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.

Tutti gli esperimenti condotti negli animali sono stati approvati dal Comitato per la cura degli animali da laboratorio e l&#39;uso della Università Nazionale Autonoma del Messico (UNAM), con il numero CICUAL-protocollo: FLC40-14. (CICUAL: &quot;Comité Institucional para el Cuidado y Uso de los Animales de Laboratorio del Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico&quot;). 1. Preparazione di embrioni Protein Extract <ol><li> Raccogliere 100 - 200 embrioni per ogni condizione di confrontare (morphants vs. wild type) allo stadio desiderato, ad esempio 72 ore dopo la fecondazione (HPF). </li><li> Luogo embrioni in piastre di Petri contenenti acqua di pesce (vedi tabella 1) e dechorionate manualmente embrioni utilizzando pinze punta fine. Evitare di utilizzare pronase durante questa fase (o qualsiasi altro proteasi durante il protocollo) come può digerire il recettore bersaglio. </li><li> Luogo embrioni in una provetta da 1,5 ml microcentrifuga e lavare gli embrioni twghiaccio con soluzione tampone 1x fosfato (PBS) (vedi Tabella 1). </li><li> Aggiungere 500 ml di tampone deyolk (vedi tabella 1). </li><li> Rilasciare la maggior parte di tuorlo pipettando gentilmente su e giù (circa 30 - 40 volte) gli embrioni nella soluzione. Per 72 HPF e 48 embrioni HPF utilizzare puntali per pipette blu e giallo, rispettivamente. Giallo suggerimenti possono avere bisogno di essere un po &#39;tagliata per evitare la distruzione di embrioni. Il rilascio tuorlo successo può essere controllato osservando gli embrioni sotto il microscopio. </li><li> Raccogliere gli embrioni per centrifugazione a 600 xg per 15 sec. </li><li> Gettare il surnatante con cura utilizzando una pipetta. </li><li> Embrioni lavare due volte con 500 microlitri tampone di lavaggio (vedi tabella 1) vortex delicatamente a velocità più bassa. </li><li> Raccogliere gli embrioni per centrifugazione a 600 xg per 15 sec. </li><li> A partire da qui, continuare la procedura a 4 ° C. </li><li> Gettare il surnatante con cura utilizzando una pipetta. </li><li> embrioni risospendere in 350 ml di tampone di lisi (vedere Tabella 1) ed omogeneizzare con un pestello in plastica. </li><li> Incubare gli embrioni lisati 30 minuti con agitazione su una sedia a dondolo provetta a 4 ° C. </li><li> Centrifugare lisato embrioni a 11.000 xg per 15 minuti per rimuovere i detriti insolubili. </li><li> Trasferimento cancellata surnatante con una pipetta in una nuova provetta. </li><li> Determinare proteine ​​totali da Metodo di Bradford 4, o altre procedure adatto. Se viene utilizzato saggio Bradford, eseguire la curva di taratura in presenza di concentrazioni equivalenti dei detergenti presenti nel tampone di lisi, in cui possono causare una sottostima della proteina normale 4. </li></ol> 2. Rilevamento di endogena proteina recettore <ol><li> Affinità Etichettatura e Immunoprecipitazione (tutti questi passaggi a 4 ° C) <ol><li> Collocare 400 - 500 mg di proteine ​​totali embrione in una provetta da 1,5 ml microcentrifuga e diluire a 1 mg / mL con tampone 1 (vedi Tabella 1). Nota: Al fine di ottenere 500 mg di proteine ​​totali dell&#39;embrione, circa 100-200 embrioni devono essere inizialmente trattati. 