Ringraziamo K. Gill per l&#39;assistenza con l&#39;allevamento del mouse e cura e per la gestione del laboratorio. Grazie anche a Mukoyama membri laboratorio per assistenza tecnica. Il finanziamento è stato fornito dal programma di ricerca intramurale degli Istituti Naitonal della Salute.

1. La raccolta degli arti pelle embrionali di topo (~ E13.5 E17.5) <ol><li> Euthanize collegato femmine con procedura approvata. Secondo il nostro protocollo animali approvati, le femmine sono eutanasia da CO 2 l&#39;esposizione e poi assicurata dalla dislocazione cervicale. Giaceva l&#39;animale al tovagliolo di carta assorbente e immergerlo completamente nel 70% EtOH / H 2 O da una bottiglia a spruzzo. </li><li> Sezionare l&#39;utero intatto e mettetelo in un piatto da 100 mm x 15 Petri contenenti soluzione equilibrata Salt ghiacciata Hanks &#39;(HBSS) per lavare nel sangue. </li><li> Separati e sezionare l&#39;embrione. Rimuovere il amnios molto sottile dall&#39;embrione. </li><li> (Opzione) Dissect un singolo embrione in un piatto x 35 10 millimetri Petri se ogni embrione deve essere genotipizzati. Coda sezionato viene trasferito in un tubo da 0,2 ml PCR per la genotipizzazione. </li><li> Tagliare le zampe anteriori di embrione ed il trasferimento arti anteriori da una pinza l&#39;anello in 24 pozzetti contenenti 2 ml di ghiacciata paraformaldeide fresca 4% (PFA) in tampone fosfato salino (PBS). </li><li> Fissare le zampe anteriori con miscelazione delicata sulla Mixer Nutator a 4 ° C durante la notte. </li><li> Il giorno seguente, togliere la PFA e lavare tre volte per 5 min in 2 ml di PBS con miscelazione delicata sulla Mixer Nutator a temperatura ambiente. </li><li> Trasferimento degli arti anteriori in 100% di metanolo (MeOH) e memorizzarli congelatore a -20 ° C enzima (il congelatore con controllo della temperatura critica e senza funzione di sbrinamento automatico). Anticorpi primari indicati nella tabella 1 lavoro dopo il trattamento MeOH 100%. </li><li> Pelle a buccia fuori dalla zampa anteriore con una pinzetta sottile. Cutanee degli arti, quando disidratato, dovrebbe separare facilmente dal arto. In primo luogo l&#39;arto con il lato ventrale (lato palmo) verso l&#39;alto. Quindi, utilizzando una pinzetta sottile, tagliare la pelle, come mostrato nel video. Avanti sezionare intorno tutto l&#39;arto, desquamazione della pelle fuori delicatamente senza alcun danno. </li></ol> 2. Tutto-mount la colorazione immunoistochimica di Skins Limb <ol><li> Reidratare le pelli arto in polipropilene da 5 ml a fondo rotondo tubo incubando attraverso la serie graduata di MeOH / PBT (PBS + 0,2% Triton X-100) (75%, 50%, 25%) per 5 minuti ciascuno. Si noti che lo scambio di soluzioni dovrebbero fare attenzione a non danneggiare le bucce degli arti. </li><li> Lavare due volte per 5 min in PBT con miscelazione delicata sulla Mixer Nutator a temperatura ambiente. </li><li> Bloccare le pelli di capra arto o con 10% di siero / PBS +0,2% TX100 tampone di bloccaggio per gli anticorpi di capra secondario o al 10% Donkey siero / PBS +0,2% TX100 tampone di bloccaggio per gli anticorpi asino secondaria di 2 ore con miscelazione delicata sulla Mixer Nutator a a temperatura ambiente. </li><li> Mettere le bucce arto su un piatto da 35 mm x 10 Petri e il trasferimento da una pinza ad anello in 2ml-microcentrifuga tubo con 800μl di anticorpi primari (diluizione appropriato tra quelli riportati nelle tabelle) nel tampone di bloccaggio (o di capra 10% di siero / PBS 0,2 TX100% o 10% Donkey siero / PBS TX100 +0,2%). Incubare le pelli arto con miscelazione delicata sulla Mixer Nutator a 4 ° C durante la notte. Si noti che più gli anticorpi primari derivati ​​da specie diverse (ad esempio, anticorpo monoclonale di ratto + anticorpo policlonale di coniglio) possono essere utilizzati contemporaneamente. </li><li> Il giorno seguente, posizionare le bucce arto su un piatto da 35 mm x 10 Petri e il trasferimento da una pinza l&#39;anello in polipropilene da 5 ml a fondo rotondo tubo con 4 ml di tampone di lavaggio (sia il 2% siero di capra / PBS +0,2% TX100 o 2 Donkey% siero / PBS TX100 +0,2%). </li><li> Lavare cinque volte per 15 minuti mescolando con delicato sulla Mixer Nutator a temperatura ambiente. </li><li> Mettere le bucce arto su un piatto da 35 mm x 10 Petri e il trasferimento da una pinza ad anello in 2ml-microcentrifuga tubo con 800μl di anticorpi secondari nel buffer di blocco (o 10% siero di capra / PBS TX100 +0,2% o al 10% Donkey siero / PBS TX100 +0,2%). Di solito si usa anticorpi secondari da Jackson ImmunoResearch (Cy3, Cy5, diluizioni 1:300) o Invitrogen (Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 633, 1:250 diluizione). Filtrare la soluzione secondaria di anticorpi utilizzando 0.22μm PVDF filtri a membrana siringa per rimuovere le particelle aggregate degli anticorpi secondari. Incubare le bucce arto al buio o avvolti con fogli di alluminio per 1 ora mescolando con delicato