholesterol in 5 ml 0.5-5% egg yolk emulsion for live imaging studies. Transfer the desired amount of cholesterol into a 1.5 ml plastic tube. Dry the lipid under a stream of N2, taking care not to blow the lipid out of the tube.</li>
		<li>Immediately, resuspend in 15 &#181;l of RT 100% ethanol by pipetting a stream down the sides of the tube and mix with 85 &#181;l of RT 1% fatty acid-free BSA in purified H2O. Protect the tube from light and store on ice.</li>
	</ol>
	</li>
	<li>Prepare Egg Yolk Liposome Feed.
	<ol>
		<li>Add a thawed aliquot of egg yolk to EM in a 50 ml conical tube. Vortex the diluted egg yolk mixture for 1-2 min; the mixture should appear to be a homogenous emulsion.</li>
		<li>Pour the mixture through a fine mesh strainer to remove protein conglomerates and collect in a new, fresh 50 ml conical tube.</li>
		<li>Pulse sonicate the egg mixture with a one-fourth inch tapered microtip (5 times (5 x 1 sec on, 1 sec off) pausing 5-10 sec between each set; output intensity: 6 W) at RT to create liposomes. Sonicator power settings are instrument dependent and therefore require optimization for each instrument and microtip. 
		Note: Continuously rock liposome mixture at RT to maintain homogeneity until use.</li>
	</ol>
	</li>
	<li>Add the fluorescent lipid analog to the egg yolk liposomes, if desired. Immediately after sonication, pipet the prepared lipids into the liposome mixture and vortex at high speed for 30 sec to incorporate into liposomes. Protect the mixture from light by wrapping the tube in foil and continue rocking at RT.</li>
</ol>
3. Feeding Paradigm
<ol>
	<li>Prepare the Larvae for Feeding.
	<ol>
		<li>Prior to beginning the feed, screen larvae for obvious morphological defects that may impede feeding. Transfer larvae to 60 mm x 15 mm Petri dishes or 6- or 12-well plates, reduce EM to a minimum, and replace with 5 ml of the prepared liposome solution.</li>
		<li>If required, transfer unfed control larvae to 5 ml EM and/or 5 ml 5% egg white in EM and treat in parallel.</li>
	</ol>
	</li>
	<li>Gently rock the larvae in an incubated rocker (29-31 &#176;C at 30 rpm) throughout the feed to encourage food intake while continuing to shield from light if fluorescent lipid analogs are present. If larvae need to be exposed to light, minimize the exposure with a tinted glass incubator cover.</li>
	<li>At the end of the feeding period, rinse the freely swimming larvae in EM 3 times. 
	Note: Larvae may begin consuming liposomes at any point during the feed. If it is critical that larvae commence feeding at the same time, screen for food intake (see 3.4) after 1 hr of feeding and return only larvae that have commenced feeding to the egg yolk emulsion to complete the feed.</li>
	<li>Screen the larvae at this point for food intake as a small number may not eat (generally ~0-5%). Examine the intestines of larvae that have been anesthetized or slightly cooled on a metal block on ice to determine if they have fed: larvae that have consumed liposomes will have a darkened intestine (Figure 1).</li>
	<li>Image or process larvae for food intake immediately. Alternately, place larvae in fresh EM and incubate at 28 &#176;C to allow metabolic processing to occur.</li>
</ol>
4. Mounting Larvae for Live Imaging
<ol>
	<li>Prepare the Mounting Media.
	<ol>
		<li>For 3% methyl cellulose: add 0.75 g methyl cellulose to 25 ml of near boiling EM, vortex, rock at RT 2-3 days until completely dissolved, and store at 4 &#176;C for years at -20 &#176;C. Repeated cycles of freezing and thawing at 4 &#176;C will remove air bubbles. Store 3% methyl cellulose on ice while mounting larvae.</li>
		<li>For 1.2% low-mount agarose: add 0.3 g of low-melt agarose to 25 mL EM and dissolve by bringing to a boil, aliquot into 2 ml tubes, and maintain at 26-28 &#176;C on a heat block. Unused aliquots can be stored at RT or -20 &#176;C and boiled again at the time of use. Be sure that agarose is cool enough to handle before exposing to larvae or they may be over-heated.</li>
	</ol>
	</li>
	<li>For low-magnification, medium-throughput live imaging, place a glass slide on a tissue on a metal block on ice and wait for the slide to cool. Apply a generous amount of 3% methyl cellulose (which has been maintained at 4 &#176;C on ice) onto the slide, transfer a larva onto the 3% methyl cellulose, using a fishing-line poker (0.41 mm diameter fishing line glued to the end of a glass capillary tube) create a troth to drain excess EM (so that methyl cellulose will not be diluted) and position the larvae as desired.</li>
	<li>For short-term (&#60;20 min), high-magnification live imaging with an upright oil emersion objective, mount larvae with the coverslip lean-to method.
	<ol>
		<li>Use cyanoacrylate based glue (Superglue) to attach a a 22 mm x 30 mm coverslip to one end of a glass slide. Use a poker to put a thin line of 3% methyl cellulose on the slide adjacent to the coverslip and transfer larvae to the slide with a wide bore Pasteur pipet. Remove excess EM so it will not dilute the methyl cellulose.</li>
		<li>Using a poker, gently position larvae in the methyl cellulose on their sides (left side up to image more of the liver, right side up to image the gallbladder) with their heads next to the coverslip.</li>
