Name: high resolution whole mount in situ hybridization within zebrafish embryos to study gene expression and function
Uploaded: 2013-10-19T18:30:00
Description: Watch this Scientific Journal Video about High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function at JoVE.com

1. Whole-mount In Situ Hybridization of Zebrafish Embryos
1.1 Preparation of Embryos
<ol>
	<li>Collect embryos at the required developmental stages. Please see Kimmel et al.5 for embryonic stage description.</li>
	<li>Remove chorions manually using forceps (Dumont Watchmakers Forceps no. 5).</li>
	<li>Fix embryos in a 4% solution of paraformaldehyde (PFA) made up in PBS (Phosphate Buffered Saline) overnight at 4 &#176;C.</li>
	<li>Wash embryos with PBS at least 3x for 5 min ea...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+and+reverse+genetic+approaches+for+the+analysis+of+vertebrate+development+in+the+zebrafish.">Forward and reverse genetic approaches for the analysis of vertebrate development in the zebrafish.</a> Dev. Cell. 21, 48-64 (2011).</li><li>Arkhipova, V., 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=Characterization+and+regulation+of+the+hb9/mnx1+beta-cell+progenitor+specific+enhancer+in+zebrafish.">Characterization and regulation of the hb9/mnx1 beta-cell progenitor specific enhancer in zebrafish.</a> Dev. Biol. 365, 290-302 (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203, 253-310 (1995).</li><li>Chitramuthu, B. P., 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=Molecular+cloning+and+embryonic+expression+of+zebrafish+PCSK5+co-orthologues:+functional+assessment+during+lateral+line+development.">Molecular cloning and embryonic expression of zebrafish PCSK5 co-orthologues: functional assessment during lateral line development.</a> Dev. Dyn. 239, 2933-2946 (2010).</li><li>Liao, E. C., 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=SCL/Tal-1+transcription+factor+acts+downstream+of+cloche+to+specify+hematopoietic+and+vascular+progenitors+in+zebrafish.">SCL/Tal-1 transcription factor acts downstream of cloche to specify hematopoietic and vascular progenitors in zebrafish.</a> Genes Dev. 12, 621-626 (1998).</li><li>Stainier, D. Y., Weinstein, B. M., Detrich, H. W., Zon, L. I., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloche,+an+early+acting+zebrafish+gene,+is+required+by+both+the+endothelial+and+hematopoietic+lineages.">Cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages.</a> Development. 121, 3141-3150 (1995).</li><li>Detrich, H. W., 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=Intraembryonic+hematopoietic+cell+migration+during+vertebrate+development.">Intraembryonic hematopoietic cell migration during vertebrate development.</a> Proc. Natl. Acad. Sci. U.S.A. 92, 10713-10717 (1995).</li><li>Liao, W., 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+zebrafish+gene+cloche+acts+upstream+of+a+flk-1+homologue+to+regulate+endothelial+cell+differentiation.">The zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial cell differentiation.</a> Development. , 124-381 (1997).</li><li>Krauss, S., Concordet, J. P., Ingham, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functionally+conserved+homolog+of+the+Drosophila+segment+polarity+gene+hh+is+expressed+in+tissues+with+polarizing+activity+in+zebrafish+embryos.">A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos.</a> Cell. 75, 1431-1444 (1993).</li><li>Akimenko, M. A., Ekker, M., Wegner, J., Lin, W., 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=Combinatorial+expression+of+three+zebrafish+genes+related+to+distal-less:+part+of+a+homeobox+gene+code+for+the+head.">Combinatorial expression of three zebrafish genes related to distal-less: part of a homeobox gene code for the head.</a> J. Neurosci. 14, 3475-3486 (1994).</li><li>Alexander, J., Rothenberg, M., Henry, G. L., 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=casanova+plays+an+early+and+essential+role+in+endoderm+formation+in+zebrafish.">casanova plays an early and essential role in endoderm formation in zebrafish.</a> Dev. Biol. 215, 343-357 (1999).</li><li>Milewski, W. M., Duguay, S. J., Chan, S. J., Steiner, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+PDX-1+structure,+function,+and+expression+in+zebrafish.">Conservation of PDX-1 structure, function, and expression in zebrafish.</a> Endocrinology. 139, 1440-1449 (1998).</li><li>Argenton, F., Walker, M. D., Colombo, L., Bortolussi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+the+trout+insulin+promoter:+implications+for+fish+as+a+favorable+model+of+pancreas+development.">Functional characterization of the trout insulin promoter: implications for fish as a favorable model of pancreas development.</a> FEBS Lett. 407, 191-196 (1997).</li><li>Biemar, F., 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=Pancreas+development+in+zebrafish:+early+dispersed+appearance+of+endocrine+hormone+expressing+cells+and+their+convergence+to+form+the+definitive+islet.">Pancreas development in zebrafish: early dispersed appearance of endocrine hormone expressing cells and their convergence to form the definitive islet.</a> Dev. Biol. 230, 189-203 (2001).</li><li>Thisse, C., Thisse, B., Schilling, T. F., Postlethwait, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+zebrafish+snail1+gene+and+its+expression+in+wild-type,+spadetail+and+no+tail+mutant+embryos.">Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos.</a> Development. 119, 1203-1215 (1993).</li><li>Cadieux, B., Chitramuthu, B. P., Baranowski, D., Bennett, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+progranulin+gene+family+and+antisense+transcripts.">The zebrafish progranulin gene family and antisense transcripts.</a> BMC Genomics. 6, 156 (2005).</li><li>Chitramuthu, B. P., Bennett, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+zebrafish+and+knockdown+technology+to+define+proprotein+convertase+activity.">Use of zebrafish and knockdown technology to define proprotein convertase activity.</a> Methods Mol. Biol. 768, 273-296 (2011).</li><li>Thisse, C., Thisse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+in+situ+hybridization+on+whole-mount+zebrafish+embryo.">High resolution in situ hybridization on whole-mount zebrafish embryo.</a> Nat. Protoc. 3, 59-69 (2008).</li></ol>