sulla Mixer Nutator a temperatura ambiente. Si noti che diversi fluorescente anticorpi coniugati secondari derivati ​​da specie diverse possono essere utilizzati contemporaneamente. </li><li> Mettere le bucce arto su un piatto da 35 mm x 10 Petri e il trasferimento da una pinza l&#39;anello in polipropilene da 5 ml a fondo rotondo tubo con 4 ml di tampone di lavaggio (sia il 2% siero di capra / PBS TX100 +0,2% o 2% Donkey siero / PBS +0,2% TX100). Lavare cinque volte per 15 minuti al buio o avvolti in fogli di alluminio con miscelazione delicata sulla Mixer Nutator a temperatura ambiente. </li><li> (Opzione per la contro-contro nucleo) Incubare le pelli degli arti con 4 ml di tampone di lavaggio (sia Capra 2% siero / PBS TX100 +0,2% o 2% Donkey siero / PBS TX100 +0,2%), con A-Pro-3 (Invitrogen T3605 , diluizione 1:3000) al buio o avvolti con fogli di alluminio per 10 minuti mescolando con delicato sulla Mixer Nutator a temperatura temperare. Poi, lavare tre volte per 5 min con 4ml del buffer di lavaggio (sia il 2% siero di capra / PBS TX100 +0,2% o 2% Donkey siero / PBS TX100 +0,2%), al buio o avvolta da un foglio di alluminio con miscelazione delicata sulla Mixer Nutator a temperatura ambiente. Si noti che la colorazione forte e specifico per i nuclei si osserva per To-pro-3 in una emissione specifica (HeNe 633 nm di eccitazione). </li></ol> 3. Montaggio del Skins Limb su Slide <ol><li> Mettere le bucce arto su un piatto x 35 10 millimetri Petri. Rimuovere polveri, cristalli, fibre dallo strato interno della pelle con una pinzetta sottile con lo stereomicroscopio, con illuminazione bassa per evitare foto esteso sbiancamento. </li><li> Trasferire le pelli arto per adesivo microscopio da una pinza ad anello. Posizionare le bucce con lo strato interno disteso verso l&#39;alto della diapositiva (cioè verso coprioggetto). Appiattire la pelle accuratamente con una pinzetta sottile e rimuovere riporto il tampone di lavaggio da Kimwipe. </li><li> Montare in supporti di montaggio anti-sbiadimento senza bolle d&#39;aria. Utilizziamo 25 coprioggetto &quot;x25&quot;. Cure su una superficie piana al buio (per esempio, i campioni montato utilizzando Prolungare reagente oro sono poste una notte al buio a temperatura ambiente prima linea). Per la conservazione a lungo termine, sigillare il coprioggetto alla diapositiva e conservare a 4 ° C. </li></ol> 4. Microscopia confocale <ol><li> Impostare laser appropriato per fluorofori. Noi usiamo microscopio confocale Leica TCS SP5 con tre sorgenti laser compresa Argon 488 nm (per Alexa Fluor 488 e GFP), DPSS 561nm (per Alexa Fluor 568 e Cy3) e HeNe 633nm (per Alexa Fluor 633, Cy5 e To-Pro-3) . </li><li> Utilizzare lo strumento di scansione sequenziale per evitare o ridurre le interferenze, in cui tutti i coloranti in campioni di doppia o tripla macchiati saranno entusiasti al tempo stesso. Nella modalità di scansione sequenziale, le immagini verranno registrate in ordine sequenziale. </li><li> Altre informazioni relative al coloranti fluorescenti e laser per l&#39;eccitazione può essere fondata in &quot;Microscopia confocale per biologi&quot; di Hibbs (2004) </li></ol> 5. Rappresentante Risultati Tutto-mount microscopia confocale tripla etichetta immnofluorescence in pelle di topo anteriore al E15.5 con anticorpi pan-marker delle cellule endoteliali PECAM-1 (Fig. 1A-C, D blu), il marker neurofilamenti 2H3 (Figura 1A verde), e le cellule muscolari lisce marcatore αSMA (Figura 1A, B rossa) ha rivelato un quadro caratteristico di ramificazione di αSMA arterie +, in linea con 2H3 + nervi periferici nella pelle degli arti. Oltre al vaso sanguigno ramificazione, questa skin modello vascolarizzazione degli arti vengono usati per studiare patterning di vasi linfatici branching tramite anticorpi per il marcatore delle cellule endoteliali linfatiche LYVE-1 (Figura 1D, E rosso) e Neuropilin2 (NRP2) (Figura 1D, F verde ). <img src="/files/ftp_upload/2620/2620fig1.jpg" alt="Figura 1" /> Figura 1. (AC) Arterie allinearsi con i nervi periferici nella pelle degli arti embrionali. Tutto-mount tripla etichetta microscopia confocale immnofluorescence con anticorpi pan-marker delle cellule endoteliali PECAM-1 (AC, blu), il marker neurofilamenti 2H3 (A, verde), e il buon marker delle cellule muscolari αSMA (A e B, rosso ) viene visualizzato. A E15.5, 2H3 + nervi periferici (frecce aperte) associare arterie (punte di freccia), che sono coperti da αSMA + cellule muscolari lisce. (DF) vasi linfatici nella pelle degli arti embrionali. Triple-label microscopia confocale immnofluorescence con anticorpi pan-marker delle cellule endoteliali PECAM-1 (D, blu), il marker delle cellule endoteliali linfatiche LYVE-1 (D ed E, rosso) e Neuropilin2 (NRP2) (D e F, verde ) viene visualizzato. Vasi linfatici sono visualizzate da entrambi LYVE-1 e NRP2, mentre LYVE-1 è espressa anche da un sottoinsieme di macrofagi (punte di freccia aperta). Scala grafica: 100μm.

