Questo lavoro è stato sostenuto dal M. Margaret Alkek Fondazione RHF.

Parte 1: fissaggio degli embrioni <ol><li> Riempire piatto dissezione di ghiaccio freddo DEPC-PBS. Tenere su ghiaccio. Aprire l&#39;uovo toccando il guscio con una pinza e rimuovere pezzi di conchiglia. </li><li> Rimuovere spessore albumina con una pinza. Tilt sacco vitellino con una pinza grossolana in modo che embrione rivolto verso l&#39;alto. </li><li> Utilizzando le forbici, tagliare un quadrato di tutto il sacco vitellino dell&#39;embrione. </li><li> Utilizzando un cucchiaio, togliere embrione dal tuorlo e luogo sul ghiaccio freddo DEPC-PBS. </li><li> Sotto il microscopio, rimuovere membrane e tuorlo e trasferimento piatto fissazione. </li><li> Definire, rimuovere DEPC-PBS. Sostituire con 4% PFA in DEPC-PBS e fissare O / N a 4 ° C. </li><li> Rimuovere fissativo. Sostituire con ghiaccio freddo DEPC-PBS. Utilizzando un ago smussato fine microcapillare o un coltello microdissezione, perforare il sistema nervoso e le cavità del cuore, per evitare cattura della sonda. </li><li> Lavare 2x 10 mn DEPC-PBS con 0,1% di Tween-20 (PBT). Disidratare 10 minuti ciascuno in 25%, 50%, 75% di metanolo in PBT. Disidratare embrioni due volte in 100% di metanolo. Conservare a -20 ° C nel metanolo. </li></ol> Parte 2: Prehybridization <ol><li> Reidratare embrioni 10 minuti ciascuna nel 75%, 50% e 25% di metanolo in PBT. Lavare 2x 10 mn in PBT. </li><li> Nel frattempo, preparare fissativo freddo ghiaccio (4% paraformaldeide in PBT). Sostituire PBT con soluzione proteinasi K (3 ml / flacone; [? 5 g / ml] finale in PBT). Assicurarsi che la fiala intera è esposta alla soluzione proteinasi K da dolci colline del flaconcino, vedere Risoluzione dei problemi per tempi di esposizione. </li><li> Utilizzando RNasi libero pipetta Pasteur rimuovere proteinasi K e aggiungere fissativo (2 ml). Immediatamente posto in ghiaccio per esattamente 20 minuti. </li><li> Tutti i lavaggi RT vengono eseguite su nutator. Lavare 2x 10 milioni nel PBT, e 1x 10mn in PBT contenente tampone di ibridazione 50%. </li><li> Lavare 1x nel buffer di ibridazione. Incubare a 65 ° C in 2 ml di buffer di ibridazione per 4-5 ore. </li></ol> Parte 3: gli embrioni ibridare con sonde <ol><li> DIG e FITC sintesi accoppiata sonda è descritto in appendice. La sonda dovrebbe dare un segnale forte, dovrebbe essere synhesized con FITC contenenti nucleotidi, la sonda più deboli dovrebbero essere sintetizzati con con DIG contenenti nucleotidi. </li><li> Preriscaldare sonde in tampone di ibridazione (500 ng / ml) utilizzando un flaconcino da 2 ml. Immediatamente sostituire il tampone di ibridazione con la soluzione della sonda. Non lasciare che gli embrioni a secco. Incubare per 16 ore a 65 ° C. </li><li> Sostituire la sonda con tampone di ibridazione, 2 lavaggi, 30 minuti ciascuna a 65 ° C. Lavare 15 minuti in tampone di ibridazione / MABT (1 / 1 v / v /) a 65 ° C. </li></ol> Parte 4: incubazione degli anticorpi DIG e colore di reazione <ol><li> Lavare 2x20 milioni nel MABT. Lavare 1 ora in 2% BBR / MABT. Lavare 5 ore nel 2% BBR/20% HISS / MABT (tampone di bloccaggio). Incubare in anticorpi 1:2000 DIG diluito in tampone di bloccaggio, O / N a 4 ° C su nutator. </li><li> Lavare 3x 10 minuti ciascuno in MABT, e 3x 1 ora ciascuno in MABT. </li><li> Lavare 2x 20 milioni nel NTMT. Aggiungi 200ul NBT / BCIP substrato NTMT a 10 ml. Rimuovere immediatamente NTMT dal flacone e sostituirlo con 1-2 ml NBT / BCIP buffer. Posto al buio. Monitorare la reazione del colore dopo 20 minuti, e ad intervalli di 20 minuti dopo. </li><li> A seguito di reazione di colore (dovrebbe apparire blu), lavare in PBS per 10 milioni di euro, da definire sulla fissazione piatto pieno di PBS, e sostituire la soluzione con 4% PFA in PBS. Fix O / N a 4 ° C. </li><li> Trasferimento a fiala di scintillazione nuovo. Lavare in PBST (PBS con 1% di Tween-20) 2x10 mn. Lavare in MABT 2x 10 mn. </li><li> Incubare in MABT 30 mn a 63 ° C. </li></ol> Parte 5: incubazione anticorpo FITC e il colore di reazione <ol><li> Lavare 2x 10 milioni nel MABT, 1 ora in 2% BBR / MABT, 5 ore in tampone di bloccaggio </li><li> Incubare in fosfatasi alcalina accoppiamento anti-FITC-anticorpo (1:500) O / N a 4 ° C. </li><li> Lavare 3x 10 minuti ciascuno in MABT, e 3x 1 ora ciascuno in MABT. </li><li> Lavare in 2x 20 milioni nel TBS pH 8,45 con 0,1% di Tween-20 (TBST). </li><li> Preparare vettore soluzione Rosso di lavoro (5 ml) in TBST utilizzando reagenti 1,2 e 3 fornito in kit. Rimuovere immediatamente TBST dal flacone e sostituirlo con tampone vettore rosso. Posto al buio. Monitorare la reazione del colore dopo 40 milioni di euro, e ad intervalli di 30 minuti dopo. </li><li> Colore seguente reazione (dovrebbe apparire rosso), il trasferimento di embrioni PBS. </li></ol> Parte 6: Fissazione ed elaborazione <ol><li> Trasferimento al piatto fissazione contenente PBS, e fissare nel 4% PFA per 20 minuti a temperatura ambiente o O / N a 4 ° C. </li><li> Lavare in PBS, 2x 10 mn. Al processo per la fotografia, il trasferimento del 20% glicerolo in PBS (questo renderà gli embrioni traslucido). Per creare una camera, taglio 2 strisce di nastro adesivo in PVC, sovrapporle sul vetrino. Tagliare una piazza del centro con una lama. Rimuovere le pinze piazza utilizzando. </li><li> Al processo per il sezionamento cera, trasferimento a PBS, lavare 2x 10 mn, disidratano in serie graduata di metanolo (25%, 50%, 75% e 100% in PBS, a 10 minuti ciascuno). </li><li> Sostituire con propanolo, 3mn. Sostituire con 1,2,3,4-tetrahydronaphthalene, 20 minuti seguita da Histoclear (2 x 1 ora). Sostituire con histoclear / cera 1 / 1 (v / v) 60 ° C per 1 ora. Sostituire con cera un processoper l&#39;esame istologico. </li></ol> Parte 7: Soluzioni Buffer di ibridazione, conservare a -20 ° C in Falcon da 50 ml: 25 formammide ml 3,25 ml (20xSSC), 0,5 ml (0,5 M EDTA), 1 ml (10% di Tween-20), 2,5 ml (1% SDS) ; 125 ul (20mg/ml tRNA);. 100ul (50 mg / ml di eparina) MABT, preparare 100mM acido maleico, il pH a 7,5 con NaOH pellet. Aggiungi 150mm NaCl e lo 0,1% Tween-20. NTMT (fatta al momento dell&#39;uso), 50 ml: 1 ml (5M NaCl), 2,5 ml (2 M Tris-HCl pH9.5), 1,25 ml (2M MgCl 2), 5 ml (10% di Tween-20). TBST: 0.1M Tris-HCl, 0,15 m di NaCl, pH 8,45, 0,1% Tween-20. Parte 8: Risoluzione dei problemi <table border="1"><tbody><tr><th> Problema </th><th> Causa </th><th> Rimedio </th></tr><tr><td valign="top"> Embrione è arrotolato </td><td valign="top"> Disidratazione insufficiente / reidratazione </td><td valign="top"> Mantenere intervalli di 10 minuti durante la successiva disidratazione / reidratazione passi </td></tr><tr><td valign="top"> Non vi è alcun segnale della sonda </td><td valign="top"> La sonda è degradata contaminazione RNasi in flaconcino </td><td valign="top"> Esegui sonda su 1% gel Assicurarsi che la fiala intera è esposta alla soluzione proteinasi K al punto 2 </td></tr><tr><td valign="top"> Sonda è cavità etichette di cuore un tubo neurale </td><td valign="top"> Sonda è intrappolato </td><td valign="top"> Al punto 2, assicurarsi che tutte le cavità sono perforate coltello microdissezione con o microcapillare </td></tr><tr><td valign="top"> Embrione disintegrato ibridazione seguente (punto 3) </td><td valign="top"> Proteinasi K passo sinistra temperatura di ibridazione troppo a lungo è troppo alta </td><td valign="top"> Proteinasi K non deve essere lasciato più lungo di 1mn (stadi 3 + e 4), 2 milioni (fase 5 a 7), 3 milioni di euro (stadio 8-10), 4 milioni di euro (stadio 11-13) Non ibridizzare a T o superiore di 65 ° C </td></tr></tbody></table> Parte 9: Risultati Rappresentante Embrioni rappresentante sono riportati di seguito: (A) Embrione (stadio HH7) è etichettato con un DIG-Sox sonda (blu) e una FITC-delta-1 sonda (rosso). (B) Embrione (stadio HH8) è etichettato con un DIG-Pax6 sonda (blu) e una FITC-Shh sonda (rosso). Nf, neurali piega, anp, piastra neurale anteriore, PSM, mesoderma presomitic, n, notocorda, nt, del tubo neurale). Doni sonda dai laboratori di Drs. C. Tabin, M. Goulding, D. Henrique, e J. Briscoe. <img src="/files/ftp_upload/old/904_2008_10_20_1/904_Figure-01.png" alt="" width="303" height="300" />