We have developed improved methods to visualize RNA with high resolution. The in situ hybridization procedures are carried out using simple custom-made baskets with porous bottoms (Figure 1). Embryos are processed using RNase-free solutions in 6-well plates at room temperature under sterile conditions.
To segregate embryos baskets are made from different colored Eppendorf tubes. Baskets with or without a rim are used for easy orientation and to identify the embryos wi...

The zebrafish (Danio rerio) has emerged as a powerful animal model for the study of vertebrate development, disease, behavior, and in-drug screening1-3. Zebrafish embryos can be obtained in large numbers from a single crossing. Fertilization and development occurs ex utero and the optically clear embryos develop rapidly. Critical developmental events occur during the first 48 hr post-fertilization (hpf). This includes the appearance of organ primordia and the initiation of cytodifferentiation. Knockdown or over-expression of proteins can be achieved through the microinjection of embryos with antisense morpholino (MO) oligonucleotides or mRNA respectively at the one or two cell stage (0.75 hpf).
The WISH of zebrafish embryos facilitates the study of the spatio-temporal expression of specific genes of interest. Application of the WISH technique following microinjection of embryos with mRNA or MO to over-express or knockdown specific protein levels reveals differential regulation of other genes.
Changes in gene expression patterns can be correlated with phenotypic changes and reveal the function of target genes during early organogenesis. Since probes can be prepared and stored in advance, the ISH technique can be applied to reveal gene expression patterns for at least 20 genes at a time using six-well plates and custom made baskets.

