The authors thank Gilberto Morales for fish care and maintenance, and Drs. Claudia Rivera and Hector Malag&#242;n (IFC-UNAM Animal Facility) for their help in rabbit immunization. This work was supported by grants from CONACYT 131226 and PAPIIT-DGAPA-UNAM IN204916.

Hayvanlarda yürütülen tüm deneyler CICUAL-Protokol numarası altında, Laboratuar Hayvanları Bakım ve México Özerk Ulusal Üniversitesi (UNAM) Kullanımı Komitesi tarafından kabul edildi: FLC40-14. (CICUAL: &quot;Comité Institucional para el Cuidado y Uso de los Animales de Laboratorio del Instituto de fizyoloji Celular, Universidad Nacional Autonoma de México&quot;). Embriyo Protein Özü 1. Hazırlık <ol><li> Her koşul örneği 72 saat sonra döllenme (hpf) için, istenilen aşamada (morphants vs yabani tip) karşılaştırmak için 200 embriyolar - 100 toplayın. </li><li> Balık su içeren Petri yerleştirin embriyolar (Tablo 1) ve elle ince uçlu forseps kullanarak embriyolar dechorionate. hedeflenen reseptör sindirmek olabilir bu aşamada (ya da protokol boyunca başka herhangi bir proteaz) sırasında pronase kullanmaktan kaçının. </li><li> 1.5 ml mikrofuge&#39;de tüp yerleştirin embriyolar ve embriyo tw yıkama1x Fosfat tampon çözeltisi (PBS) ile buz (Tablo 1 e bakınız). </li><li> Deyolk tampon 500 ul ekle (Tablo 1 e bakınız). </li><li> (- 40 kez yaklaşık 30) çözelti içinde embriyolar yavaşça yukarı ve aşağı pipetleme sarısı en bırakın. 72 hpf ve 48 hpf embriyolar için sırasıyla mavi ve sarı pipet uçları kullanın. Sarı ipuçları biraz embriyoların yok önlemek için kesmek gerekebilir. Başarılı sarısı bırakma mikroskop altında embriyoların gözlemleyerek kontrol edilebilir. </li><li> 15 saniye boyunca 600 x g&#39;de santrifüj ile embriyolar toplayın. </li><li> Bir pipet kullanarak dikkatlice süpernatant atın. </li><li> Yıkama embriyolar iki kez 500 ul yıkama tamponu ile hafifçe düşük hızda karıştırın (Tablo 1 e bakınız). </li><li> 15 saniye boyunca 600 x g&#39;de santrifüj ile embriyolar toplayın. </li><li> Buradan itibaren, 4 ° C&#39;de prosedürü devam etmektedir. </li><li> Bir pipet kullanarak dikkatlice süpernatant atın. </li><li> lizis tamponu 350 ul süspanse embriyolar (Tablo 1 &#39;e bakınız) ve plastik bir havan tokmağı kullanılarak homojen hale getirilir. </li><li> 4 ° C de bir test tüpü rocker lize embriyolar çalkalama ile 30 dakika kuluçkalayın. </li><li> Santrifüj çözünmeyen artık çıkarmak için 15 dakika 11,000 x g&#39;de embriyolar parçalanmıştır. </li><li> Transferi yeni bir tüp bir pipet kullanarak süpernatant temizledi. </li><li> Bradford protein deneyi 4 veya başka bir uygun yöntem vasıtasıyla, toplam protein belirler. Bradford tahlili kullanıldığında, bunlar 4 Standart proteinin tahmin edilmesine neden olarak, liziz tamponunda deterjanlar sunar eşdeğer konsantrasyonlarının varlığında kalibrasyon eğrisi gerçekleştirin. </li></ol> 2. Tespit Endojen Reseptör Protein <ol><li> Affinity Etiketleme ve Immunoprecipitation (bütün bu adımlar 4 o C&#39;de) <ol><li> 1.5 ml mikrofuge&#39;de tüp içinde toplam embriyo protein, 500 mg ve tamponu 1 (bakınız Tablo 1) ile 1 mcg / ul sulandırmak - 400 yerleştirin. Not: yaklaşık 100 toplam embriyo proteinin 500 ug elde etmek için - 200 embriyo ilk işlenmelidir. 