72 HPF embrioni di routine resa ~ 5 mg di proteine ​​totali dell&#39;embrione, che è sufficiente per il rilevamento betaglycan. </li><li> Aggiungere un volume sufficiente di TGF-β2 etichettati stock di raggiungere una concentrazione finale di 150 PM e incubare con l&#39;agitazione su una provetta rocker 2 ore a 4 ° C. TGF-β2 deve essere etichettato prima AFLIP viene avviato, con 125 I con il metodo T chloramine come descritto da Cheifetz et al. 5. ATTENZIONE: l&#39;utilizzo di schermatura per ridurre al minimo l&#39;esposizione durante la manipolazione 125 ho etichettato ligando. </li><li> Aggiungere un volume sufficiente di anticorpo non diluito contro il recettore di interesse al fine di raggiungere una diluizione 1: 100 e continuare incubazione per altri 2 ore a 4 ° C (incubazione può essere O / N). Questa è la diluizione ottimale di antisiero # 31, utilizzato in questo studio e descritto altrove 3, ma a seconda della qualità del untibody, una diluizione maggiore o minore può essere ottimale. </li><li> Aggiungere 50 ml di perline vincolanti adatto immunoglobuline (ad esempio, proteina G-Sepharose, precedentemente equilibrata in TNTE e risospeso 1: 5 del suo volume originale in TNTE) e incubare per 50 min sotto agitazione su un bilanciere provetta a 4 ° C. </li><li> Recuperare le perle di microcentrifugation a 11.000 xg per 20 sec. </li><li> Gettare il surnatante in un contenitore di rifiuti radioattivi appropriata. </li><li> Lavare le IP-perle tre volte con 1 ml di tampone di lavaggio IP (vedi Tabella 1), con vortex e microcentrifugation a 11.000 xg per 20 secondi ogni volta. </li><li> Risospendere IP-perline in 250 ml di tampone di lavaggio IP. </li><li> Aggiungere 1,5 ml di disuccinimidyl suberato (DSS, sciolti in DMSO a 10 mg / ml). Preparare la soluzione DSS appena prima del suo uso in questa fase. </li><li> Incubare per 15 minuti a 4 ° C con agitazione. </li><li> Al fine di placare la reazione di reticolazione, aggiungere 500 microlitri otampone di lavaggio f IP, integrato con abbastanza Tris-Cl pH 7,4 magazzino per raggiungere 25 mM Tris-Cl. I gruppi amminici liberi in Tris catturano DSS che non ha reagito. </li><li> Centrifugare a 11.000 xg per 20 secondi per raccogliere IP-perline e scartare il surnatante. </li><li> Risospendere IP-perline in 30 ml di ridurre Laemmli buffer. </li><li> campioni bollire 5 min a 94 ° C. </li><li> Opzionalmente, analizzare i campioni in un contatore gamma utilizzando il protocollo del produttore. </li></ol></li><li> Analisi dei campioni <ol><li> campioni oggetto di denaturazione SDS-PAGE. Utilizzare poliacrilammide al percentuale appropriata per la massa del recettore ed eseguire gel secondo la procedura standard. </li><li> Immergere il gel in soluzione fissativo (vedi Tabella 1) per 30 minuti a RT. </li><li> Lavare il gel con acqua distillata per 15 min. </li><li> Mettere il gel in carta da filtro precedentemente idratata e coprire con pellicola involucro di saran. </li><li> gel secco a 80 ° C per 1 ora. </li><li> Esporre il gel su un phosphorimager scre biancoit a RT O / N. </li><li> Scansione schermo esposto utilizzando un phosphorimager utilizzando il protocollo del produttore. </li></ol></li></ol>

