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>
	<li>Kuhlman, J., Eisen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+screen+for+mutations+affecting+development+and+function+of+the+enteric+nervous+system.">Genetic screen for mutations affecting development and function of the enteric nervous system.</a> Dev. Dyn. 236, 118-127 (2007).</li><li>Pack, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+development+of+zebrafish+digestive+organs.">Mutations affecting development of zebrafish digestive organs.</a> Development. 123, 321-328 (1996).</li><li>Wallace, K. N., Pack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+and+conserved+aspects+of+gut+development+in+zebrafish.">Unique and conserved aspects of gut development in zebrafish.</a> Dev. Biol. 255, 12-29 (2003).</li><li>Wallace, K. N., Akhter, S., Smith, E. M., Lorent, K., Pack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+growth+and+differentiation+in+zebrafish.">Intestinal growth and differentiation in zebrafish.</a> Mech. Dev. 122, 157-173 (2005).</li><li>Ng, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+digestive+system+in+zebrafish:+III.+Intestinal+epithelium+morphogenesis.">Formation of the digestive system in zebrafish: III. Intestinal epithelium morphogenesis.</a> Dev. Biol. 286, 114-135 (2005).</li><li>Hama, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+zebrafish+digestive+organ+function+using+multiple+quenched+fluorescent+reporters.">In vivo imaging of zebrafish digestive organ function using multiple quenched fluorescent reporters.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 296, 445-453 (2009).</li><li>Carten, J. D., Bradford, M. K., Farber, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+digestive+organ+morphology+and+function+using+differential+fatty+acid+metabolism+in+live+zebrafish.">Visualizing digestive organ morphology and function using differential fatty acid metabolism in live zebrafish.</a> Dev. Biol. 360, 276-285 (2011).</li><li>Rich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+high-content+model+system+for+studies+of+gastrointestinal+transit:+the+zebrafish.">A new high-content model system for studies of gastrointestinal transit: the zebrafish.</a> Neurogastroenterol Motil. 21, 225-228 (2009).</li><li>Bagnat, M., Cheung, I. D., Mostov, K. E., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+control+of+single+lumen+formation+in+the+zebrafish+gut.">Genetic control of single lumen formation in the zebrafish gut.</a> Nat. Cell Biol. 9, 954-960 (2007).</li><li>Bagnat, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cse1l+is+a+negative+regulator+of+CFTR-dependent+fluid+secretion.">Cse1l is a negative regulator of CFTR-dependent fluid secretion.</a> Curr. Biol. 20, 1840-1845 (2010).</li><li>Shepherd, I., Eisen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+zebrafish+enteric+nervous+system.">Development of the zebrafish enteric nervous system.</a> Methods Cell Biol. 101, 143-160 (2011).</li><li>Brugman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxazolone-induced+enterocolitis+in+zebrafish+depends+on+the+composition+of+the+intestinal+microbiota.">Oxazolone-induced enterocolitis in zebrafish depends on the composition of the intestinal microbiota.</a> Gastroenterology. 137, 1757-1767 (2009).</li><li>Oehlers, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammatory+bowel+disease+(IBD)+susceptibility+genes+NOD1+and+NOD2+have+conserved+anti-bacterial+roles+in+zebrafish.">The inflammatory bowel disease (IBD) susceptibility genes NOD1 and NOD2 have conserved anti-bacterial roles in zebrafish.</a> Dis. Model Mech. 4, 832-841 (2011).</li><li>Oehlers, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+enterocolitis+model+in+zebrafish+larvae+that+is+dependent+on+microbiota+and+responsive+to+pharmacological+agents.">A chemical enterocolitis model in zebrafish larvae that is dependent on microbiota and responsive to pharmacological agents.</a> Dev Dyn. 240, 288-298 (2011).</li><li>Faro, A., Boj, S. F., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fishing+for+intestinal+cancer+models:+unraveling+gastrointestinal+homeostasis+and+tumorigenesis+in+zebrafish.">Fishing for intestinal cancer models: unraveling gastrointestinal homeostasis and tumorigenesis in zebrafish.</a> Zebrafish. 6, 361-376 (2009).