72 HPF embriyolar rutin Betaglikan tarama için yeterli olana toplam embriyo proteininin ~ 5 ug verim. </li><li> 150 pM&#39;lik bir nihai konsantrasyon elde edildi ve 4 ° C &#39;de, bir test tüpü külbütör 2 saat ajitasyonla inkübasyona etiketli TGF-β2 stokunun yeterli hacim. AFLIP başlatılmadan önce Cheifetz ve ark., 5 ile tarif edildiği gibi, TGF-β2 kloramin T yöntemiyle 125I ile etiketlenmesi gerekir. DİKKAT: Ben ligand etiketli 125 taşıma sırasında maruziyeti en aza indirmek için koruyucu. </li><li> 100 seyreltme ve (kuluçka O / N olabilir), 4 ° C&#39;de başka bir 2 saat daha inkübasyona devam: 1 ulaşmak için ilgi alıcısına karşı seyreltilmemiş antikorun yeterli hacim. Bu, bu çalışmada kullanılan ve başka bir yerde tarif edilen 3, ama kalitesine bağlı olarak, antiserum # 31 optimal dilüsyonudurtibody daha büyük veya daha küçük bir seyreltme uygun olabilir. </li><li> Uygun bir immünoglobulin bağlama, boncuk tanelerin 50 ul ekle (örneğin, daha önce TNTE içinde dengelenmiş 1 yeniden süspanse edildi, G-proteini-Sefaroz: TNTE orjinal hacminin 5) ve 4 ° bir test tüpü rocker çalkalama ile 50 dakika boyunca inkübe C. </li><li> 20 saniye boyunca 11.000 xg mikrosantrifüj boncuk kurtarın. </li><li> Uygun bir radyoaktif çöp konteyner içinde süpernatant atın. </li><li> 20 saniye, her seferinde 11,000 xg, vorteks ve mikro santrifüjleme IP yıkama tamponu (bakınız Tablo 1) 1 ml IP boncuk üç kez yıkayın. </li><li> Süspanse IP-boncuk, IP yıkama tamponu 250 ul. </li><li> (10 mg / ml DMSO içinde çözülmüş DSS) disüksinimidil suberat 1.5 ul ekle. Sadece bu aşamada kullanılmadan önce DSS çözeltisi hazırlayın. </li><li> çalkalama ile 4 ° C&#39;de 15 dakika boyunca inkübe edin. </li><li> Çapraz bağlama reaksiyonu söndürmek için 500 ul o eklemeYeterince Tris-Cl, pH 7.4 stok ile desteklenmiş F IP yıkama tamponu, 25 mM Tris-Cl ulaşmak için. Tris serbest amino grupları reaksiyona girmemiş DSS yakalamak. </li><li> 20 saniye boyunca 11.000 xg&#39;de Santrifüj IP boncuk toplamak ve süpernatant atmak için. </li><li> Süspanse IP boncuk Laemmli tamponu indirgeme 30 ul. </li><li> Kaynama örnekleri 94 ° C &#39;de 5 dak. </li><li> İsteğe bağlı olarak, imalatçının protokolü kullanılarak bir gamma sayacında örnekleri analiz eder. </li></ol></li><li> numune Analizi <ol><li> SDS-PAGE denatüre Konu örnekler. reseptör kütlesi için uygun oranda poliakrilamid kullanın ve standart prosedür altında jel çalıştırın. </li><li> Sabitleştirici çözeltide jel daldırın oda sıcaklığında 30 dakika boyunca (bakınız Tablo 1). </li><li> 15 dakika boyunca damıtılmış su ile jel yıkayın. </li><li> Daha önce sulu filtre kağıdına jel yerleştirin ve Saran wrap film ile kaplayın. </li><li> 1 saat boyunca 80 ° C&#39;de kuru jeli. </li><li> Beyaz Fosfor bağırıyorum üzerinde jel AçığaRT O / N en-. </li><li> Üreticinin protokolü kullanılarak bir fosforimager kullanılarak maruz ekranı tarayın. </li></ol></li></ol>

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The authors have nothing to disclose.

