Name: high resolution whole mount in situ hybridization within zebrafish embryos to study gene expression and function
Uploaded: 2013-10-19T18:30:00.000+00:00
Duration: 10 min 6 s
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

<ol>
	<li>Lieschke, G. J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat. Rev. Genet. 8, 353-367 (2007).</li><li>Wolman, M., Granato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+genetics+in+larval+zebrafish:+learning+from+the+young.">Behavioral genetics in larval zebrafish: learning from the young.</a> Dev. Neurobiol. 72, 366-372 (2010).</li><li>Lawson, N. D., Wolfe, 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=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>

Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Biz yüksek çözünürlüklü RNA görselleştirmek için geliştirilmiş yöntemler geliştirdik. In situ hibridizasyon prosedürlerinde gözenekli alt (Şekil 1) ile basit bir ölçüye sepet ile gerçekleştirilmektedir. Embriyolar, steril koşullar altında, oda sıcaklığında 6 oyuklu plakalarda RNase içermeyen çözeltiler kullanılarak işlenir. Ayırmak için embriyolar sepetleri farklı renkli Eppendorf tüpleri yapılır. Ya da bir ağız olmadan Sepetler kolay yönlendirme için kullanılan ve bir sonda olarak tekabül sepet içinde embriyolar tespit etmek. 65 Ön-hibritleme 70 ° C sıcaklık ve% 50 hibridizasyon ° deiyonize formamid C yüksek çözünürlüklü elde etmek için sinyallerin özellikle önemli olduğu bulunmuştur. Buna ek olarak 15 dakika sonrası için PBST ile alkalin Tris tamponu ile iki kere embriyo inkübe bir anti-DIG antikoru ve BCIP varlığında, inkübasyon öncesi ile inkübasyondan sonra yıkar veNBT yüksek kaliteli net sinyallerin görünümünü kolaylaştırır. Bizim ellerde biz 70 en uygun zaman ve hibridizasyon için embriyo permeabilization ve sabitleme ° C net sinyaller elde çok kritik olduğunu yaşadı. Yetersiz geçirgenliği yüksek arka plan sinyalleri üretti. Uzun sabitleme sinyal algılama inhibe ise Genişletilmiş permeabilization veya yetersiz sabitleme embriyoların bozulması sonuçlandı. 55-60 arasında Hibridizasyon ° C de yüksek arka plan sinyalleri (Şekil 7) üretti. Ilgi gen antisens (AS) transkript kodlar ise anlamda prob transkript AS için bir sinyal algılar. Boyama Reaksiyon, 30 dakika boyunca metanol içinde embriyo yerleştirme durdurulduğunda ya da daha uzun bir gerçek mor renge sinyaline dönüştüren ve non-spesifik boyama kaldırır sonra. Sabit embriyolar birkaç ay boyunca 4 ° C&#39;de durdurma çözeltisi karanlıkta saklanabilir. Avantajları ve disadvantagDİLERİZ es DİLERİZ embriyogenez sırasında hedef genlerin zamansal ve mekansal ifade ve larva aşamalarını hem karakterize etmek için etkili bir tekniktir. Bu protokol, aynı zamanda, deney hedeflerine bağlı olarak uygun bir modifikasyon ile iki gen, ya da aynı zamanda RNA ve protein ekspresyonu co-lokalizasyon RNA ifade görselleştirme izin verir. Bu yüksek çözünürlük protokol birçok doku ve hücre tipi içinde RNA ekspresyonu tespit etmek için kullanılabilir. Bu, en az arka plan sinyali ile çok düşük bol RNA türlerinin tespiti için de kullanılabilir. Ancak, dokuların iç bölmeleri içinde genlerin doğru ifade görselleştirmek için doku bölümleri kullanarak in situ hibridizasyon gerektirir.