<ol>
	<li>Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+nerves+determine+the+pattern+of+arterial+differentiation+and+blood+vessel+branching+in+the+skin.">Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin.</a> Cell. 109, 693-705 (2002).</li><li>Mukouyama, Y. S., Gerber, H. P., Ferrara, N., Gu, C., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve-derived+VEGF+promotes+arterial+differentiation+via+neuropilin+1-mediated+positive+feedback.">Peripheral nerve-derived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback.</a> Development. 132, 941-952 (2005).</li><li>Wang, H. U., Chen, Z. F., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+distinction+and+angiogenic+interaction+between+embryonic+arteries+and+veins+revealed+by+ephrin-B2+and+its+receptor+Eph-B4.">Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4.</a> Cell. 93, 741-753 (1998).</li><li>Gerety, S. S., Wang, H. U., Chen, Z. F., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetrical+mutant+phenotypes+of+the+receptor+EphB4+and+its+specific+transmembrane+ligand+ephrin-B2+in+cardiovascular+development.">Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development.</a> Mol Cell. 4, 403-414 (1999).</li></ol>

Nessun conflitto di interessi dichiarati.

Il sistema vascolare è fondamentale per lo sviluppo degli organi durante l&#39;embriogenesi, nonché per la manutenzione degli organi e delle funzioni riproduttive negli adulti, in quanto fornisce sufficiente ossigeno e nutrienti agli organi. Adeguata rete vascolare è ben stabilita con processi complessi e multi-passo per l&#39;angiogenesi in cui viene riorganizzato pre-esistente rete capillare di strutture fortemente ramificate e gerarchica. Sebbene numerose opere sono state dimostrato che una varietà di molecole è...