Ilgi konusu bir proteinine karşı spesifik bir antikor ile Western lekeleri kullanımı embriyojenez sırasında ifadesini 7 incelemek için değerli bir araçtır. Bununla birlikte, yüksek-glikosile proteinlerin immünoblot yetersizlikler nedeniyle transfer ve nitroselüloz ya da PVDF zarlarına 8,9 zayıf bağlanma için çok başarılı olmamıştır. Proteoglikanlar çünkü negatif yüklü ve polistiren yüzeyler veya hidrofobik lekeleme membranların ya iyi bağlamaz onların kovalent bağlı glikozaminoglikan zincirleri (GAG) nedeniyle, bu eksiklik bir iyi bir örnektir. Ayrıca GAG zincirlerinin boyutu heterojen etkili leke alanı başına düşen miktarı azaltarak, bir jel sektörü üzerinde protein &quot;yayılır.&quot; İlgili protein ekspresyonunun düşük seviyelerde bulunur Buna ek olarak, düzenli western blotlar tarafından tespit zor bir iştir. reseptörü (TGFBR3) ya da Betaglikan β tip III transforme edici büyüme faktörü(BG), Zebra balığı 3 iki GAG bağlanma memelilerde 1 ve sitelerin biriyle zar reseptörü, bu özelliklerin hepsi var. Bu engellerin üstesinden gelmek için protokoller geliştirilmiştir edilmiştir rağmen, avidin-biotin kompleks (ABC) sistemi 10,11 veya pozitif yüklü membranların 12 proteoglikanların imüno-lekeleme ile algılama gibi, aynı protokolde hem sorunları çözemez. doğal ligandı TGFβ2 için ve belirli bir antikorun mevcudiyeti BG yüksek afinite yararlanarak, AFLIP afinite etiketi ve immüno-avantajlarını birleştiren bir teknik yukarıda Western blot algılama sınırlamaları ortadan edilebilir ele aldı. Bunların radyo etiketli ligandları ile membrana bağlı reseptörlerin afinite etiketlemesi çok önemli bir büyüme tanımlanması ve karakterizasyonu için vesile kültürlenmiş hücreler 13-16 reseptörleri faktörleri olan bir de kullanılan bir araçtır.hücrelerin tek bir tabakası etiketleme konvansiyonel afinite doku kültürü tabağına ligand etiketleme kovalent tabi tutulur ve daha sonra lize edilir olsa da, AFLIP ligand reseptör kompleksleri ilk önce embriyo lizatlarında, o zaman, immüno-çökeltme ile saflaştırılmış ve son olarak da, kovalent çapraz bağlanmış oluşturulur. , Radyo etiketli ligandların kullanımı bölgesinin AFLIP başka büyüme faktörleri veya etiketli bir ligand ve iyi bir antikor, mevcut olduğu sitokin reseptörlerine uyarlanabilir çok hassas ve prensip olarak. AFLIP bu potansiyel bu düşük bol ama fizyolojik ilgili moleküllerin tespit yardımcı olacaktır. Çünkü afinite etiketi arasında çapraz bağlama aşamasında doğal değişkenlik, AFLIP kantitatif olarak tayin reseptörleri seviyelerinin saptanması için kullanılamaz. Yeterli paralel kontrolleri ile gerçekleştirilen Ancak, onların ifade göreli seviyeleri oldukça doğru ölçümün sağlayabilir. AFLIP toplam protein özleri kullanmak gibi, restri değildisseke yetişkin örneklerinin uygulandığı takdirde reseptörünün bir spesifik hücresel konuma cted, o tam bir birey ya da organda kendi ifadesini ortaya koymaktadır. gerekli AFLIP diğer biyokimyasal protokollere bağlı olabilir Son olarak, karbonhidrat enzimatik sindirim gibi, daha da ilgi reseptörü karakterize etmek. Geleneksel afinite etiketleme benzer şekilde, taze 125-etiketli ligand şiddetle tavsiye edilir. 125 Ben kısa yarı ömrü göz önüne alındığında, ligand taze izotop ile etiketleme sonra ilk 2 hafta içinde kullanılmalıdır. protokol uyarılar en mukabil adımlarda açıklanan olmasına rağmen, özel bir söz mümkün olduğu kadar tam ve düzgün olmalıdır sarısı dikkatli uzaklaştırılması verilir. sarısı düzensiz miktarda bırakılması lizatları iyi niyetli embriyo proteinin yanlış kantitatif neden olacaktır.