Zebra balığı (Br.rerio) omurgalı geliştirme, hastalık, davranış çalışma için güçlü bir hayvan modeli olarak ortaya, ve-ilaç tarama 1-3 oldu. Balığı embriyolar, tek bir geçiş yerinden çok sayıda temin edilebilir. Gübreleme ve geliştirme eski rahim ve optik açık embriyoların hızla geliştirmek oluşur. Kritik gelişimsel olaylar ilk 48 saat sonrası fertilizasyon (hpf) meydana gelir. Bu organın taslakları ortaya ve hücre farklılaşması ve başlanması. Proteinlerin demonte veya aşırı ifadesi, morfolino antisens (MO) oligonükleotidlerle embriyoların mikroenjeksiyon yoluyla elde ya da bir ya da iki hücreli aşamada (0,75 HPF) sırasıyla mRNA olabilir. Zebra balığı embriyoların DİLERİZ ilgi belirli genlerin uzay-zamansal ifade çalışma kolaylaştırır. DİLERİZ tekniğin uygulama aşırı ifade için mRNA veya MO ile embriyo mikroenjeksiyon takipess veya demonte özel protein düzeyleri diğer genlerin diferansiyel düzenleme ortaya koymaktadır. Gen ekspresyonu değişiklikleri fenotipik değişikliklerle ilişkili ve erken organogenez sırasında hedef genlerin işlevini ortaya olabilir. Probes hazırlanmış ve önceden depolanmış olabilir, çünkü ISH tekniği altı oyuklu plakalar ve özel yapılmış sepet ile aynı anda en az 20 gen için gen ekspresyonu ortaya çıkarmak için uygulanabilir.

<table><tbody><tr><td>Taq Polymerase</td><td>Invitrogen</td><td>10342053</td><td></td></tr><tr><td>TopoTA Cloning Kit- Dual Promoter pCRII-TOPO</td><td>Invitrogen</td><td>K4650-01</td><td></td></tr><tr><td>HQ Mini Plasmid Purification Kit</td><td>Invitrogen</td><td>K2100-01</td><td></td></tr><tr><td>Agarose</td><td>Roche</td><td>11 685 660 001</td><td></td></tr><tr><td>Not1 and HindIII Restriction Enzymes</td><td>Invitrogen</td><td>15441-025, 15207-012</td><td></td></tr><tr><td>Buffer Saturated Phenol, ultrapure</td><td>Invitrogen</td><td>15513039</td><td>Store at 4 &amp;deg;C. Eyes, skin, and respiratory tract irritant and suspected carcinogen. Care should be taken while handling.</td></tr><tr><td>Chloroform</td><td>Fisher</td><td>C607-1</td><td>Toxic and suspected carcinogen. Work under fume hood.</td></tr><tr><td>RNase-free DNAse I</td><td>Roche</td><td>4716728001</td><td>Prepare small aliquots and store at -20&amp;deg;C.</td></tr><tr><td>DIG-RNA Labeling Mix with Sp6, T7 and T3 RNA Polymerase</td><td>Roche</td><td>11277073910</td><td></td></tr><tr><td>RNase Inhibitor</td><td>Roche</td><td>10777-019</td><td></td></tr><tr><td>3.0 M Sodium Acetate (pH 5.5)</td><td>Fisher</td><td>50-751-7355</td><td></td></tr><tr><td>100% Ethanol</td><td>Commercial alcohols</td><td></td><td></td></tr><tr><td>Diethyl Pyrocarbonate (DEPC)</td><td>Sigma</td><td>40718-25ML</td><td>DEPC is an eye, skin, and respiratory irritant. Avoid contact with skin and eyes. Use safety glasses, gloves, and mask.