<ol>
	<li>Streit, A., Berliner, A. J., Papanayotou, C., Sirulnik, A., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+neural+induction+by+FGF+signaling+before+gastrulation.">Initiation of neural induction by FGF signaling before gastrulation.</a> Nature. 406, 74-78 (2000).</li><li>Rodr&#237;guez Esteban, C., Capdevila, J., Economides, A. N., Pascual, J., Ortiz, A., Izpis&#250;a Belmonte, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+novel+Cer-like+protein+Caronte+mediates+the+establishment+of+embryonic+left-right+asymmetry.">The novel Cer-like protein Caronte mediates the establishment of embryonic left-right asymmetry.</a> Nature. 401, 243-251 (1999).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+the+organizer+after+radical+ablation+of+Hensen's+node+and+the+anterior+primitive+streak+in+the+chick+embryo.">Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo.</a> Development. 122, 3263-3273 (1996).</li><li>Kawakami, M., Nakanishi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+an+endogenous+PKA+inhibitor,+PKIalpha,+in+organizing+left-right+axis+formation.">The role of an endogenous PKA inhibitor, PKIalpha, in organizing left-right axis formation.</a> Development. 128, 2509-2515 (2001).</li><li>Basch, M. L., Bronner-Fraser, M., Garcia-Castro, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+the+neural+crest+occurs+during+gastrulation+and+requires+Pax7.">Specification of the neural crest occurs during gastrulation and requires Pax7.</a> Nature. 441, 218-222 (2006).</li></ol>

The authors have nothing to disclose.

Il 2-colore montare tutto nel metodo di ibridazione in situ è ​​utilizzato per determinare sia i modelli spaziali e temporali di espressione genica, utilizzando uno o due geni di interesse. Le applicazioni includono la valutazione dei pattern di espressione genica di nuovi geni (ad esempio 1,2, così come i cambiamenti nel gene insulto seguente espressione (manipolazione embrionale 3, perline 4, ed elettroporazione di RNA o DNA costruisce 5).