Anticorpi Pan-marker delle cellule endoteliali <table border="1"><tbody><tr><td> Anticorpo </td><td> Specie </td><td> Azienda </td><td> Catalogo # </td><td> Condizione di lavoro </td></tr><tr><td> PECAM-1 </td><td> Criceto armeno (M) </td><td> Chemicon </td><td> MAB1398Z </td><td> Diluizione 1:100 # 1 </td></tr><tr><td> PECAM-1 </td><td> Rat (M) </td><td> BD </td><td> 553369 </td><td> Diluizione 1:300 </td></tr><tr><td> VEGFR2 </td><td> Rat (M) </td><td> eBioscience </td><td> 14-5821-82 </td><td> Diluizione 1:200 </td></tr><tr><td> CD34 </td><td> Rat (M) </td><td> eBioscience </td><td> 13-0341 </td><td> Diluizione 1:300 </td></tr><tr><td> Collagene IV </td><td> Coniglio (P) </td><td> ABD Serotec </td><td> 2150-1470 </td><td> Diluizione 1:300 # 2 </td></tr></tbody></table> Arterioso marker delle cellule endoteliali <table border="1"><tbody><tr><td> Anticorpo </td><td> Specie </td><td> Azienda </td><td> Catalogo # </td><td> Condizione di lavoro </td></tr><tr><td> Neuropilin1 </td><td> Coniglio (P) </td><td> Laboratorio del Kolodkin Alex # 3 </td><td></td><td> Diluizione 1:3000 </td></tr><tr><td> Unc5H2 </td><td> Capra (P) </td><td> R &amp; S </td><td> AF1006 </td><td> Diluizione 1:200 </td></tr></tbody></table> Venoso marker delle cellule endoteliali <table border="1"><tbody><tr><td> Anticorpo </td><td> Specie </td><td> Azienda </td><td> Catalogo # </td><td> Condizione di lavoro </td></tr><tr><td> EphB4 </td><td> Capra (P) </td><td> R &amp; S </td><td> AF446 </td><td> Diluizione 1:100 </td></tr></tbody></table> Linfatico marker delle cellule endoteliali <table border="1"><tbody><tr><td> Anticorpo </td><td> Specie </td><td> Azienda </td><td> Catalogo # </td><td> Condizione di lavoro </td></tr><tr><td> LYVE-1 # 4 </td><td> Coniglio (P) </td><td> Abcam </td><td> ab14917 </td><td> Diluizione 1:200 </td></tr><tr><td> LYVE-1 # 4 </td><td> Rat (M) </td><td> MBL </td><td> D225-3 </td><td> Diluizione 1:200 </td></tr><tr><td> Prox-1 </td><td> Coniglio (P) </td><td> Chemicon </td><td> AB5475 </td><td> Diluizione 1:1000 </td></tr><tr><td> Prox-1 </td><td> Capra (P) </td><td> R &amp; S </td><td> AF2727 </td><td> Diluizione 1:50 </td></tr><tr><td> Neuropilin2 </td><td> Coniglio (P) </td><td> Cell Signaling </td><td> 3366 </td><td> 1; 100 diluizione </td></tr><tr><td> Podoplanin </td><td> Criceto siriano (M) </td><td> Ibridomi Banca </td><td> 8.1.1 </td><td> Diluizione 1:200 </td></tr></tbody></table> Delle cellule muscolari lisce / periciti marcatore <table border="1"><tbody><tr><td> Anticorpo </td><td> Specie </td><td> Azienda </td><td> Catalogo # </td><td> Condizione di lavoro </td></tr><tr><td> αSMA-Cy3 </td><td> Mouse (M) # 5 </td><td> Sigma </td><td> c-6198 </td><td> Diluizione 1:500 # 6 </td></tr><tr><td> NG2 </td><td> Coniglio (P) </td><td> Chemicon </td><td> AB5320 </td><td> Diluizione 1:200 </td></tr><tr><td> SM22α </td><td> Coniglio (P) </td><td> Abcam </td><td> ab14106 </td><td> Diluizione 1:200 </td></tr></tbody></table> Anticorpi per