</td></tr><tr><td>NaOH</td><td>Fisher</td><td>SS255-1</td><td></td></tr><tr><td>EDTA 0.5 M (pH 8.0)</td><td>Fisher</td><td>50-751-7404</td><td></td></tr><tr><td>Instant Ocean Salt</td><td>Aquarium Systems</td><td>N/A</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Fisher</td><td>FL-03-0900</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma</td><td>P6148-1KG</td><td>Prepare and store at -20&amp;deg;C as 40 ml aliquots for later use. PFA is toxic. Use safety glasses, gloves, and dust mask.</td></tr><tr><td>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><td>Methanol</td><td>Fisher</td><td>A947-4</td><td></td></tr><tr><td>Dumont (Watchmaker&amp;rsquo;s) Forceps pattern no. 5</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Six-well Cell Culture Cluster</td><td>Sarstedt</td><td>83.1839</td><td></td></tr><tr><td>Baskets are made of nylon mesh and Eppendorf tubes</td><td>In-house preparation</td><td></td><td>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&amp;nbsp;the cut ends of the Eppendorf tubes. Place tubes with the nylon mesh covering the cut end on an electrical&amp;nbsp;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><td>Polyoxyethylenesorbitan Monolaurate (Tween 20)</td><td>Sigma</td><td>P1379-500ML</td><td></td></tr><tr><td>Proteinase K</td><td>Fermentas</td><td>EO0491</td><td></td></tr><tr><td>Acetic Anhydride</td><td>Sigma</td><td>242845</td><td></td></tr><tr><td>Triethanolamine</td><td>Sigma</td><td>90279-100ML</td><td></td></tr><tr><td>Formamide, high purity grade</td><td>Sigma</td><td>F9037</td><td></td></tr><tr><td>AG501-X8 Resin</td><td>Bio Rad</td><td>142-6424</td><td></td></tr><tr><td>Citric Acid monohydrate</td><td>Sigma</td><td>C0706-500G</td><td></td></tr><tr><td>Heparin Sodium Salt</td><td>Sigma</td><td>H3393-25KU</td><td></td></tr><tr><td>Saline-sodium Citrate Buffer (SSC)</td><td>Sigma</td><td>S6639</td><td></td></tr><tr><td>tRNA from bakers yeast type X</td><td>Sigma</td><td>R5636-1ML</td><td></td></tr><tr><td>NaCl</td><td>Fisher</td><td>S640-500</td><td></td></tr><tr><td>Tris-HCl</td><td>Fisher</td><td>BP153-1</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>S25403</td><td></td></tr><tr><td>Levamisole Hydrochloride</td><td>Sigma</td><td>31742</td><td></td></tr><tr><td>N,N-Dimethylformamide&lt;br/&gt; anhydrous (DMF)</td><td>Sigma</td><td>227056</td><td>Irritant, toxic, combustible, and suspected teratogen. Handle with proper safety attire including gloves and goggles.</td></tr><tr><td>5-Bromo-4-chloro-3-indolyl phosphate (BCIP)</td><td>Sigma</td><td>N6639</td><td>Protect this solution from light.</td></tr><tr><td>Nitroblue tetrazolium (NBT)</td><td>Sigma</td><td>840W</td><td>Protect this solution from light.</td></tr><tr><td>Albumin from Bovine Serum, purified fraction V (BSA)</td><td>Sigma</td><td>A8806</td><td></td></tr><tr><td>Sheep Anti-digoxigenin-AP Fab Fragments</td><td>Roche</td><td>1093274910</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516-500ML</td><td></td></tr><tr><td>96-well cell culture plates</td><td>Sarstedt</td><td>83.1835</td><td></td></tr><tr><td>Tg(mnx1:GFP)ml2/Tg(hb9:GFP)ml2</td><td>ZIRC</td><td>Tg(mnx1:GFP)ml2</td><td></td></tr><tr><td>Instant Ocean</td><td>Aquarium Systems</td><td>N/A</td><td></td></tr></tbody></table>