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Chem. 263 (32), 16984-16991 (1988).</li><li>Kamaid, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Betaglycan+knock-down+causes+embryonic+angiogenesis+defects+in+zebrafish.">Betaglycan knock-down causes embryonic angiogenesis defects in zebrafish.</a> Genesis. 53, 583-603 (2015).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rapid+and+Sensitive+Method+for+the+Quantitation+of+Microgram+Quantities+of+Protein+Utilizing+the+Principle+of+Protein-Dye+Binding.">A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Cheifetz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+transforming+growth+factor-b+receptor+subsets+as+determinants+of+cellular+responsiveness+to+three+TGF-b+isoforms.">Distinct transforming growth factor-b receptor subsets as determinants of cellular responsiveness to three TGF-b isoforms.</a> J. Biol. Chem. 265, 20533-20538 (1990).</li><li>L&#243;pez-Casillas, F., Wrana, J. 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The authors have nothing to disclose.

L&#39;uso di Western blot con un anticorpo specifico contro una proteina di interesse è uno strumento prezioso per studiare la sua espressione 7 durante l&#39;embriogenesi. Tuttavia, immunoblotting di proteine ​​altamente glicosilata, non ha avuto molto successo a causa del loro trasferimento inefficiente e debole legame di nitrocellulosa o PVDF membrane 8,9. I proteoglicani sono un buon esempio di questa lacuna, a causa delle loro catene di glicosaminoglicani (GAG covalente allegate) che sono caricati negativamente e non si legano bene alle superfici sia di polistirolo o membrane idrofobiche assorbente. Inoltre, l&#39;eterogeneità dimensioni delle catene GAG ​​&quot;spread&quot; la proteina su un settore del gel, riducendone suo importo per ogni area della macchia. Inoltre, se la proteina di interesse ha bassi livelli di espressione, la sua individuazione da western blot regolari è un compito difficile. Il fattore di crescita trasformante tipo III β del recettore (TGFBR3) o betaglycan(BG), un recettore di membrana con due siti di attacco di GAG nei mammiferi 1 e uno in zebrafish 3, ha tutte queste proprietà. Sebbene, protocolli per superare questi ostacoli sono stati concepiti, come il rilevamento da parte del sistema avidina-biotina complesso (ABC) 10,11 o immunobloting di proteoglicani sulle membrane cariche positivamente 12, non possono risolvere i problemi sia nella stessa protocollo. Sfruttando l&#39;elevata affinità di BG per il suo naturale ligando TGFβ2 e della disponibilità di un anticorpo specifico, AFLIP, una tecnica che combina i vantaggi di etichettatura affinità e immunoprecipitazione, sopra discusso limiti di rilevamento western blot possono essere superati. Etichettatura affinità dei recettori di membrana con i loro ligandi radioattivi è uno strumento ben utilizzato che è stato determinante per l&#39;identificazione e la caratterizzazione di alcuni importanti fattori di crescita recettori in cellule in coltura 13-16.Mentre in affinità convenzionale etichettare un monostrato di cellule è sottoposto a covalenti etichettatura ligando sul piatto di coltura tissutale e quindi lisate, in AFLIP i complessi ligando-recettore vengono prima formati in lisati embrione, quindi purificati mediante immunoprecipitazione e, infine, in modo covalente cross-linked. A causa del suo uso di ligandi radioattivi, AFLIP è molto sensibile e in linea di principio può essere adattato ad altri fattori di crescita o recettori citochine per i quali sono disponibili un ligando marcato e una buona anticorpi. Questo potenziale di AFLIP aiuterebbe l&#39;individuazione di queste basse abbondanti ma fisiologici molecole rilevanti. A causa della variabilità intrinseca nella fase di reticolazione di etichettatura affinità, AFLIP non può essere utilizzato per determinare quantitativamente i livelli dei recettori misurati. Tuttavia, se eseguita con adeguati controlli paralleli, può fornire una misura abbastanza preciso dei livelli relativi di loro espressione. Come utilizzare AFLIP estratti totali di proteine, non è restriCTED ad uno specifico localizzazione cellulare del recettore, rivela la sua espressione in tutta la persona o organo, se applicata a esemplari adulti sezionati. Infine, se necessario AFLIP potrebbe essere collegato ad altri protocolli biochimici, come digestioni enzimatiche di carboidrati, per caratterizzare ulteriormente il recettore di interesse. Simile a etichettatura affinità convenzionale, un ligando appena marcato 125 I è fortemente raccomandato. Data la breve emivita di 125 I, il ligando deve essere utilizzato entro le prime 2 settimane dopo la marcatura con isotopo fresca. Anche se la maggior parte degli avvertimenti del protocollo sono stati menzionati nelle fasi corrispondenti, una menzione speciale deve essere data al accurata rimozione del tuorlo, che deve essere il più completo e uniforme possibile. Lasciando quantità irregolari di tuorlo comporterebbe quantificazione errata di bona fide proteine ​​dell&#39;embrione nei lisati.