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Eggs</td>
<td>Animal</td>
<td>Charles River </td>
<td>Premium Fertile</td>
<td>&nbsp;</td>
<tr>
<td>Stereomicroscope</td>
<td>Microscope</td>
<td>Leica Microsystems</td>
<td>MZ9.5</td>
<td>&nbsp;</td>
<tr>
<td>Marsh Automatic Incubator</td>
<td>Tool</td>
<td>Lyon</td>
<td>RX</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization Incubator</td>
<td>Tool</td>
<td>Hybaid</td>
<td>Micro-4</td>
<td>&nbsp;</td>
<tr>
<td>Adams Nutator orbital mixer</td>
<td>Tool</td>
<td>Fisher</td>
<td>14-062</td>
<td>&nbsp;</td>
<tr>
<td>Curved Forceps (1)</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>72991-4C</td>
<td>&nbsp;</td>
<tr>
<td>Fine scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14161-10</td>
<td>&nbsp;</td>
<tr>
<td>Spoon</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>10370-18</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Capillary Pipette</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>70950-12</td>
<td>&nbsp;</td>
<tr>
<td>Microcapillary tube</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1049-1PAK</td>
<td>Pull using vertical micropipette puller; blunt end with fine forceps</td>
</tr>
<tr>
<td>Microdissecting knife</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10056-12</td>
<td>Use to puncture cavities prior to in situ hybridization</td>
</tr>
<tr>
<td>Minuten pins 0.2mm diam</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-20</td>
<td>Keep pins together using a magnetic stirrer;</td>
</tr>
<tr>
<td>Sylgard 184 Silicon Elastomer Curing Agent and Base</td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>0001986475</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>Diethylpyrocarbonate (depc)</td>
<td>Reagent</td>
<td>Acros Organics</td>
<td>10025025</td>
<td>Add 1ml depc to 1l PBS; shake; autoclave</td>
</tr>
<tr>
<td>16% PFA</td>
<td>Reagent</td>
<td>Electron Microscopy Sciences</td>
<td>15710</td>
<td>&nbsp;</td>
<tr>
<td>Tween-20</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P1379-100ML</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P2308-5MG</td>
<td>Stock at 10 mg/ml in depc-H2O in 10ul aliquots. Store at -20C.</td>
</tr>
<tr>
<td>Formamide, deionized</td>
<td>Reagent</td>
<td>Ambion</td>
<td>9342</td>
<td>&nbsp;</td>
<tr>
<td>Yeast tRNA-25 mg</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15401-011</td>
<td>Stock at 20 mg/ml in depc-H2O in 125ul aliquots. Store at -20C</td>
</tr>
<tr>
<td>Heparin Sodium salt</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H4784-250MG</td>
<td>Stock at 50 mg/ml in depc-H2O in 100ul aliquots. Store at -20C.</td>
</tr>
<tr>
<td>SSC x20 pH5.5</td>
<td>Reagent</td>
<td>Fisher</td>
<td>PR-V4261</td>
<td>&nbsp;</td>
<tr>
<td>EDTA (0.5M)</td>
<td>PR-V4231</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>SDS</td>
<td>Reagent</td>
<td>Fisher</td>
<td>BP166-100</td>
<td>&nbsp;</td>
<tr>
<td>Maleic acid</td>
<td>Reagent</td>
<td>Fluka</td>
<td>63189</td>
<td>&nbsp;</td>
<tr>
<td>Boehringer Blocking Reagent</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11096176001</td>
<td>Use at 2% in MABT. To make, heat with MABT for 20 mn at 60C. Can make a stock and store in 1-5 ml aliquots at 20C.</td>
</tr>
<tr>
<td>Heat inactivated sheep serum</td>
<td>Reagent</td>
<td>Jackson Immuno</td>
<td>013-000-121</td>
<td>Dissolve in 10 ml H2O; heat 550C, 30 mn. Aliquot at 1ml, -20C.</td>
</tr>
<tr>
<td>Anti-Digoxigenin-AP, Fab fragments</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11093274910</td>
<td>&nbsp;</td>
<tr>
<td>NBT/BCIP stock solution</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11681451001</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Fluorescein-AP, Fab fragments</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11426338910</td>
<td>&nbsp;</td>
<tr>
<td>2M Tris</td>
<td>BP1759-500</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vector Red Alkaline Phosphatase Substrate Kit</td>
<td>Reagent</td>
<td>Vector Laboratories</td>
<td>SK-5100</td>
<td>&nbsp;</td>
<tr>
<td>1,2,3,4-tetrahydronaphthalene</td>
<td>Reagent</td>
<td>Sigma</td>
<td>429325-100ML</td>
<td>Under hood</td>
</tr>		
</tbody>
</table>
</div>