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

Bu makale Zebra balığı embriyolarının in situ hibridizasyon (DİLERİZ) in tüm montaj odaklanır. WISH teknoloji doku dağılım ve gelişim aşamasında açısından hem de gen ifadesinin değerlendirilmesi kolaylaştırır. Protokoller digoksijenin ile etiketlenmiş antisens RNA probları kullanılarak zebrabalıkları embriyoların WISH kullanımı tarif edilmektedir. Problar klonlanmış ve lineerize edilmiş gen şablonlar in vitro transkripsiyon yoluyla digoksigenin-bağlı nükleotit ilave edilerek oluşturulur. Belirlenen gelişim aşamalarında hasat embriyoların chorions spesifik problar ile inkübasyondan önce kaldırılır. Aşırı prob kaldırmak için bir yıkama işlemi sonrasında, embriyolar alkalin fosfataz ile konjuge edilmiş anti-digoksigenin antikor ile inkübe edilir. Alkalin fosfataz için bir kromojenik substrat kullanılarak, belirli bir gen ekspresyonu tespit edilebilir. Gen ifade seviyesine bağlı olarak tüm işlem 2-3 gün içinde tamamlanabilir.

Sepet başına 50 embriyo (gen / başına deneysel koşul için) söz konusu genin ifade deseni bir deneyde elde edilebilir olan protokolü kullanarak. Hemen hemen tüm embriyolar belirli bir gen için benzer ekspresyonu göstermektedir. In situ hibridizasyon boyama için temsili örnekler Şekil 3-6 &#39;de gösterilmektedir. Anlamında ve anti-anlamda hem riboprobes PC5.1 karşılık gelen cDNAs, PC5.2 6, SCL/tal-1 7, gata-1 8,9, flk / kdrl 10, shh 11, dlx2 12, fkd7 gelen sentezlendi / foxa1 13, insülin 14,15, 16 ve tripsin, Taq DNA polimeraz kullanılarak, tam uzunluktaki bir mRNA kademeli amplifikasyonuyla inşa edilmiş ve pCRII-TOPO (Invitrojen) içine klonlanmıştır. Bireysel amplikonların orijinal dizi analizi ile teyit edilmiştir. Klon yönelim doğrulama sonra, ilgili antisens riboprobes sentezlendiya da SP6 ya da T7 RNA polimeraz kullanılarak burada tarif edilen değişiklikler 18,19 ile 17 protokolleri kullanarak. In situ hibridizasyon bütün montaj ile ilgili aşamalar, Şekil 2&#39;de kısaca gösterilmiştir. Ilgi Spastine Özgü Riboprop ile hibridizasyon sonrası gözlenen mor boyama bolluk ve özellikle gen ifade siteleri hem de temsil eder. Örneğin PC5.1 için 24hpf de otik vezikül ve yanal çizgi primordium ön ve arka parçası içinde çok ayrık ifade gösterir ve güçlü 72 hpf (Şekil 3) ile ön ve arka yanal çizgi neuromasts yerleşmiştir. Aksine PC5.2 gösterir Somitlerin, otik vezikül ve Pronephric kanal içinde ve farklı bölgeselleşme ile CNS içinde her yerde ifade yüksek 96 hpf (Şekil 4) de karaciğer ve bağırsak içinde ifade edilir. İlgili anlamda riboprobes kullanılarak gen ifadesinin bulunmaması negatif kontrol olarak hizmet vermektedir. ( <strOng&gt; Şekiller 3b ve 4b). Kan belirteçlerinin ekspresyon analizleri ikili kafatası hücreleri içinde ve ara hücre kütlesi (ICM) SCL/tal-1 bir boyama gösterdi. Gata-1 için boyama de ICM içinde görülür rağmen ifade deseninin farklıdır. FLK / kdrl ifadesi arka beyin, ana ve bölümler arası damarları içinde ve ICM (Şekil 5) gözlendi. Shh için boyama (Şekil 5) Notokordun gözlendi. 34 hpf dlx2 ifadesi lokalize telencephalon, diencephalon, hipotalamus ve kranial ectomesenchymal kemerler ve 24 hpf fkd7/foxa1 ifade ağırlıklı olarak taban plakası ve hypochord görülür. Pankreas belirteçlerin ekspresyonu pankreas (Şekil 6), endokrin pankreas ve tripsin insülin boyanma gösterdi. Yüksek çözünürlükte ekspresyon modelini gösteren Bu sonuçlar, belirtilen protokolün başarısına temsil ederyüksek ve düşük bol genler hem ifadesinin saptanması için. Daha fazla netlik görüntüler için bir mikroskopik slayt 20 embriyo montaj sonra konfokal mikroskopi kullanılarak alınabilir. Deney protokolü optimizasyon esnasında alt-optimal koşullar altında yapıldı zaman elde edilen sonuçlar örneklerinden bazıları Şekil 7 &#39;de gösterilmiştir. Kötü Shh ifade ile yüksek bir arka plan sinyali 55-60 ° C arasında yeterli geçirgenliği ve hibridizasyon altında fark edildi <img alt="Şekil 1" fo:content-width="6in" fo:src="/files/ftp_upload/50644/50644fig1large.jpg" src="/files/ftp_upload/50644/50644fig1.jpg" width="500px" /> Şekil 1. Tutmak için hazırlık ve Eppendorf-naylon kullanımı örgü sepet embriyolar düzenledi. Bir iyi altı Eppendorf-naylon örgü sepet tutabilir. Dosyasını altı Eppendorf naylon örgü sepet ihtiva eden 6-çukurlu steril plakalar gösterirPBS içinde metanol art arda seyreltmeleri için de kullanılır. <img