		<li>Spot superglue in the corners of a second coverslip and gently place one edge of the coverslip on the mounted coverslip and the other on the slide, bridging the larvae. Take care not to apply excess pressure during this step or with the objective while imaging so that larvae remain uninjured.</li>
	</ol>
	</li>
	<li>For long-term, high-magnification imaging with an upright immersion objective, mount larvae in agarose. Add a larva to a small aliquot of 1.2% low melt agarose then transfer the larva in a small volume of agarose to a petri dish.
	<ol>
		<li>Quickly position the larvae with a poker before the agarose solidifies. Allow the agarose to dry for 2-3 min and cover the agarose fully with fresh EM to prevent it from drying.</li>
	</ol>
	</li>
	<li>For long-term, high-magnification imaging with an inverted objective, mount larvae on a glass-bottom dish in agarose.
	<ol>
		<li>Cool a metal block on ice. Place the dish on the metal block separated by a tissue to prevent excessive cooling and larval freezing. Transfer a larva to the dish and remove the EM by wicking with a tissue.</li>
		<li>Place a drop of 28 &#176;C low-melt agarose (1.2%) on top of the larva and immediately position with a poker (left side down to best image the liver, right side down to image the gallbladder). Remove dish from the cold block, allow to dry for 2-3 min, and add a sufficient volume of fresh EM to cover the agarose (~2-5 ml).</li>
	</ol>
	</li>
</ol>
5. Food Intake Assay
<ol>
	<li>At the end of an egg yolk liposome feed containing 6.4 &#181;M BODIPY C16, pool a minimum of 10 washed larvae in a plastic 1.5 or 2 ml tube, remove EM, and snap freeze. The samples may be stored at -80 &#176;C protected from light for several months. 
	Note: If possible, collect multiple replicates. It is important to collect unfed larvae at this step to normalize for background lipid fluorescence (ideally larvae from the same clutch are used).</li>
	<li>Perform a modified Bligh-Dyer lipid extraction3,21.
	<ol>
		<li>Add 100 &#181;l homogenization buffer (20 mM Tris-Cl, 1 mM EDTA) to the sample on ice (alternatively purified H2O can be used). Homogenize on ice with a microtip sonicator (5 sec total: 1 sec on 1 sec off). Check that larvae are totally homogenized; if not, repeat. Transfer to a 13 ml disposable glass culture tube (other glass tubes may be used). 
		Note: Chloroform is hazardous; perform all subsequent lipid extraction steps at RT in a chemical hood.</li>
		<li>For each 100 &#181;l of homogenization buffer used, add 375 &#181;l of 1:2 (chloroform:methanol). Vortex 30-60 sec, incubate 10 min, add 125 &#181;l of chloroform per 100 &#181;l homogenization buffer used, and vortex 30 sec.</li>
		<li>Add 125 &#181;l of 200 mM Tris pH 7.5 per 100 &#181;l homogenization buffer used, vortex 30 sec, and centrifuge (2,000 x g, 5 min, RT, protected from light). Carefully remove the samples from the centrifuge. They will be separated into two phases: an upper phase is aqueous, an interface of larval debris, and a lower organic phase (contains lipids).</li>
		<li>With a clean glass pipette collect the bottom, organic phase and transfer it into a clean 13 ml glass tube. 
		Note: Take care to avoid the aqueous phase and larval debris at the interface; if contamination occurs, repeat centrifugation step and recollect. Discard the upper, aqueous phase; it may be helpful to remove and discard this phase before collecting the organic phase. The sample can be stored at -80 &#176;C for up to 1 month.</li>
		<li>Dry the organic phase under vacuum (0.12 atm) while protecting from light. Do not continue to dry the samples after the liquid has evaporated as it will reduce lipid solubility. Resuspend the lipids in 2:1 (chloroform:methanol) and store on ice. The optimal volume of the chloroform:methanol solution depends on the detection method used; start low and continue to dilute as needed. A general guideline is to use 10 &#181;l for 10 pooled larvae.</li>
	</ol>
	</li>
	<li>Spot the entire sample volumes in evenly spaced intervals onto a channeled TLC plate and scan the plate with a laser scanner routinely used for biomolecular imaging (e.g., a fluorescent plate reader). For the fluorescent lipid analogs included in the materials list that have an excitation maxima from 500-650 nm and emission maxima from 510-665 nm, use a 488 nm laser and with emission collection at 520 nm.
	<ol>
		<li>Quantify the total fluorescence of each spot with imaging software. 
		Note: Correct for naturally occurring background lipid fluorescence by subtracting the fluorescence of paired, unfed larval samples.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The techniques described here allow researchers to treat larval zebrafish with a lipid-rich feed, visualize dietary lipid processing in live larvae, and quantify larval food intake. To ensure success, special attention should be given to several critical steps. Commercial chicken eggs vary; to minimize potential variability we perform all assays on organic eggs from cage-free chickens that have not been enriched for omega-3 fatty acids. Lower feeding rates may be observed in fish younger than 6 dpf with remaining endogen...