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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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.

La figura 1 mostra un risultato rappresentativo ottenuto con AFLIP. Segnali in corsia 1 provengono dalla 125 I-ligando covalentemente legati sia alla zebrafish proteina betaglycan nucleo (core BG, al di sotto del marcatore 150 kDa), o il nucleo BG che è stato elaborato nella sua forma proteoglicani dall&#39;attaccamento di glicosaminoglicani (GAG, striscio vanno da 170 kDa all&#39;inizio del gel). Questo modello di migrazione, una proteina nucleo tagliente più un proteoglicano spalmato (causa eterogeneità nella lunghezza delle catene GAG), è caratteristica del TGFBR3 2. Poiché il DSS non covalentemente collegare ogni singolo complesso ligando-recettore formata, c&#39;è la connessione 125 I-ligando che appare nella parte anteriore migrazione del gel (125 I-TGF-β2). Questo legante gratuito è stato legato al recettore, e rimase legato durante la immunoprecipitazione, ma dal momento che non è stato covalentemente attaccata, staccato durante la procedura di SDS-PAGE. Nonetheless, la forza del suo segnale correlato ancora a quella data dal recettore marcato. Questo può essere apprezzata confrontando corsie 2 e 3. Al 48 HPF embrioni zebrafish presentano minori quantità di BG di 72 embrioni HPF 3, che può essere visto dai segnali deboli al GAG e nucleo BG, corrispondenti alla intensità della segnali dati dal legante libero compare alla parte anteriore del gel (corsia 2). Dato che gli anticorpi contro ratto BG 6 non cross-reagisce con il ZF BG, gli anticorpi quando utilizzato in AFLIP dato alcun segnale, quindi, che serve come controllo negativo (Corsia 3). Risultati negativi simili sono stati ottenuti con il siero pre-immune dell&#39;anticorpo anti-ZF BG 3. La Figura 2 mostra come AFLIP può essere utilizzato per misurare i livelli di espressione di un dato recettore embrioni sottoposti a una manipolazione sperimentale, in questo caso, la diminuzione del betaglycan causa morfolino ininiezione 3. Corsia 1 mostra il livello di TGFBR3 in un hpf tipo selvaggio embrione 72, mentre Corsia 2 mostra l&#39;effetto della somministrazione 7 ng di un morfolino diretto al confine exon2-intron2 del trascritto primario ZF BG. Si noti che il regolamento BG giù ottenuto con questo morfolino non viene riprodotto dal suo controllo mis-abbinato (Lane 3). Questi risultati hanno confermato che il fenotipo ottenuto con questo morfolino era specifico per il colpo del TGFBR3 come descritto in precedenza 3. <img alt="Figura 1" src="/files/ftp_upload/54405/54405fig1.jpg" /> Figura 1. AFLIP Rilevamento di BG in embrioni di zebrafish. Estratto proteico totale da embrioni a 72 ore dopo la fecondazione (HPF) (corsie 1 e 3) e 48 HPF (corsia 2) sono stati sottoposti a etichettatura affinità 125 I-TGF-β2 seguita da IP con coniglio anti-zebrafish BG (corsie 1 e 2, ZBG) o di coniglio anti-topo BG (Lane 3, RBG) Come controllo. Nel autoradiografia BG può essere rilevato senza GAG (nucleo BG) o come BG glicosaminoglicani modificato (GAG). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54405/54405fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/54405/54405fig2.jpg" /> Figura 2. BG Morpholino Knockdown rilevato dal AFLIP. 