alt="Şekil 2," fo:content-width="6in" fo:src="/files/ftp_upload/50644/50644fig2large.jpg" src="/files/ftp_upload/50644/50644fig2.jpg" width="500px" /> Şekil 2. In situ hibridizasyon tekniği tüm montaj dahil adımları gösteren diyagram. Tespit aşamalı sonra, kullanılıncaya kadar embriyolar, -20 ° C&#39;de metanol içinde saklanabilir. Sondalar önceden sentezlendi ve -80 tutulabilir ° C Onlar uzun süre kullanılan istiyorsak küçük hacimde depolama sondalar tavsiye edilir. In situ hibridizasyon deneylerinde genel olarak tüm montaj 3 veya 4 gün içinde tamamlanabilir. Zayıf ifade genler için boyama reaksiyon boyama reaksiyon 4 de yapılmaktadır zaman artık oda sıcaklığında 16 saat kadar sürebilir ya da olacak ° C <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/50644/50644fig2large.jpg" target="_blank">büyük f görmek için buraya tıklayın</a>ŞEKIL. <img alt="Şekil 3," fo:content-width="6in" fo:src="/files/ftp_upload/50644/50644fig3large.jpg" src="/files/ftp_upload/50644/50644fig3.jpg" width="500px" /> Şekil 3. Situ hibridizasyon analizlerinde tüm montaj tarafından PC5.1 ifadesi 24 hpf ve 72 hpf sahnelenen embriyoları kullanılarak gerçekleştirilmiştir. 24 hpf embriyoların (a) Yanal görünümü ön içinde PC5.1 çok ayrık ifade göstermek ve PC5.1 antisens Spastine Özgü Riboprop kullanarak otik vezikül ve yanal çizgi primordium posterior kısmı. 72 hpf spesifik boyanması ön ve arka yanal çizgi neuromasts içinde gözlenmiştir. (B) PC5.1 anlamda Spastine Özgü Riboprop kullanarak gen ifadesinin olmaması bir negatif kontrol olarak hizmet vermektedir. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/50644/50644fig3large.jpg" target="_blank">büyük rakam görmek için buraya tıklayın</a> . gether.within-page = &quot;her zaman&quot;&gt; <img alt="Şekil 4," fo:content-width="6in" fo:src="/files/ftp_upload/50644/50644fig4large.jpg" src="/files/ftp_upload/50644/50644fig4.jpg" width="500px" /> Şekil 4. PC5.2 in situ hibridizasyon analizleri 24 hpf ve 96 hpf tüm montaj anda PC5.2 anti-duyu ve duyu riboprobes kullanılarak gerçekleştirilmiştir. (A) 24 hpf embriyoların Yanal görünümü CNS içinde her yerde ifade göstermek, otik vezikül ve Pronephric kanal Somitlerin. Karaciğer ve bağırsak belirli boyama PC5.2 antisens Spastine Özgü Riboprop kullanarak 96 hpf görülmektedir. (B) PC5.2 anlamda Spastine Özgü Riboprop kullanarak gen ifadesinin olmaması bir negatif kontrol olarak hizmet vermektedir. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/50644/50644fig4large.jpg" target="_blank">büyük rakam görmek için buraya tıklayın</a> . &quot;oad/50644/50644fig5.jpg&quot; width = &quot;500px /&gt; . Şekil 5 SCL/tal-1 Spastine Özgü Riboprop kullanarak 24 hpf situ hibridizasyon analizlerinde Tüm montaj ikili kafatası hücreleri içinde ve ara hücre kütlesi (ICM) boyama gösterdi; gata-1 Spastine Özgü Riboprop boyama ICM görülür; flk Beyin, ana ve intersegmental damarları içinde ve ICM / kdrl ifade. Shh için boyama Notokordun gözlenmiştir. <img alt="Şekil 6," fo:content-width="5in" fo:src="/files/ftp_upload/50644/50644fig6large.jpg" src="/files/ftp_upload/50644/50644fig6.jpg" width="500px" /> . Şekil 6 dlx2 Spastine Özgü Riboprop kullanarak 34 hpf situ hibridizasyon analizlerinde Tüm montaj telencephalon, diensefalon, hipotalamus, ve kranial ectomesenchymal kemerlerin dlx2 ifade ortaya; 24 hpf fkd7/foxa1 ifade zemin p ağırlıklı olarak görülürgeç ve hypochord, insülin boyama ekzokrin pankreas endokrin pankreas ve tripsin ifadesinde görülmektedir. <img alt="Şekil 7" fo:content-width="6in" fo:src="/files/ftp_upload/50644/50644fig7large.jpg" src="/files/ftp_upload/50644/50644fig7.jpg" width="500px" /> Şekil 7. Yeterli geçirgenliği ve uygun hibridizasyon sıcaklık situ hibridizasyon sinyalleri net için önemlidir. Embriyolar tarafından permeabilize zaman bir shh Spastine Özgü Riboprop kullanarak 24 hpf ve 48 hpf situ hibridizasyon analizlerinde Tüm montaj arka plan sinyali olmadan Notokordun ve taban içinde ifade ortaya 12-15 dakika ve 70 ° C&#39;de hibridize için proteinaz K tarafından sindirilmeye Embriyoların yetersiz sindirim (5 dk proteinaz K tedavisi) veya düşük sıcaklıkta melez maruz zaman eksik ekspresyonu ve yüksek arka plan sinyalleri gözlendi (60 ° C). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/50644/50644fig7large.jpg" target="_blank">büyük rakam görmek için buraya tıklayın</a> .