The mechanisms by which the intestine regulates dietary lipid processing, the liver controls complex lipid synthesis and lipoprotein metabolism, and how these organs work with the central nervous system to control food intake are incompletely understood. It is of biomedical interest to elucidate this biology in light of the current epidemics of obesity, cardiovascular disease, diabetes, and non-alcoholic fatty liver disease. Studies in cell culture and mice have provided the majority of our understanding of the mechanistic relationships between dietary lipids and disease, and zebrafish (Danio rerio) are emerging as an ideal model to complement this work.
Zebrafish have similar gastrointestinal (GI) organs, lipid metabolism, and lipoprotein transport to higher vertebrates 1,2, develop rapidly, and are genetically tractable. The optical clarity of the larval zebrafish facilitates in vivo studies, a particular advantage for study of the GI system as its extracellular milieu (i.e., bile, microbiota, endocrine signaling) is virtually impossible to model ex vivo. In accordance, a body of research combining the genetic tractability and conduciveness to live imaging of zebrafish larvae with a variety of dietary manipulations (high-fat3,4, -cholesterol5, and -carbohydrate diets6,7), and models of cardiovascular disease8, diabetes9,10, hepatic steatosis11-13, and obesity14-16, are emerging to provide a host of metabolic insights.
An essential aspect of transitioning the larval zebrafish into metabolic research is the optimization of techniques developed in other model animals to the zebrafish and the development of novel assays that exploit the unique strengths of the zebrafish. This protocol presents techniques developed and optimized to feed larval zebrafish a lipid-rich meal, visualize dietary lipid processing from whole body to subcellular resolution, and measure food intake. Chicken egg yolk was chosen to compose the lipid-rich meal as it contains high levels of fats and cholesterol (lipids compose ~58% of chicken egg yolk, of which ~5% is cholesterol, 60% are triglycerides, and 35% are phospholipids). Chicken egg yolk provides more fat than typical commercial zebrafish micropellet foods (~15% lipids) and the advantage that it is a standardized feed with known percentages of specific fatty acids species, as zebrafish diets and feeding regiments have not been standardized across labs17. Moreover, fluorescent lipid analogs provided in the egg yolk visualize transport and accumulation of dietary lipids18, image cellular components including lipid droplets by acting both as vital dyes3 and through covalent incorporation into complex lipids, investigate metabolism through thin layer chromatography (TLC)19 and high performance liquid chromatography (HPLC) (S.A.F. unpublished data), and provide a quantitative assay for total food intake20.

