Gli autori ringraziano Leif Oxburgh e Derek Adams per aver generosamente condiviso la loro conoscenza, Leif Oxburgh per i commenti utili sul manoscritto e Stefan Wölfl e Ulrike Müller per il loro sostegno tecnico e per Saskia Schmitteckert, Julia Gobbert, Sascha Weyer e Viola Mayer laboratorio. Questo lavoro è stato sostenuto da Development, The Company of Biologists (a CP).

I topi sono stati mantenuti secondo la normativa svedese e la legislazione dell&#39;Unione europea (2010/63 / UE). Tutte le procedure sono state eseguite seguendo le linee guida del comitato etico svedese (autorizza C79 / 9, C248 / 11 e C135 / 14). Le procedure presso l&#39;Università di Heidelberg che coinvolgono soggetti di origine animale sono stati approvati dal Regierungspräsidium Karlsruhe e dagli Ufficiali di Benessere degli animali presso l&#39;Università di Heidelberg. 1. Preparazione di reagenti e materiali per la cultura NOTA: utilizzare una cappa a flusso laminare per ridurre al minimo la contaminazione. <ol><li> Nel giorno della dissezione, pre-cluster Fc umano solo o ricombinante proteina chimerica efrina fusa con Fc umana mescolandola con anticorpi caprini anti-umani Fc a un rapporto molare di 1: 5 in soluzione salina fosfata tamponata sterile (PBS). Incubare per 1 ora a 37 ° C 5 . </li><li> Preparare il mezzo di coltura integrando il Modified Eagle Medium di Dulbecco: NutrienteLa miscela F-12 (DMEM / F12) con 1% di penicillina / streptomicina (v / v), 1% di glutammina (v / v), 1% di insulina transferrin-selenio (v / v), 50μg / Transferrin e 1,5 μg / mL di amfotericina. NOTA: 10 ml di terreno è sufficiente per 16 embrioni. </li><li> Aggiungere una soluzione efrina ricombinata raggruppata a una concentrazione finale di 20 μg / mL al terreno di coltura. Usa Fc umano cluster come controllo. </li><li> Preparare le piastre di coltura aggiungendo 500 μl di terreno di coltura a ciascun pozzetto di una piastra a 4 pozzetti in polistirolo sterile e non rivestita. </li><li> Utilizzando pinze sterili, posizionare con cura un filtro a membrana in policarbonato con una dimensione di pori di 5 μm per pozzetto in cima al mezzo. Trasferire la piastra preparata all&#39;incubatore (37 ° C / 5% CO 2 ). Assicurarsi che il filtro rimanga asciutto sulla superficie superiore e fluttua. NOTA: Un filtro è sufficiente per due anlagen renali. </li><li> Usando una lama di rasoio, tagliare circa 30 punte pipetta (volume: 100 μL) approssimativamenteY 1 mm dalla punta per provocare un&#39;ampia apertura. Riposizionare le punte in un rack di pipetta. </li></ol> 2. Dissezione dei Rudiments di Metanephric a E11.5 <ol><li> Sacrificare un mouse in gravidanza a E11.5 con dislocazione cervicale (o utilizzando un metodo approvato dal comitato etico locale). </li><li> Rimuovere l&#39;utero e gli embrioni, come descritto da Brown e altri 6 . Da questo punto in avanti, lavorare sotto un microscopio di dissezione. </li><li> Usando la pinza da orologeria n. 5, tagliate l&#39;embrione direttamente sotto i piedini anteriori. Usa un piatto di Petri fresco per ogni embrione. Rimuovere le gambe e la coda dell&#39;embrione. Per fare questo, afferrare il tessuto da rimuovere con una coppia di pinze da orologeria n. 5 e utilizzare le punte di una seconda coppia da tagliare. </li><li> Per rimuovere la massa degli organi ventrali, utilizzare le punte di una coppia di pinze per aprire lateralmente la parete del corpo dell&#39;embrione in una direzione rostrale-caudale. <ol><li> Tagliare superficialmente, parallelo a tL&#39;aorta dorsale. Utilizzare l&#39;aorta dorsale piena di sangue e quindi visibile per orientarsi. Separare con cautela la parte ventrale della parete del corpo dalla parte dorsale utilizzando le punte delle pinze chiuse e far girare l&#39;embrione. Tagliare lateralmente la parete del corpo in una direzione rostrale-caudale e utilizzare l&#39;aorta dorsale per l&#39;orientamento, come per il lato precedente. </li></ol></li><li> Una volta che la parete ventrale del corpo è stata separata dal corpo dorsale, afferrarlo con una