125 I-TGF-β2 etichettatura affinità di BG da embrioni non trattati WT (lane1), microiniettati con particolare BG morfolino (corsia 2) o un controllo mancata corrispondenza (Lane 3). I conteggi per milione (CPM) recuperato dopo l&#39;immunoprecipitazione e la fase di reticolazione sono indicati. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54405/54405fig2large.jpg" target="_blank">Si prega di cliccare qui per vedere una versione più grande di questa figura.</a> <table border="1" fo:keep-together.within-page="1" fo:keep-wesimo-next.within-page = &quot;always&quot;&gt; <tbody><tr><td> Buffer </td><td> Composizione </td></tr><tr><td> Buffer 1 </td><td> 50 mM Tris-HCl pH 7,5, 150 mM NaCl, 0,1% Triton X-100 </td></tr><tr><td> tampone Deyolk </td><td> 55 mM NaCl, KCl 1,8 mm, 1,25 mm NaHCO 3 </td></tr><tr><td> acqua Pesce </td><td> Vedi Tabella Materiali </td></tr><tr><td> soluzione fissativa </td><td> 50% CH 3 OH, il 12% CH3COOH, 0,185% HCHO </td></tr><tr><td> tampone di lavaggio IP </td><td> 10 mM Na 2 HPO 4, 2 mM KH 2 PO 4, 137 mM NaCl, 2,7 mM KCl, 0,1% Triton X-100, 0,02% SDS, pH 7,4 </td></tr><tr><td> tampone Laemmli </td><td> 2% SDS, 10% glicerolo, 100 mM DTT, 60 mM Tris (pH 6,8), 0,001% bromofenolo blu. </td></tr><tr><td> tampone di lisi </td><td> 50 mM Tris-HCl pH7.4, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0,5% Triton X-100, 0,1% SDS, 0,5% sodio DeoxychOlate </td></tr><tr><td> PBS </td><td> 137 mM NaCl, 2,7 mM KCl, 10 mM Na 2 HPO 4, 2 mM KH 2 PO 4, pH7.4 </td></tr><tr><td> TNTE </td><td> 50 mM Tris-HCl pH7.4, 150 mM NaCl, 1 mM EDTA, 0,1% Triton X-100 </td></tr><tr><td> tampone di lavaggio </td><td> 110 mm NaCl, KCl 3,5 mm, 2,7 mM CaCl 2, 10 mM Tris-HCl pH 8,5 </td></tr></tbody></table> Tabella 1: Composizioni Buffer

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.

Affinità Etichettatura Rilevazione di endogena Recettori da embrioni di zebrafish

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 Views.  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.

Mechanical Injury Vessel in embrioni di zebrafish

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

Ricerca

Biologia dello sviluppo

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.

Applicazione del DNA-Specific Stain metile Verde al Fluorescent Etichettatura degli embrioni

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.

Isolamento e caratterizzazione di singole cellule da embrioni di zebrafish

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.

Generazione di Parabiotic embrioni di zebrafish da fusione chirurgica di Sviluppo Blastulae

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.

Manipolazione Ploidia di embrioni di zebrafish con Heat Shock 2 Trattamento

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.

Biosensing potenziale del membrane del neurone del motore in embrioni di Zebrafish in tensione

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.

Induzione di ipossia in rana vivente e embryos zebrafish

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.

Visualizzazione dell'attività elettrica cellulare in Zebrafish embrioni in anticipo e tumori

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.

Un metodo di montaggio a strati per la microscopia confocale Time-Lapse estesa di embrioni di pesci zebra interi

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.

Intero Monte Immunohistochimica in Embrioni e Larve di pesce zebra

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