<table border="0" cellpadding="0" cellspacing="0" width="832">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">Tricaine (ethyl 3-aminobenzoate methanesulofnate salt)</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:127px;">A5040-25G</td>
			<td style="width:335px;">Anesthesia for larval zebrafish</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Chicken eggs</td>
			<td style="width:143px;">N/A</td>
			<td style="width:127px;">N/A</td>
			<td>Organic, cage-free eggs, not enriched for omege-3 fatty acids</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Ultrasonic processor 3000 sonicator</td>
			<td style="width:143px;">Misonix, Inc.</td>
			<td style="width:127px;">S-3000</td>
			<td>To make egg yolk liposomes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Sonabox acoustic enclosure</td>
			<td style="width:143px;">Misonix, Inc.</td>
			<td style="width:127px;">432B</td>
			<td>To make egg yolk liposomes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">1/8&#8221; tapered microtip</td>
			<td style="width:143px;">Misonix, Inc.</td>
			<td style="width:127px;">419</td>
			<td>To make egg yolk liposomes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Amber vials (4 ml, glass)</td>
			<td style="width:143px;">National Scientific</td>
			<td style="width:127px;">13-425</td>
			<td>Lipid storage; includes vials, open-top caps, and cap septa</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Incu-Shaker Mini&#160;</td>
			<td style="width:143px;">Benchmark</td>
			<td style="width:127px;">1222U12</td>
			<td>Incubated shaker for feeds</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">BODIPY FL C16&#160;</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">D3821</td>
			<td>Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">BODIPY FL C12&#160;</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">D3822</td>
			<td>Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">BODIPY FL C5&#160;</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">D3834</td>
			<td>Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Pentanoic Acid)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">BODIPY FL C5</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">D2183</td>
			<td>Fluorescent lipid analog; (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Propionic Acid)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">TopFluor cholesterol&#160;</td>
			<td style="width:143px;">Avanti Polar Lipids Inc.</td>
			<td style="width:127px;">810255</td>
			<td>Fluorescent lipid analog; 23-(dipyrrometheneboron difluoride)-24-norcholesterol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fatty acid-free BSA</td>
			<td>Sigma-Aldrich</td>
			<td style="width:127px;">A0281-1G</td>
			<td>For TopFluor cholesterol solubilization</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Methyl cellulose</td>
			<td>Sigma-Aldrich</td>
			<td style="width:127px;">M0387</td>
			<td>Mounting media for live larval imaging; 75 x 25 x 1 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">Low melt agarose</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">BP165-25</td>
			<td>Mounting media for live larval imaging; 22 x 30</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">VWR microscope slides&#160;</td>
			<td style="width:143px;">VWR&#160;</td>
			<td style="width:127px;">16004-422</td>
			<td>Mounting larvae for live imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Coverslips&#160;</td>
			<td style="width:143px;">Cover Glass</td>
			<td style="width:127px;">12-544A</td>
			<td>Mounting larvae for live imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Super glue</td>
			<td style="width:143px;">Loctite</td>
			<td style="width:127px;">LOC01-30379</td>
			<td>Mounting larvae for live imaging</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">FluoroDish (glass bottom dish)</td>
			<td style="width:143px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:127px;">FD35-100</td>
			<td>Mounting larvae for live imaging; 35 mm dish, 23 mm glass, 0.17 mm glass thickness&#160;&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Confocal microscope</td>
			<td>Leica Microsytems</td>
			<td style="width:127px;">SP-2, SP-5</td>
			<td>Microscope for high magnification live imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Stereoscope</td>
			<td style="width:143px;">Nikon</td>
			<td style="width:127px;">SM21500</td>
			<td>Microscope for low magnification live imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Glass culture tubes&#160;</td>
			<td style="width:143px;">Kimble</td>
			<td style="width:127px;">73500-13100</td>
			<td>Lipid extraction; (13 x 100 mm; 13 ml)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Savant SpeedVac Plus&#160;</td>
			<td style="width:143px;">ThermoQuest</td>
			<td style="width:127px;">SC210A</td>
			<td>Lipid extraction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Channeled TLC plates</td>
			<td style="width:143px;">Whatman Scientific</td>
			<td style="width:127px;">WC4855-821</td>
			<td>Food intake assay; LK5D Silica Gel 150 A, 20 x 20 cm, 250 um thick; Discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">Channeled TLC plates</td>
			<td style="width:143px;">Analtech, Inc.</td>
			<td style="width:127px;">66911</td>
			<td>Food intake assay; Direct replacement for Whatman Scientific TLC plates</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Typhoon 9410 Variable Mode Imager</td>
			<td style="width:143px;">GE Healthcare</td>
			<td style="width:127px;">9410</td>
			<td>Fluorescent plate reader for food intake assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:231px;">ImageQuant software</td>
			<td style="width:143px;">GE Healthcare</td>
			<td>29000605</td>
			<td>Analysis of food intake assay</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">5 3/4&#8217; Wide bore, borosilicate disposable pasteur pipets&#160;&#160;&#160;</td>
			<td style="width:143px;">Kimble</td>
			<td style="width:127px;">63A53WT</td>
			<td>Transfering larvae</td>
		</tr>
	</tbody>
</table>