giornalista GFP <table border="1"><tbody><tr><td> Anticorpo </td><td> Specie </td><td> Azienda </td><td> Catalogo # </td><td> Condizione di lavoro </td></tr><tr><td> GFP </td><td> Coniglio (P) </td><td> Invitrogen </td><td> A11122 </td><td> Diluizione 1:300 </td></tr><tr><td> GFP </td><td> Rat (M) </td><td> Nacalai tesque </td><td> 04404-84 </td><td> Diluizione 1:1000 </td></tr><tr><td> GFP </td><td> Chick (P) </td><td> Chemicon </td><td> P42212 </td><td> Diluizione 1:300 </td></tr></tbody></table> Anticorpi per giornalista LacZ <table border="1"><tbody><tr><td> Anticorpo </td><td> Specie </td><td> Azienda </td><td> Catalogo # </td><td> Condizione di lavoro </td></tr><tr><td> β-gal </td><td> Coniglio (P) </td><td> MP Biomedica </td><td> 55976 </td><td> Diluizione 1:5000 </td></tr><tr><td> β-gal </td><td> Capra (P) </td><td> ABD Serotec </td><td> 4600-1409 </td><td> Diluizione 1:500 </td></tr><tr><td> β-gal </td><td> Chick (P) </td><td> Abcam </td><td> ab9361 </td><td> Diluizione 1:200 </td></tr></tbody></table> Anticorpi per assoni periferici <table border="1"><tbody><tr><td> Anticorpo </td><td> Specie </td><td> Azienda </td><td> Catalogo # </td><td> Condizione di lavoro </td></tr><tr><td> 2H3 </td><td> Mouse (M) # 7 </td><td> Ibridomi Banca </td><td> 2H3 </td><td> Diluizione 1:200 </td></tr><tr><td> Tuj1 </td><td> Mouse (M) # 8 </td><td> Covance </td><td> MMS-435P </td><td> Diluizione 1:500 </td></tr><tr><td> Peripherin </td><td> Coniglio (P) </td><td> Chemicon </td><td> AB1530 </td><td> Diluizione 1:1000 </td></tr></tbody></table> Anticorpi per la migrazione di cellule di Schwann <table border="1"><tbody><tr><td> BFABP </td><td> Coniglio (P) </td><td> Il laboratorio di Thomas Müller # 9 </td><td></td><td> Diluizione 1:3000 </td></tr></tbody></table> (P): anticorpo policlonale, (M): anticorpo monoclonale # 1: capra anti-armeno criceto-Cy3 (Jackson ImmunoResearch 127-165-160) anticorpo deve essere usato come anticorpo secondario. # 2: l&#39;anticorpo collagene IV può essere utilizzato per rilevare i vasi sanguigni dopo ibridazione in situ. # 3: L&#39;anticorpo Neuropilin1 è gentilmente fornito da laboratorio del Kolodkin di Alex nella Johns Hopkins University. Pecora anti-umano degli anticorpi Neuropilin1 è disponibile in R &amp; S (AF3870), anche se non l&#39;abbiamo ancora testato. # 4: Il LYVE-1 anticorpi anche rilevare un sottoinsieme dei macrofagi nella pelle embrionale. # 5: L&#39;anti-αSMA anticorpo monoclonale di topo IgG2a. # 6: Il Cy3-anticorpo coniugato αSMA è incubato per 1 ora a temperatura ambiente insieme al secanticorpi secondaria per gli altri anticorpi primari. # 7: 2H3 anticorpo è topo monoclonale IgG1 contro neurofilamenti. # 8: Tuj1 anticorpo è topo IgG2a anticorpi monoclonali contro Neuron specifica classe III beta-tubulina. # 9: (cervello-specifiche di acidi grassi legame con le proteine) BFABP anticorpo è gentilmente fornito da laboratorio del Müller Thomas in Max-Delbrück-Centro di Medicina Molecolare.