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

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

Zebra balığı, küçük tropikal balık, omurgalı geliştirme ve hastalık sırasında gen fonksiyonu çalışmak için popüler bir model haline gelmiştir. Hedef genlerin uzaysal ve zamansal ifade ile tespit edilebilir<em&gt; In situ</em&gt; Melezleme. Bizim geliştirilmiş protokol düşük spesifik olmayan arka plan sinyal düşük bol transkript tespiti için izin verir.

Yüksek Çözünürlüklü Tüm Dağı<em&gt; Yerinde</emGen İfade ve Fonksiyon Eğitim için Zebra balığı Embriyolar içinde&gt; Hibridizasyon

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

high-resolution-whole-mount-situ-hybridization-within-zebrafish

Nanyang Technological University

Research

JoVE Journal

Biology

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

22.1K Görüntüleme.  Royal Victoria Hospital. Zebra balığı, küçük tropikal balık, omurgalı geliştirme ve hastalık sırasında gen fonksiyonu çalışmak için popüler bir model haline gelmiştir. Hedef genlerin uzaysal ve zamansal ifade ile tespit edilebilir In situ Melezleme. Bizim geliştirilmiş protokol düşük spesifik olmayan arka plan sinyal düşük bol transkript tespiti için izin verir.

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

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

Tüm Dağı Zebra balığı Embriyolar kirpikli Organları boyanmasına Fosfo-epitoplar

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

Kimya

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

Zebra balığı bütün Dağı Floresan Situ hibridizasyon ve ayirt kombine bir protokol aracılığıyla Multiciliated hücrelerde görüntülenmesi

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Gelişim Biyolojisi

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebra balığı Tüm Dağı Yüksek Çözünürlüklü Çift Floresan Yerinde Hibridizasyon

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Biyoloji

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

Zebrabalığında Situ Hibridizasyon Boyamada Genotibrasyon ve Nicelik

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Tüm Zebrabalığı Embriyolarının Uzun Zaman Atlamalı Konfokal Mikroskobu için Katmanlı Montaj Yöntemi

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Zebrabalığı Embriyoları ve Larvalarında Tüm Mount Immunohistochemistry

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at 2 days post fertilization. Consisting of only two nephrons, the pronephros serves as the sole kidney during larval life until more renal function is required due to the increasing body mass. To cope with this higher demand, the mesonephros (adult kidney) begins to form during metamorphosis. The new primary nephrons fuse to the pronephros and form connected lumens. Then, secondary nephrons fuse to primary ones (and so on) to create a branching network in the mesonephros. The vast majority of research is focused on the pronephros due to the ease of using embryos. Thus, there is a need to develop techniques to study older and larger larvae and juvenile fish to better understand mesonephros development. Here, an in situ hybridization protocol for gene expression analysis is optimized for probe penetration, washing of probes and antibodies, and bleaching of pigments to better visualize the mesonephros. The Tg(lhx1a-EGFP) transgenic line is used to label progenitor cells and the distal tubules of nascent nephrons. This protocol fills a gap in mesonephros research. It is a crucial model for understanding how new kidney tissues form and integrate with existing nephrons and provide insights into regenerative therapies.

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish is an important model for understanding kidney development. Here, an in situ hybridization protocol is optimized to detect gene expression in zebrafish larvae and juveniles during mesonephros development.

In Situ Mesonephros Gelişimi Sırasında Zebra Balığı Larvaları ve Yavrularında Melezleme

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Tüm Dağı In situ Moleküler Biyoloji ve organizma Biyoloji Link Hibridizasyon

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

Tüm Dağı ile Zebra balığı Embriyo örnekler Vitray gözlenmesi ve Analiz Daire Dağı Hazırlık<em&gt; In situ</em&gt; Melezleme

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry of zebrafish embryonic sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. It is useful to detect the expression patterns of two genes in zebrafish if there is a paucity of antibodies.

In Situ Kriyokesitli Zebra Balığı Embriyolarında İmmünohistokimya ile Kombine Hibridizasyon

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a ...

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