high-fat feeding paradigm for larval zebrafish: feeding, live imaging, and quantification of food intake

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that larvae that fail to feed will not confound experimental results. The chicken egg yolk liposomes can be spiked with fluorescent lipid analogs, including fatty acids and cholesterol, enabling both systemic and subcellular visualization of dietary lipid processing. Several methods are described to mount larvae that are conducive to short- and long-term live imaging with both upright and inverted objectives at high and low magnification. Additionally presented is an assay to quantify larval food intake by extracting the lipids of larvae fed fluorescent lipid analogs, spotting the lipids on a thin layer chromatography plate, and quantifying the fluorescence. Finally, critical aspects of the procedures, important controls, options for modifying the protocols to address specific experimental questions, and potential limitations are discussed. These techniques can be applied not only to focused, hypothesis driven inquiries, but also to a variety of screens and live imaging techniques to study dietary lipid metabolism and the control of food intake.

When fed on a rocker at 29-31 &#176;C, the majority of healthy larvae (&#8805;95%) will eat within 1 hr. Upon consuming the egg yolk emulsion, the larval intestine darkens in color. Very dark intestines can be observed at 2 hr (Figure 1). If larvae are unfed or fail to feed, the intestine remains clear. Larvae fed egg white exhibit a distended intestinal lumen that does not darken in color.
<img alt="Figure 1" src="/files/ftp_upload/54735/54735...

Watch this Scientific Journal Video about High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake at JoVE.com

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques.

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich ...

high-fat-feeding-paradigm-for-larval-zebrafish-feeding-live-imaging

Research

JoVE Journal

Biology

10.4K Views.  Carnegie Institution for Science. Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques. 

Video: High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been conducted to investigate the effect of gut microbes as a whole; however, different microbes could exert distinct effects toward the host. This article provides the methodology to study the effect of each specific mosquito gut microbe and the potential mechanism.
This protocol contains two parts. The first part introduces how to dissect the mosquito midgut, isolate cultivable bacteria colonies, and identify bacteria species. The second part provides the procedure to generate antibiotic-treated mosquitoes and reintroduce one specific bacteria species.

This article presents a protocol to investigate the effect of individual mosquito gut bacteria, including isolation and identification of mosquito midgut cultivable microbes, antibiotic depletion of mosquito gut bacteria, and reintroduce one specific bacteria species.

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been ...

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease transmitted through an infectious bite from the female Anopheles mosquito. Malaria control will eventually rely on a multitude of approaches, which includes ways to block transmission to, through and from mosquitoes. To study mosquito stages of malaria parasites in the laboratory, we have optimized a protocol to culture highly infectious Plasmodium falciparum gametocytes, a parasite stage required for transmission from the human host to the mosquito vector. P. falciparum gametocytes mature through five morphologically distinct steps, which takes approximately 1-2 weeks. Gametocyte culture described in this protocol is completed in 15 days and are infectious to mosquitoes from days 15-18. These protocols were developed to maintain a continuous cycle of infection competent gametocytes and to maintain uninterrupted supply of mosquito stages of the parasite. Here, we describe the methodology of gametocyte culture and how to infect mosquitoes with these parasites using glass membrane feeders.

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Detailed investigations on mosquito stages of malaria parasites are critical to design effective transmission blocking strategies. This protocol demonstrates how to effectively culture infectious gametocytes and then feed these gametocytes to mosquitoes to generate mosquito stages of P. falciparum.

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease ...

Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment failure is mainly due to the invasiveness of GBM cells, which speaks for the need for a better understanding of GBM motile properties. To investigate the molecular mechanism supporting GBM invasion, new physiological models enabling in-depth characterization of protein dynamics during invasion are required. These observations would pave the way to the discovery of novel targets to block tumor infiltration and improve patient outcomes. This paper reports how an orthotopic xenograft of GBM cells in the zebrafish brain permits subcellular intravital live imaging. Focusing on microtubules (MTs), we describe a procedure for MT labeling in GBM cells, microinjecting GBM cells in the transparent brain of 3 days post fertilization (dpf) zebrafish larvae, intravital imaging of MTs in the disseminating xenografts, altering MT dynamics to assess their role during GBM invasion, and analyzing the acquired data.

We report a technique permitting live imaging of microtubule dynamics in glioblastoma (GBM) cells invading a vertebrate brain tissue. Coupling the orthotopic injection of fluorescently tagged GBM cells into a transparent zebrafish brain with high-resolution intravital imaging allows the measurement of cytoskeleton dynamics during in situ cancer invasion.

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment ...