Name: microgavage of zebrafish larvae
Uploaded: 2013-02-20T13:00:00
Description: Watch this Scientific Journal Video about Microgavage of Zebrafish Larvae at JoVE.com

We thank members of the Rawls laboratory for helpful suggestions on content and Dr. Alan Fanning for valuable discussions on tight junction size permeability and disruption methods. We also thank Dr. Michael Chua and Dr. Neal Kramarcy of the Michael Hooker Microscopy Facility for confocal microscope support. This work was supported by National Institutes of Health grants T32 DK007737-15 (J.L.C. trainee), F32 DK094592 (to J.L.C.), and R01 DK081426 (to J.F.R.).

We use published protocols for standard zebrafish husbandry and maintenance to obtain larvae for microgavage27. We primarily use natural breeding to propagate fish, and they are kept on a 14 hr light/10 hr dark cycle. This protocol has been optimized for microgavage of zebrafish larvae at 6-7 days post-fertilization (dpf). We anticipate that these methods will be generally applicable to younger and older developmental stages with minor modifications. Unless otherwise noted, we used the wild-type TL zebrafish s...

<ol>
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In this work, we describe a novel protocol for direct delivery of materials to the larval zebrafish intestine by microgavage. There are several critical steps throughout the procedure that should be kept in mind. First, the zebrafish larvae should be in good health before the gavage experiment to prevent death unrelated to treatment. Another important factor is the quality of the gavage needle. One of the most difficult parts of the protocol is clipping the needle at the appropriate location without creating sharp, jagge...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Finquel (Tricaine methanesulfonate)</td>
			<td>Argent Chemical Laboratories</td>
			<td>MS-222</td>
			<td>Larvae anesthesia</td>
		</tr>
		<tr>
			<td>Phenol Red Solution</td>
			<td>Sigma-Aldrich</td>
			<td>P0290-100 ml</td>
			<td>0.5% in DPBS, cell-culture tested</td>
		</tr>
		<tr>
			<td>Mineral oil, light, white (high purity)</td>
			<td>Amresco</td>
			<td>J217-500 ml</td>
			<td>For needle backfill and volume testing</td>
		</tr>
		<tr>
			<td>TxRed-Dextran, 10,000 MW, lysine fixable</td>
			<td>Invitrogen/ Molecular Probes</td>
			<td>D-1863</td>
			<td>1% suspension in 1x PBS/phenol red</td>
		</tr>
		<tr>
			<td>FM4-64FX</td>
			<td>Invitrogen/ Molecular Probes</td>
			<td>F34653</td>
			<td>5 mM stock in water</td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>MP Biochemicals</td>
			<td>0215549590</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Drummond Scientific</td>
			<td>3-000-203-G/X</td>
			<td>OD 1.14 mm, ID 0.53 mm, 3.5 in length</td>
		</tr>
		<tr>
			<td>Plastic form for mold making</td>
			<td>Adaptive Science Tools</td>
			<td>TU-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nanoject II microinjection unit</td>
			<td>Drummond Scientific</td>
			<td>3-000-204</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Flaming Brown Micropipette Puller, 3.0 mm wide-trough filament</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97, FT330B</td>
			<td>Needle fabrication</td>
		</tr>
		<tr>
			<td>Student Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td>0.1 x 0.06 mm, needle clipping</td>
		</tr>
		<tr>
			<td>Eyepiece Graticle, 5 mm - 100 divisions</td>
			<td>Leica</td>
			<td>10394771</td>
			<td>Needle clipping</td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige</td>
			<td>MF-900</td>
			<td>Needle clipping and fire-polishing</td>
		</tr>
		<tr>
			<td>Tuberculin SlipTip syringe needle</td>
			<td>Becton Dickinson</td>
			<td>309626</td>
			<td>1 ml, 25G 5/8-needle</td>
		</tr>
		<tr>
			<td>Fluoresbrite YG Microspheres (2.0 &mu;m)</td>
			<td>Polysciences, Inc.</td>
			<td>18338</td>
			<td>Intestinal motility analysis</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Leica</td>
			<td>S6E</td>
			<td>Gavage procedure</td>
		</tr>
		<tr>
			<td>Fluorescence Stereomicroscope</td>
			<td>Leica</td>
			<td>M205C</td>
			<td>Dextran barrier assay</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM510</td>
			<td>Imaging of dextran in circulation</td>
		</tr>
	</tbody>
</table>

microgavage of zebrafish larvae

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe interactions17-25. Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae26. Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results. We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system.

When gavage is performed properly, the delivered material should be entirely contained within the anterior intestine with little to no residual material in the esophagus (Figure 3). A delivery volume should be chosen that can be accommodated within the anterior bulb and does not leak out through cloaca or esophagus. If the volume of material or pressure of delivery is too high, then the physical force of gavage may lead to damage of or leakage through the epithelium. It may also push out or alter other c...

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Microgavage of Zebrafish Larvae

We present a novel method for microgavage of larval zebrafish utilizing standard embryo microinjection and stereomicroscopy equipment. We demonstrate that microgavage is a safe and efficient technique useful for delivering controlled amounts of diverse materials specifically into the larval zebrafish intestinal lumen.

Microgavage من يرقات اسماك الزرد

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe ...

Research

JoVE Journal

Biology

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تشريح أقراص تخيلي من يرقات ذبابة الفاكهة طور مرحلي 3

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عالية الدقة قياس سلوك رائحة يحركها يرقات ذبابة الفاكهة في

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

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بروتوكولات العدوى عن طريق الفم من اليرقات Lepidopteran مع الفيروسة العصوية

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be ...

Protocols for Microapplicator-assisted Infection of Lepidopteran Larvae with Baculovirus

بروتوكولات لMicroapplicator بمساعدة العدوى من اليرقات Lepidopteran مع الفيروسة العصوية

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. . This video shows how lepidopteran larvae can be ...

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

الكهربية طرق تسجيل الإمكانيات متشابك من يرقات ذبابة الفاكهة من NMJ

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

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في الوريد من يرقات اسماك الزرد Microinjections لدراسة إصابة الكلى الحاد

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

في التصوير من يرقات ذبابة الفاكهة فيفو سليمة الفرعية الخلوية في القرار

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Immunostaining of Dissected Zebrafish Embryonic Heart

Immunostaining تشريح القلب الجنينية اسماك الزرد

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

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في الجسم الحي من Electroporation Morpholinos في شبكية العين اسماك الزرد الكبار

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

في Electroporation المجراة من Morpholinos في الكبار التجديد زعنفة الذيل الزرد

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...