Specific detection of proteins expressed during embryonic development is required to validate expression profiles obtained by measuring their cognate mRNAs with RT-PCR or in situ hybridization (ISH). This is commonly achieved by a western blot of embryo extracts followed by detection with specific antibodies. However, this approach is hard to apply to proteins that are in very low abundance, or that have properties that hamper their quantitative transfer during their blotting. Betaglycan, also known as the type III transforming growth factor &#946; (TGF-&#946;) receptor (TGFBR3), is an example of these difficulties. TGFBR3 is a part time membrane proteoglycan that binds TGF-&#946; through its core protein1, with notably higher affinity for the isoform TGF-&#946;2, a property that distinguishes it from any other TGF-&#946; binding protein2. TGFBR3 in the zebrafish is expressed from 8 hpf on, reaching a maximum by 72 hpf, as detected by RT-PCR of its mRNA3. 
However, despite the availability of a very specific antibody3, every attempt to detect its translated product by western blot proved unsuccessful. Reckoning that TGFBR3's proteoglycan nature, as well as putative low abundance may be accountable for this failure, a detection method, AFLIP, which takes advantage of TGFBR3 high affinity for TGF-&#946;2 was devised. In this method a protein extract from pooled embryos is allowed to specifically bind 125I-labeled TGF-&#946;2 and the receptor-ligand complexes are purified by immunoprecipitation and cross-linked before separation by SDS-PAGE. The migration patterns observed by autoradiography of the gels revealed the presence and nature of the labeled receptor species. This approach combines the ligand specificity of affinity labeling with immunoprecipitation by specific antibodies, increasing detection range, avoiding the inefficient transfer blotting of TGFBR3. Due to its inherent properties, the AFLIP assay is not a quantitative assay but can be used to confidently gauge relative experimental differences in the analyzed receptor.

<table border="0" cellpadding="0" cellspacing="0" width="670">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Disuccinimidyl suberate (DSS)</td>
			<td style="width:168px;">ThermoFisher Scientific</td>
			<td style="width:119px;">21555</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Protein G Sephraose 4 Fast Flow</td>
			<td style="width:168px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">17-0618-01</td>
			<td style="width:167px;"></td>
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			<td height="20" style="height:20px;width:216px;">Gel Dryer Model 583&#160;</td>
			<td style="width:168px;">BIO-RAD</td>
			<td style="width:119px;">1651745</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Typhoon 9400</td>
			<td style="width:168px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">63-0055-78</td>
			<td style="width:167px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cobra II Auto gamma counter</td>
			<td style="width:168px;">Packard</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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			<td height="20" style="height:20px;width:216px;">Exposure Cassette</td>
			<td style="width:168px;">Molecular Dynamics</td>
			<td style="width:119px;">63-0035-44</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Na2HPO4</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">K2HPO4</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3246</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CH4O</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">9070</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">C2H4O2</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">9508</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CH2O</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">2106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">L4509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">ED</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">T9284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">C3306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaHCO3</td>
			<td style="width:168px;">Fisher Scientific</td>
			<td style="width:119px;">S233</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PMSF</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7626</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Crystal Sea Marine Mix</td>
			<td style="width:168px;">Marine Enterprises International</td>
			<td style="width:119px;">http://www.meisalt.com/Crystal-Sea-Marinemix</td>
			<td></td>
		</tr>
	</tbody>
</table>

affinity labeling detection of endogenous receptors from zebrafish embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