double whole mount in situ hybridization of early chick embryos

L&#39;embrione di pollo è uno strumento prezioso per lo studio dello sviluppo embrionale precoce. La sua trasparenza, accessibilità e facilità di manipolazione, lo rendono uno strumento ideale per studiare l&#39;espressione genica nel cervello, del tubo neurale, somite e il cuore la formazione di primordi. Questo video mostra le varie fasi a 2 colori montare tutto ibridazione in situ; In primo luogo, l&#39;embrione è sezionato dall&#39;uovo e fissati in paraformaldeide. In secondo luogo, l&#39;embrione è trasformato per prehybridization. L&#39;embrione viene quindi ibridato con due sonde diverse, una accoppiata a scavare, e uno accoppiato al FITC. A seguito di ibridazione durante la notte, l&#39;embrione viene incubato con l&#39;anticorpo DIG accoppiati. Reazione del colore per substrato DIG viene eseguita, e la regione di interesse appare blu. L&#39;embrione viene quindi incubato con anticorpo FITC accoppiati. L&#39;embrione è trasformato per una reazione a colori con FITC, e la regione di interesse appare rosso. Infine, l&#39;embrione è fisso e trattati per phtograph e sezionamento. Una guida alla risoluzione è anche presentato.

Watch this Scientific Journal Video about Double Whole Mount in situ Hybridization of Early Chick Embryos at JoVE.com

Double Whole Mount in situ Hybridization of Early Chick Embryos

Questo video dimostra a 2 colori montare tutto ibridazione in situ, un metodo con cui il modello di espressione spaziale e temporale dei 2 geni differenti possono essere visualizzati in embrioni di pollo giovani. Questo metodo è stato originariamente introdotto da David Wilkinson, Domingos Henrique, Phil Ingham e David Ish-Horowicz.

Doppia Monte intero ibridazione in situ di embrioni di pollo primi

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

double-whole-mount-in-situ-hybridization-of-early-chick-embryos

Research

JoVE Journal

Biology

12.3K Views.  Institute of Biosciences and Technology - Texas A&M Health Science Center. Questo video dimostra a 2 colori montare tutto ibridazione in situ, un metodo con cui il modello di espressione spaziale e temporale dei 2 geni differenti possono essere visualizzati in embrioni di pollo giovani. Questo metodo è stato originariamente introdotto da David Wilkinson, Domingos Henrique, Phil Ingham e David Ish-Horowicz.

Video: Double Whole Mount in situ Hybridization of Early Chick Embryos

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Metodo per la Cultura di Chick primi embrioni ex vivo (Nuova Cultura)

Ricerca

Biologia

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Metodo per la colorazione intero anticorpi Monte a Chick

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.

Zebrafish intero monte ad alta risoluzione doppia fluorescente In Situ Ibridazione

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

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

Cella Transfection singola in embrioni di pollo

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

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.

Utilizzando il monte intero In situ Ibridazione a Link Biologia Molecolare e organismal

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

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Tutto il monte In Situ</emIbridazione> di E8.5 a E11.5 embrioni di topo

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Monitoraggio Deformazioni tessuto morfogenetica in embrione di pollo primi

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Tutta la Monte RNA fluorescente<em&gt; In situ</em&gt; L&#39;ibridazione di<em&gt; Drosophila</em&gt; Embrioni

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em&gt; In situ</emIbridazione&gt; in embrioni Mount interi e istologia Adattato per Elasmobranchi Marine

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualizzazione e l&#39;analisi di molecole di mRNA mediante fluorescenza<em&gt; In Situ</em&gt; Ibridazione in<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Methods Article

Doppia Monte intero ibridazione in situ di embrioni di pollo primi