</li><li>Fleming, A., Jankowski, J., Goldsmith, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+gut+function+and+disease+changes+in+a+zebrafish+larvae+model+of+inflammatory+bowel+disease:+a+feasibility+study.">In vivo analysis of gut function and disease changes in a zebrafish larvae model of inflammatory bowel disease: a feasibility study.</a> Inflamm. Bowel Dis. 16, 1162-1172 (2010).</li><li>Kanther, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+colonization+induces+dynamic+temporal+and+spatial+patterns+of+NF-kappaB+activation+in+the+zebrafish+digestive+tract.">Microbial colonization induces dynamic temporal and spatial patterns of NF-kappaB activation in the zebrafish digestive tract.</a> Gastroenterology. , (2011).</li><li>Rawls, J. F., Samuel, B. S., Gordon, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gnotobiotic+zebrafish+reveal+evolutionarily+conserved+responses+to+the+gut+microbiota.">Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 4596-4601 (2004).</li><li>Rawls, J. F., Mahowald, M. A., Goodman, A. L., Trent, C. M., Gordon, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+and+genetic+analysis+link+bacterial+motility+and+symbiosis+in+the+zebrafish+gut.">In vivo imaging and genetic analysis link bacterial motility and symbiosis in the zebrafish gut.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 7622-7627 (2007).</li><li>Kanther, M., Rawls, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-microbe+interactions+in+the+developing+zebrafish.">Host-microbe interactions in the developing zebrafish.</a> Curr. Opin. Immunol. 22, 10-19 (2010).</li><li>Bates, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+signals+from+the+microbiota+promote+different+aspects+of+zebrafish+gut+differentiation.">Distinct signals from the microbiota promote different aspects of zebrafish gut differentiation.</a> Dev. Biol. 297, 374-386 (2006).</li><li>Cheesman, S. E., Guillemin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=We+know+you+are+in+there:+conversing+with+the+indigenous+gut+microbiota.">We know you are in there: conversing with the indigenous gut microbiota.</a> Res Microbiol. 158, 2-9 (2007).</li><li>Bates, J. M., Akerlund, J., Mittge, E., Guillemin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+alkaline+phosphatase+detoxifies+lipopolysaccharide+and+prevents+inflammation+in+zebrafish+in+response+to+the+gut+microbiota.">Intestinal alkaline phosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish in response to the gut microbiota.</a> Cell Host Microbe. 2, 371-382 (2007).</li><li>Camp, J. G., Jazwa, A. L., Trent, C. M., Rawls, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intronic+cis-regulatory+modules+mediate+tissue-specific+and+microbial+control+of+angptl4/fiaf+transcription.">Intronic cis-regulatory modules mediate tissue-specific and microbial control of angptl4/fiaf transcription.</a> PLoS Genetics. , (2012).</li><li>Cheesman, S. E., Neal, J. T., Mittge, E., Seredick, B. M., Guillemin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+proliferation+in+the+developing+zebrafish+intestine+is+regulated+by+the+Wnt+pathway+and+microbial+signaling+via+Myd88.">Epithelial cell proliferation in the developing zebrafish intestine is regulated by the Wnt pathway and microbial signaling via Myd88.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 4570-4577 (2011).</li><li>Field, H. A., Kelley, K. A., Martell, L., Goldstein, A. M., Serluca, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+gastrointestinal+physiology+using+a+novel+intestinal+transit+assay+in+zebrafish.">Analysis of gastrointestinal physiology using a novel intestinal transit assay in zebrafish.</a> Neurogastroenterol. Motil. 21, 304-312 (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book. , (2000).</li><li>Pham, L. N., Kanther, M., Semova, I., Rawls, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+generating+and+colonizing+gnotobiotic+zebrafish.">Methods for generating and colonizing gnotobiotic zebrafish.</a> Nat. Protoc. 3, 1862-1875 (2008).</li><li>Shen, L., Weber, C. R., Raleigh, D. R., Yu, D., Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+junction+pore+and+leak+pathways:+a+dynamic+duo.">Tight junction pore and leak pathways: a dynamic duo.</a> Annu. Rev. Physiol. 73, 283-309 (2011).</li><li>Van Itallie, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+density+of+small+tight+junction+pores+varies+among+cell+types+and+is+increased+by+expression+of+claudin-2.">The density of small tight junction pores varies among cell types and is increased by expression of claudin-2.