whole-mount immunohistochemical analysis for embryonic limb skin vasculature: a model system to study vascular branching morphogenesis in embryo

Tutto-mount analisi immunoistochimica per l&#39;imaging la vascolarizzazione intera è fondamentale per la comprensione dei meccanismi cellulari di ramificazione morfogenesi. Abbiamo sviluppato la pelle modello vascolarizzazione degli arti per lo studio dello sviluppo vascolare in cui viene riorganizzato un preesistente primitivo plesso capillare in una rete ramificata gerarchicamente vascolare. Tutto-mount microscopia confocale con etichettatura più permette l&#39;imaging con robusto dei vasi sanguigni intatti così come i loro componenti cellulari, comprese le cellule endoteliali, periciti e cellule muscolari lisce, utilizzando specifici marcatori fluorescenti. I progressi in questo modello vascolarizzazione della pelle degli arti con studi genetici hanno migliorato la comprensione dei meccanismi molecolari di sviluppo vascolare e patterning. La pelle modello vascolarizzazione degli arti è stato utilizzato per studiare come i nervi periferici fornire un modello spaziale per la differenziazione ed il modello delle arterie. In questo articolo video descrive un protocollo semplice e robusto per colorare i vasi sanguigni intatti vascolare con anticorpi specifici e anticorpi secondari fluorescenti, che è applicabile per vascolarizzato organi embrionali dove siamo in grado di seguire il processo di sviluppo vascolare.

Watch this Scientific Journal Video about Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo at JoVE.com

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Introduciamo un tutto-mount immunoistochimica e microscopia confocale a scansione laser con l&#39;etichettatura per analizzare più intricata la formazione della rete vascolare nella pelle degli arti embrionali di topo.

Con tutto il supporto per l&#39;analisi immunoistochimica vasi embrionali pelle degli arti: un sistema modello per studiare morfogenesi vascolare diramazione in embrione

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

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Biology

17.5K Views.  National Heart, Lung, and Blood Institute, National Institutes of Health. Introduciamo un tutto-mount immunoistochimica e microscopia confocale a scansione laser con l'etichettatura per analizzare più intricata la formazione della rete vascolare nella pelle degli arti embrionali di topo.

Video: Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Double Whole Mount in situ Hybridization of Early Chick Embryos

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  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and 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 ...

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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 ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Embrionali di topo Cultura Lung, un sistema per valutare i meccanismi molecolari della Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself dispositivo per il recupero di campioni crioconservati cadere accidentalmente nelle cisterne criogeniche

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un sistema per<em&gt; Ex vivo</emColtura&gt; del pancreas embrionale

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

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 ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Embrionali di topo di sviluppo in un embrione di tutta la cultura di sistema privo di siero

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Analisi di apoptosi in embrioni di zebrafish da Whole-mount immunofluorescenza per rilevare caspasi 3 attivata

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...