coppia di pinze e tirare attentamente per rimuoverla insieme alla massa dell&#39;organo. NOTA: L&#39;anlagen di metanufricoltura di solito rimane attaccato alla parete del corpo dorsale. Sono strutture ovali, ~ 200 μm situate a livello degli arti posteriori. </li><li> Tenere la parete del corpo dorsale con una coppia di pinze, con il lato ventrale rivolto verso l&#39;alto. Utilizzando l&#39;altra coppia di pinze, afferrare l&#39;aorta dorsale alla sua estremità rostrale e sbucchiare attentamente l&#39;aorta dorsale dalla parete del corpo dorsale. NOTA: entrambi i reni metanfricari di solito rimangono attaccatiL&#39;aorta dorsale. </li><li> Usando una coppia di pinze, tagliare rostrally al rene anlagen per rimuovere l&#39;aorta. Quindi, separare con attenzione i due anlagen renali utilizzando le punte delle pinze. </li><li> Scorri con cautela il tessuto indesiderato dal rene anlagen usando le punte delle pinze. Fare attenzione a non danneggiare il mesenchima, in quanto i danni possono provocare una scarsa crescita ed escludere l&#39;esplante dall&#39;ulteriore analisi. </li><li> Trasferire separatamente il rene anlagen usando una pipetta di microlitro e una punta di pipetta ampiamente aperta in 10 μL di PBS al filtro a membrana in policarbonato in una piastra a 4 pozzetti. NOTA: a causa della tensione superficiale, l&#39;esplante sarà coperto con uno strato sottile di media. Un filtro è sufficiente per due anlagen renale. <ol><li> Posizionare ciascuno dei due anlagen al centro di ciascuna rispettiva metà del filtro. Trasferire la piastra all&#39;incubatore a 37 ° C / 5% CO 2 . Lasciare la piastra nell&#39;incubatrice finché non si avvicina il metanufricoLe imitazioni dell&#39;embrione successivo sono pronte per essere poste sul filtro. </li></ol></li><li> Ripetere i passaggi 2.3-2.9.1 per ogni embrione. NOTA: con una certa pratica, la procedura di dissezione richiederà circa 5 minuti per embrione. </li><li> Incubare il rene anlagen per 3 giorni a 37 ° C / 5% CO 2 . </li></ol> 3. Preparazione dei reagenti per la fissazione e la colorazione <ol><li> Per preparare 4% di paraformaldeide (PFA) in PBS senza calcio e magnesio (p / v), sciogliere il PFA a 60 ° C in PBS, continuando a mescolare. Riscaldare a temperatura ambiente e utilizzare un filtro di carta per rimuovere le particelle rimanenti. Aliquota e conservare a 4 ° C per uso immediato o congelare per l&#39;immagazzinamento a lungo termine. ATTENZIONE: PFA è tossico. Indossare l&#39;attrezzatura di protezione individuale specificata negli standard locali di sicurezza e lavorare in una cappa di fumo. Scartare i rifiuti seguendo le linee guida istituzionali e utilizzare contenitori di rifiuti designati. </li><li> Per preparare la soluzione di permeabilizzazione, diluire Triton X-100In PBS senza calcio e magnesio allo 0,3% (v / v). </li><li> Per preparare la soluzione di blocco, aggiungere il siero di capra del 5% (v / v) e lo 0,1% di Triton X-100 a PBS senza calcio e magnesio. Aggiungere azidi di sodio ad una concentrazione finale di 0,02% (w / v). Conservare a 4 ° C. ATTENZIONE: l&#39;azido di sodio è tossico. Maneggiare l&#39;azide di sodio in conformità alle normative locali in materia di sicurezza e ambiente. NOTA: quando si utilizzano anticorpi per la colorazione, utilizzare il siero del 5% della specie che è stato derivato dall&#39;anticorpo secondario per realizzare una soluzione di blocco. </li><li> Diluire l&#39;agglutinina Dolichorus biflorus biotinilata 1: 200 in soluzione di blocco. Diluire streptavidina coniugata Alexa488 1: 200 in soluzione di blocco. </li><li> Per ottenere il mezzo di incisione pronto per l&#39;uso (vedere la tabella dei materiali), aggiungere 0,1 g di glicerolo al 25% (w / v) in 0,1 m di Tris-HCl (pH 8,5) . Riscaldare a 50 ° C sotto agitazione continua per 1 h, raffreddare e regolare il pH a 8.0-8.5 usando 1M NaOH. Evitare concentrazioni più elevate di NaOH. Aggiungere 100 μg / mL N-propilgallato e timerosale ad una concentrazione finale di 0,02% (w / v). Mescolare per 30 minuti a temperatura