</a> J. Cell. Sci. 121, 298-305 (2008).</li><li>Van Itallie, C. M., Anderson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+size-dependent+permeability+of+the+tight+junction+using+PEG+profiling.">Measuring size-dependent permeability of the tight junction using PEG profiling.</a> Methods Mol. Biol. 762, 1-11 (2011).</li><li>Watson, C. J., Rowland, M., Warhurst, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+modeling+of+tight+junctions+in+intestinal+cell+monolayers+using+polyethylene+glycol+oligomers.">Functional modeling of tight junctions in intestinal cell monolayers using polyethylene glycol oligomers.</a> Am. J. Physiol. Cell Physiol. 281, 388-397 (2001).</li><li>Rodgers, L. S., Fanning, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+epithelial+permeability+by+the+actin+cytoskeleton.">Regulation of epithelial permeability by the actin cytoskeleton.</a> Cytoskeleton (Hoboken). 68, 653-660 (2011).</li><li>Gonzalez-Mariscal, L., Chavez de Ramirez, B., Cereijido, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+junction+formation+in+cultured+epithelial+cells+(MDCK).">Tight junction formation in cultured epithelial cells (MDCK).</a> J. Membr. Biol. 86, 113-125 (1985).</li><li>Palant, C. E., Duffey, M. E., Mookerjee, B. K., Ho, S., Bentzel, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++regulation+of+tight-junction+permeability+and+structure+in+Necturus+gallbladder.">Ca2+ regulation of tight-junction permeability and structure in Necturus gallbladder.</a> Am. J. Physiol. 245, C203-C212 (1983).</li><li>Holmberg, A., Schwerte, T., Pelster, B., Holmgren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+the+gut+motility+control+system+in+zebrafish+Danio+rerio+embryos+and+larvae.">Ontogeny of the gut motility control system in zebrafish Danio rerio embryos and larvae.</a> J. Exp. Biol. 207, 4085-4094 (2004).</li><li>Rich, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kit-like+immunoreactivity+in+the+zebrafish+gastrointestinal+tract+reveals+putative.">Kit-like immunoreactivity in the zebrafish gastrointestinal tract reveals putative.</a> 236, 903-911 (2007).</li><li>Holmberg, A., Olsson, C., Hennig, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TTX-sensitive+and+TTX-insensitive+control+of+spontaneous+gut+motility+in+the+developing+zebrafish+(Danio+rerio)+larvae.">TTX-sensitive and TTX-insensitive control of spontaneous gut motility in the developing zebrafish (Danio rerio) larvae.</a> J. Exp. Biol. 210, 1084-1091 (2007).</li><li>Hennig, G. W., Costa, M., Chen, B. N., Brookes, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+peristalsis+in+the+guinea-pig+small+intestine+using+spatio-temporal+maps.">Quantitative analysis of peristalsis in the guinea-pig small intestine using spatio-temporal maps.</a> J. Physiol. 517 (Pt 2), 575-590 (1999).</li><li>Flynn, E. J., Trent, C. M., Rawls, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+and+nutritional+control+of+adipogenesis+in+zebrafish+(Danio+rerio.">Ontogeny and nutritional control of adipogenesis in zebrafish (Danio rerio.</a> J. Lipid Res. 50, 1641-1652 (2009).</li><li>Clifton, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+inhibitors+of+dietary+lipid+absorption+using+zebrafish.">Identification of novel inhibitors of dietary lipid absorption using zebrafish.</a> PLoS One. 5, e12386 (2010).</li><li>Jin, S. W., Beis, D., Mitchell, T., Chen, J. N., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+analyses+of+vascular+tube+and+lumen+formation+in+zebrafish.">Cellular and molecular analyses of vascular tube and lumen formation in zebrafish.</a> Development. 132, 5199-5209 (2005).</li><li>Berghmans, S., Hunt, J., Roach, A., Goldsmith, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+offer+the+potential+for+a+primary+screen+to+identify+a+wide+variety+of+potential+anticonvulsants.">Zebrafish offer the potential for a primary screen to identify a wide variety of potential anticonvulsants.</a> Epilepsy Res. 75, 18-28 (2007).</li></ol>

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...

Watch this Scientific Journal Video about Microgavage of Zebrafish Larvae at JoVE.com

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.

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

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