Şekil 1, AFLIP ile elde edilen temsili bir sonuç göstermektedir. 125 I-ligand gelen Lane 1 Sinyaller kovalent (150 kDa işaretleyici altında BG çekirdek) zebrabalıkları Betaglikan çekirdek protein veya glikozaminoglikan eki (GAG, smear tarafından proteoglikan forma işlendikten BG çekirdek ya bağlı ), 170 kDa, jelin en arasında değişmektedir. Göç Bu model, keskin çekirdek proteini ek olarak (nedeniyle GAG zincirlerinin uzunluğu heterojenliği) bir bulaşmış proteoglikan, TGFBR3 2 özelliğidir. DSS kovalent her ligand-reseptör kompleksi oluşmuş bağlantı olmadığından, jel (125 I-TGF-β2) göç önünde görünen 125 I-ligand ücretsiz. Bu serbest ligand reseptöre bağlanmış, ve immüno-çökeltme sırasında bağlı kalan, ancak kovalent bağlı değildi, çünkü SDS-PAGE işlemi sırasında ayrılır. Yok hayırnetheless olarak, sinyalin gücü hala işaretli reseptör ile verilen bir karşı gelir. Bu yoğunluğuna karşılık gag ve BG özünde hafif sinyalleri, görülebilir 72 HPF embriyolar 3 daha BG düşük miktarlarda, zebra balığı embriyolar HPF 48 &#39;de kuşak 2 ve 3 karşılaştırarak gösteren takdir edilebilir jel ön görülen serbest ligand tarafından verilen sinyalleri (Şerit 2). AFLIP kullanıldığında sıçan karşı antikorlar BG 6 ZF BG ile çapraz reaksiyona girmemektedir göz önüne alındığında, antikorlar, bir negatif kontrol (Şerit 3) olarak görev bu nedenle, herhangi bir sinyal verdi. Benzer negatif sonuçlar, anti-ZF BG antikor 3 ön-immün serum ile elde edilmiştir. Şekil 2, AFLIP bağlı olarak morfolino, bu durumda, bir deney manipülasyona tabi embriyolar Betaglikan azalması, belirli bir reseptör ekspresyon düzeylerini ölçmek için nasıl kullanılabileceğini göstermektedirjection 3. Şerit 2 ZF BG primer transkriptin exon2-intron2 sınır yönelik bir morfolino 7 ng tatbik etkisini gösterirken Şerit 1, 72 HPF vahşi tip embriyoda TGFBR3 seviyesini göstermektedir. Bu morfolino ile elde edilen BG aşağı regülasyon hatalı eşleştirilmiş kontrol (Lane 3) tarafından yeniden unutmayın. Bu sonuçlar, 3 daha önce tanımlandığı gibi, bu morfolino elde fenotipi TGFBR3 demonte için spesifik olduğunu teyit etti. <img alt="Şekil 1" src="/files/ftp_upload/54405/54405fig1.jpg" /> Zebra balığı Embriyolar BG Şekil 1. AFLIP Algılama. 72 saat sonrası fertilizasyon (hpf) de embriyolardan total protein ekstresi (Şerit 1 ve 3) ve 48 hpf (Lane 2) takip 125l-TGF-β2 afinite etiketleme tabi tutuldu BG (Lane 3 Rbg tavşan anti-zebra balığı BG (kulvarlar 1 ve 2, ZBG) veya tavşan anti-fare ile IP tarafından) Bir kontrol olarak. Otoradyografi BG GAG (BG çekirdek) olmadan tespit edilebilir ya da bir glikozaminoglikan modifiye BG (GAG) olarak. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54405/54405fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="şekil 2" src="/files/ftp_upload/54405/54405fig2.jpg" /> Şekil 2. BG Morfolino demonte. AFLIP tarafından algılanan belirli BG morfolino (Şerit 2) ya da bir uyumsuzluk kontrolü (Şerit 3) ile microinjected muamele edilmemiş WT embriyolar (Lane1), BG 125 I-TGF-β2 afinite etiketi. Milyon (cpm) başına sayımları immünopresipitasyon ve çapraz bağlama adımı belirtilmiştir sonra. Iyileşti <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54405/54405fig2large.jpg" target="_blank">bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız.</a> <table border="1" fo:keep-together.within-page="1" fo:keep-wi&#39;inci-next.within-page = &quot;always&quot;&gt; <tbody><tr><td> Tampon </td><td> kompozisyon </td></tr><tr><td> Tampon 1 </td><td> pH 7.5, 50 mM Tris-HCI, 150 mM NaCI,% 0.1 Triton X-100 </td></tr><tr><td> Deyolk tamponu </td><td> 55 mM NaCI, 1.8 mM KCI, 1.25 mM NaHCO 3 </td></tr><tr><td> balık su </td><td> Malzeme Tablosu görün </td></tr><tr><td> fiksatif çözüm </td><td> % 50 CH3OH,% 12 CH3COOH, 0.185% HCHO </td></tr><tr><td> IP yıkama tamponu </td><td> 10 mM Na-2 HPO 4, 2 mM KH 2 PO 4, 137 mM NaCI, 2.7 mM KCI,% 0.1 Triton X-100,% 0.02 SDS, pH 7.4 </td></tr><tr><td> Laemmli tamponu </td><td> % 2 SDS,% 10 gliserol, 100 mM DTT, 60 mM Tris (pH 6.8),% 0.001 bromfenol mavisi. </td></tr><tr><td> liziz tamponu </td><td> 50 mM Tris-HCI pH 7.4, 150 mM NaCI, 1 mM EDTA, 1 mM PMSF,% 0.5 Triton X-100,% 0.1 SDS,% 0.5 sodyum DeoxychOlate </td></tr><tr><td> PBS </td><td> HPO 4 137 mM NaCI, 2.7 mM KCI, 10 mM Na-2, 2 mM KH 2 PO 4, pH 7.4 </td></tr><tr><td> TNTE </td><td> 50 mM Tris-HCI pH 7.4, 150 mM NaCI, 1 mM EDTA,% 0.1 Triton X-100 </td></tr><tr><td> Yıkama tamponu </td><td> 110 mM NaCI, 3.5 mM KCI, 2.7 mM CaCl2, 10 mM Tris-HCI, pH 8.5 </td></tr></tbody></table> Tablo 1: Tampon bileşimler