ambiente. ATTENZIONE: Thimerosal è tossico. Maneggiarlo conformemente alle normative locali in materia di sicurezza e ambiente. <ol><li> Riempire il supporto di incorporazione in tubi da 50 ml, bilanciare il rotore e centrifugare a 3.200 xg e la temperatura ambiente per 10 min. Decantare la soluzione chiara in 15 mL tubi e scartare il pellet. Aliquota e conservare a -20 ° C per diverse settimane. Una volta scongelata, mantenere la soluzione a 4 ° C. Riscaldare a ~ 30 ° C immediatamente prima dell&#39;uso. </li></ol></li><li> Preparare il maggior numero di diapositive in vetro come filtri con esplanti. Per questo, incollare due piccole copertine, 18 x 18 mm, su entrambi i lati del vetrino con l&#39;adesivo istantaneo, lasciare uno spazio di circa 15 mm per il filtro. </li></ol> 4. Fissazione e colorazione <ol><li> Dopo un periodo di coltura di 3Giorni in vitro (div), togliere le piastre dall&#39;incubatore e aspirare attentamente il mezzo di coltura da ciascun pozzetto utilizzando una micropipetta. Assicurarsi di non toccare il filtro e mantenerla aperta verso la parete del pozzetto. </li><li> Aggiungere 500 μl di 4% di PFA / PBS a ciascun pozzetto. I filtri galleggiano sul fissativo. Utilizzando una micropipetta, aggiungete con cautela il fissativo PFA per sottomettere i filtri. Incubare a temperatura ambiente per 30 min. NOTA: Per tutte le fasi successive, occorre prestare attenzione a non lavare le esplosioni dal filtro. Tenere l&#39;apertura della punta della pipetta verso la parete del pozzetto. </li><li> Rimuovere il PFA e risciacquare due volte con 500 μl di PBS, sommersando il filtro. Aggiungere a ciascun pozzetto 500 μL di soluzione di permeabilizzazione e incubare a temperatura ambiente per 1 h. </li><li> Sciacquare due volte con 500 μl di PBS e aggiungere a ciascun pozzetto 500 μL di soluzione di blocco. Incubare a temperatura ambiente per 1 ora. </li><li> Sostituire la soluzione di blocco con250 μl di agglutinina biflorus biotinilata Dolichorus diluita 1: 200 in soluzione di blocco e incubare a 4 ° C per 24 ore. </li><li> Rimuovere la soluzione di agglutinina Doliforum biflorus e risciacquare due volte con 500 μL di PBS per pozzetto. </li><li> Aggiungere 250 μL di streptavidina coniugata con Alexa488, diluita 1: 200 in soluzione di blocco e incubare a 4 ° C per 24 ore. In alternativa, incubare a temperatura ambiente per 2 ore. NOTA: si ottiene un migliore rapporto segnale-rumore quando incubato a 4 ° C. </li><li> Sciacquare due volte con 500 μl di PBS per pozzetto. Utilizzando la pinza, trasferire i filtri sulle vetrate preparate, con gli esplanti rivolti verso l&#39;alto. Montare con ~ 50-100 μL di supporto incorporante. Copertura con coperchio da 1,5 mm di lunghezza da 60 mm. Lasciare che la soluzione di media incorporante si solidifichi per 2 ore a temperatura ambiente al buio. Procedere all&#39;immagine o conservare le diapositive, avvolte in fogli, a 4 ° C fino a quando non sarà pronto per l&#39;immagine. </li><li> Immagine con un ampio campo di epifluorescenza microscopio 7 &lt;/ Sup&gt; accoppiato ad una lampada Hg e utilizzare una unità a specchio per eccitazione blu (banda di eccitazione, 460 - 495 nm, specchio bicromato, 505 nm e banda di emissione, 510 - 550 nm) e 20x Plan-Apochromat lenti, 0,75 numeri apertura. </li></ol>

<ol>
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O., 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=Integrated+&#946;-catenin,+BMP,+PTEN,+and+Notch+signalling+patterns+the+nephron.">Integrated &#946;-catenin, BMP, PTEN, and Notch signalling patterns the nephron.</a> eLife. 4, e04000 (2015).</li><li>Peuckert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+Eph/Ephrin+signaling+controls+several+phases+of+urogenital+development.">Multimodal Eph/Ephrin signaling controls several phases of urogenital development.</a> Kidney Int. 90 (2), 373-388 (2016).</li><li>Pasquale, E. 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(50), e2555 (2011).