<table border="0" cellpadding="0" cellspacing="0" width="1117">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:440px;">Taq Polymerase</td>
			<td style="width:159px;">Invitrogen</td>
			<td align="right" style="width:137px;">10342053</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TopoTA Cloning Kit- Dual Promoter pCRII-TOPO</td>
			<td>Invitrogen</td>
			<td>K4650-01</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HQ Mini Plasmid Purification Kit</td>
			<td>Invitrogen</td>
			<td>K2100-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Roche</td>
			<td style="width:137px;">11 685 660 001</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Not1 and HindIII Restriction Enzymes</td>
			<td>Invitrogen</td>
			<td>15441-025, 15207-012</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;">Buffer Saturated Phenol, ultrapure</td>
			<td>Invitrogen</td>
			<td align="right">15513039</td>
			<td>Store at 4 &#176;C. Eyes, skin, and respiratory tract irritant and suspected carcinogen. Care should be taken while handling.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Chloroform</td>
			<td>Fisher</td>
			<td>C607-1</td>
			<td>Toxic and suspected carcinogen. Work under fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase-free DNAse I</td>
			<td>Roche</td>
			<td align="right">4716728001</td>
			<td>Prepare small aliquots and store at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DIG-RNA Labeling Mix with Sp6, T7 and T3 RNA Polymerase</td>
			<td>Roche</td>
			<td align="right">11277073910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase Inhibitor</td>
			<td>Roche</td>
			<td>10777-019</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3.0 M Sodium Acetate (pH 5.5)</td>
			<td>Fisher</td>
			<td>50-751-7355</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">100% Ethanol</td>
			<td>Commercial alcohols</td>
			<td style="width:137px;"></td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Diethyl Pyrocarbonate (DEPC)</td>
			<td>Sigma</td>
			<td>40718-25ML</td>
			<td style="width:383px;">DEPC is an eye, skin, and respiratory irritant. Avoid contact with skin and eyes. Use safety glasses, gloves, and mask.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH</td>
			<td>Fisher</td>
			<td>SS255-1</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA 0.5 M (pH 8.0)</td>
			<td>Fisher</td>
			<td>50-751-7404</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Instant Ocean Salt</td>
			<td>Aquarium Systems</td>
			<td style="width:137px;">N/A</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline (PBS)</td>
			<td>Fisher</td>
			<td style="width:137px;">FL-03-0900</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Paraformaldehyde (PFA)</td>
			<td>Sigma</td>
			<td>P6148-1KG</td>
			<td>Prepare and store at -20&#176;C as 40 ml aliquots for later use. PFA is toxic. Use safety glasses, gloves, and dust mask.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Phenylthiocarbomide (PTC)</td>
			<td>Sigma</td>
			<td>P7629-25G</td>
			<td>PTC is highly toxic. Use safety glasses, gloves, and dust mask.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Fisher</td>
			<td>A947-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont (Watchmaker&#8217;s) Forceps pattern no. 5</td>
			<td>Fine Science Tools</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Six-well Cell Culture Cluster</td>
			<td>Sarstedt</td>
			<td align="right" style="width:137px;">83.1839</td>
			<td></td>
		</tr>
		<tr height="182">
			<td height="182" style="height:182px;">Baskets are made of nylon mesh and Eppendorf tubes</td>
			<td>In-house preparation</td>
			<td style="width:137px;"></td>
			<td style="width:383px;">To make the baskets, cut Eppendorf tubes (1.5 ml) with or without rims to remove the conical end. Cut nylon mesh into small pieces corresponding to the size of&#160;the cut ends of the Eppendorf tubes. Place tubes with the nylon mesh covering the cut end on an electrical&#160;hot plate until both the tube and nylon mesh stick together (carry out in fume hood). Cut off the excess mesh from the baskets and store them in 100% methanol until used.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Polyoxyethylenesorbitan Monolaurate (Tween 20)</td>
			<td>Sigma</td>
			<td>P1379-500ML</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td>Fermentas</td>
			<td style="width:137px;">EO0491</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetic Anhydride</td>
			<td>Sigma</td>
			<td align="right" style="width:137px;">242845</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triethanolamine</td>
			<td>Sigma</td>
			<td>90279-100ML</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formamide, high purity grade</td>
			<td>Sigma</td>
			<td style="width:137px;">F9037</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">AG501-X8 Resin</td>
			<td>Bio Rad</td>
			<td style="width:137px;">142-6424</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citric Acid monohydrate</td>
			<td>Sigma</td>
			<td>C0706-500G</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin Sodium Salt</td>
			<td>Sigma</td>
			<td>H3393-25KU</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline-sodium Citrate Buffer (SSC)</td>
			<td>Sigma</td>
			<td style="width:137px;">S6639</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tRNA from bakers yeast type X</td>
			<td>Sigma</td>
			<td>R5636-1ML</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:440px;">NaCl</td>
			<td>Fisher</td>
			<td>S640-500</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl</td>
			<td>Fisher</td>
			<td>BP153-1</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgCl2</td>
			<td>Fisher</td>
			<td>S25403</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Levamisole Hydrochloride</td>
			<td>Sigma</td>
			<td align="right" style="width:137px;">31742</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:440px;">N,N-Dimethylformamide 
			anhydrous (DMF)</td>
			<td>Sigma</td>
			<td align="right" style="width:137px;">227056</td>
			<td style="width:383px;">Irritant, toxic, combustible, and suspected teratogen. Handle with proper safety attire including gloves and goggles.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-Bromo-4-chloro-3-indolyl phosphate (BCIP)</td>
			<td>Sigma</td>
			<td style="width:137px;">N6639</td>
			<td>Protect this solution from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitroblue tetrazolium (NBT)</td>
			<td>Sigma</td>
			<td style="width:137px;">840W</td>
			<td>Protect this solution from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from Bovine Serum, purified fraction V (BSA)</td>
			<td>Sigma</td>
			<td style="width:137px;">A8806</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sheep Anti-digoxigenin-AP Fab Fragments</td>
			<td>Roche</td>
			<td align="right">1093274910</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma</td>
			<td>G5516-500ML</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well cell culture plates</td>
			<td>Sarstedt</td>
			<td align="right" style="width:137px;">83.1835</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tg(mnx1:GFP)ml2/Tg(hb9:GFP)ml2</td>
			<td>ZIRC</td>
			<td style="width:137px;">Tg(mnx1:GFP)ml2</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Instant Ocean</td>
			<td>Aquarium Systems</td>
			<td style="width:137px;">N/A</td>
			<td style="width:383px;"></td>
		</tr>
	</tbody>
</table>

high resolution whole mount in situ hybridization within zebrafish embryos to study gene expression and function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

Using the protocol with 50 embryos per basket (per gene/per experimental condition) the expression pattern of that gene can be achieved in one experiment. Almost all the embryos show similar expression patterns for a particular gene. Representative examples of the in situ hybridization staining are shown in Figures 3-6.
Both the sense and anti-sense riboprobes were synthesized from the cDNAs corresponding to PC5.1, PC5.26, SCL/tal-17, gata-1...

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High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

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Biology

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