Watch this Scientific Journal Video about Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos at JoVE.com

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

Zebra balığı Embriyolar Endojen Reseptör Affinity Etiketleme Tespiti

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

affinity-labeling-detection-endogenous-receptors-from-zebrafish

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

5.6K Görüntüleme.  Universidad Nacional Autónoma de México. A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

Video: Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Zebra balığı Embriyolar Mekanik Gemi Sakatlık

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Gelişim Biyolojisi

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored until recently. In this article, we illustrate the method for preparing a methyl green aqueous stock solution, that when diluted can be used as a very convenient fluorescent nuclear label for fixed cells and tissues. Easy procedures to label whole zebrafish and chick embryos are detailed, and examples of images obtained shown. Methyl green is maximally excited by red light, at 633 nm, and emits with a relatively sharp spectrum that peaks at 677 nm. It is very inexpensive, non-toxic, highly stable in solution and very resistant to photobleaching when bound to DNA. Its red emission allows for unaltered high resolution scanning confocal imaging of nuclei in thick specimens. Finally, this methyl green staining protocol is compatible with other cell staining procedures, such as antibody labeling, or actin filaments labeling with fluorophore-conjugated phalloidin.

A method for fluorescent staining of fixed biological material with the specific DNA label methyl green is described. Methyl green is used in a diluted aqueous solution and is very resistant to photobleaching. Its far-red emission allows for deep specimen imaging, making it particularly adequate for whole embryos.

Embriyolar Floresan Etiketleme DNA Özgü Leke Metil Green Uygulaması

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

İzolasyon ve Zebra balığı Embriyolar gelen Tek Hücre Karakterizasyonu

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

Blastulae Gelişmekte Cerrahi Fusion tarafından Parabiyotik balığı Embriyolar Üretimi

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

Isıl Şok 2 Tedavi ile Zebra balığı Embriyolar ploidisi Manipülasyon

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

Canlı Zebra balığı Embriyolarında Biosense Motor Nöron Membranı Potansiyeli

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

Kurbağa ve Zebra balığı Embriyolarında Hipoksinin İndüklenmesi

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Zebra balığı erken embriyo ve tümör hücresel elektrik aktivitesinin görselleştirme

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

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. ...