</li><li>Grobstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inductive+interaction+in+the+development+of+the+mouse+metanephros.">Inductive interaction in the development of the mouse metanephros.</a> J Exp Zool. 130, 319-340 (1955).</li><li>Sax&#233;n, L., Toivonen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Primary Embryonic Induction. , (1962).</li><li>Sax&#233;n, L., Koskimies, O., Lahti, A., Miettinen, H., Rapola, J., Wartiovaara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+kidney+mesenchyme+in+an+experimental+model+system.">Differentiation of kidney mesenchyme in an experimental model system.</a> Adv Morphog. 7, 251-293 (1968).</li><li>Dudley, A. T., Godin, R. E., Robertson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+FGF+and+BMP+signaling+pathways+regulates+development+of+metanephric+mesenchyme.">Interaction between FGF and BMP signaling pathways regulates development of metanephric mesenchyme.</a> Genes Dev. 13, 1601-1613 (1999).</li><li>Per&#228;l&#228;, N., 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=Sema4C-Plexin+B2+signalling+modulates+ureteric+branching+in+developing+kidney.">Sema4C-Plexin B2 signalling modulates ureteric branching in developing kidney.</a> Differentiation. 81 (2), 81-91 (2011).</li><li>Thesleff, I., Ekblom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+transferrin+in+branching+morphogenesis,+growth+and+differentiation+of+the+embryonic+kidney.">Role of transferrin in branching morphogenesis, growth and differentiation of the embryonic kidney.</a> J Embryol exp Morph. 82, 147-161 (1984).</li><li>Watanabe, T., Costantini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+analysis+of+ureteric+bud+branching+morphogenesis.">Real-time analysis of ureteric bud branching morphogenesis.</a> Dev Biol. 271, 98-108 (2004).</li><li>Sebinger, D. 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A., Unbekandt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA-mediated+RNA+interference+in+embryonic+kidney+organ+culture.">siRNA-mediated RNA interference in embryonic kidney organ culture.</a> Methods Mol Biol. 886, 295-303 (2012).</li><li>Sax&#233;n, L., Lehtonen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+kidney+in+organ+culture.">Embryonic kidney in organ culture.</a> Differentiation. 36 (1), 2-11 (1987).</li><li>Bard, J. B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+mouse+kidney+embryogenesis+writ+small.">The development of the mouse kidney embryogenesis writ small.</a> Curr Opin Genet Dev. 2, 589-595 (1992).</li><li>Davies, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+cold+storage+and+transport+of+viable+embryonic+kidney+rudiments.">A method for cold storage and transport of viable embryonic kidney rudiments.</a> Kidney Int. 70 (11), 2031-2034 (2006).</li><li>Batourina, E., 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=Distal+ureter+morphogenesis+depends+on+epithelial+cell+remodeling+mediated+by+vitamin+A+and+Ret.">Distal ureter morphogenesis depends on epithelial cell remodeling mediated by vitamin A and Ret.</a> Nat Genet. 32 (1), 109-115 (2002).</li></ol>

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Questo manoscritto descrive un metodo per isolare l&#39;anlagen metanofrico in sviluppo dall&#39;embrione del topo e per coltivare i rudimenti degli organi. Questo metodo è una tecnica standard, come sviluppato da Grobstein 8 e Saxén 9 , 10 ed è stato adattato e modificato da molti altri 11 , 12 . Il successo del metodo dipende principalmente dalla durata della dissezione, in q...

The mammalian kidney is derived from two primordial structures with mesodermal origin: the tubular epithelial ureteric bud and the metanephric mesenchyme. During nephrogenesis, the ureteric bud invades the metanephric mesenchyme and branches to form the collecting system. The metanephric mesenchyme gives rise to the epithelial elements of the nephrons. These processes occur in a precisely timed and spatially coordinated manner and are initiated by reciprocal inductive mechanisms. Both tissue components communicate and affect the other's cell morphogenesis. 
In the 1920s, it was Boyden who performed the in vivo obstruction of the mesonephric duct in chicken, providing the first indication of inductive interactions as separated nephric blastema fail to differentiate1. At about the same time, the first successful attempts to culture chicken nephric rudiments in a hanging drop were published. Subsequently, the organ culture was developed to study tissue interactions in mammalian organogenesis. In the 1950s, Grobstein developed a technique in which metanephric rudiments could be cultured on a filter. This technique was modified by Sax&#233;n, who placed the filter on a Trowell-type screen in a culture dish1. Over the years, many modifications and applications for organ culture have emerged. The method described here is based on Sax&#233;n's technique but is simplified, as the filters float free on the medium and the diameter of the culture well only slightly exceeds the diameter of the filter, limiting unwanted movement of the filter.
Whole-organ culture is a classical, cheap, and simple but powerful tool to investigate cellular processes and intercellular communication during organogenesis. Organ culture allows for treatment with biological agents, such as growth factors, antibodies, antisense oligonucleotides, viruses, and peptides, as well as with pharmaceutical compounds and other chemicals. Also, gene function may be studied using explants derived from genetically modified mice or using inducible gene inactivation technology, such as the Cre-loxP system. This allows for the study of genetic mutations that cause embryonic lethality prior to the development of the kidney. Organ culture can also be combined with fluorescent tagging for gene function or lineage tracing and modern imaging techniques, which enable real-time monitoring of cell behavior2.
In the specific example provided here, the effect of EphrinB2-activated Eph-receptor signaling on the branching morphology of the ureteric bud was investigated. The morphology of the EphA4/EphB2 double-knockout mice suggested several severe defects in kidney development, which were detectable as early as embryonic day 11 (E11) and involved the ureteric bud, the ureter, and the common nephric duct3. Signaling via Eph receptors requires the clustering of the ligand-receptor dimer4. To over-activate Eph signaling, the kidney rudiments from E11.5 mouse embryos were cultured in the presence of clustered recombinant EphrinB2-Fc. EphrinB2 is a known ligand for the EphA4 receptor, which is expressed in the ureteric bud tips3.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM/F-12</td>
			<td>Thermo Fisher Scientific</td>
			<td>21331020</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, calcium, magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14040117</td>
			<td>use for dissection</td>
		</tr>
		<tr>
			<td>holo-Transferrin human</td>
			<td>Sigma-Aldrich</td>
			<td>T0665</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium (ITS -G) (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>41400045</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B solution</td>
			<td>Sigma-Aldrich</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S8032</td>
			<td></td>
		</tr>
		<tr>
			<td>Thimerosal</td>
			<td>Sigma-Aldrich</td>
			<td>T5125</td>
			<td></td>
		</tr>
		<tr>
			<td>Propyl gallate</td>
			<td>Sigma-Aldrich</td>
			<td>2370</td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol 4-88</td>
			<td>Sigma-Aldrich</td>
			<td>81381</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated Dolichorus Biflorus Agglutinin</td>
			<td>Vector Laboratories</td>
			<td>B-1035</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 conjugated Streptavidin</td>
			<td>Jackson Immuno Research</td>
			<td>016-540-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse Ephrin-B2 Fc Chimera Protein, CF</td>
			<td>R&#38;D Systems</td>
			<td>496-EB</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IgG1 Fc, CF</td>
			<td>R&#38;D Systems</td>
			<td>110-HG-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Human IgG Fc Antibody</td>
			<td>R&#38;D Systems</td>
			<td>G-102-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline tablets</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td>use for fixation and immunostaining</td>
		</tr>
		<tr>
			<td>Dumont #5, biologie 
			tips, INOX, 11cm</td>
			<td>agnthos.se</td>
			<td>0208-5-PS</td>
			<td>2 pairs of forceps are needed</td>
		</tr>
		<tr>
			<td>Iris scissors, straight, 12cm</td>
			<td>agnthos.se</td>
			<td>03-320-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Dressing Forceps, 
			straight, delicate, 13cm</td>
			<td>agnthos.se</td>
			<td>08-032-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes Nunclo Delta treated</td>
			<td>Thermo Fisher Scientific</td>
			<td>150679</td>
			<td></td>
		</tr>
		<tr>
			<td>TMTP01300 Isopore Membrane Filter, polycarbonate, Hydrophilic, 5.0 &#181;m, 13 mm, white, plain</td>
			<td>MerckMillipore</td>
			<td>TMTP01300</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunclon Multidishes 
			4 wells, flat bottom</td>
			<td>Sigma-Aldrich</td>
			<td>D6789-1CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass24x50mm thickn. No.1.5H 0.17+/-0.005mm</td>
			<td>nordicbiolabs</td>
			<td>107222</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glasses No.1.5, 18x18mm</td>
			<td>nordicbiolabs</td>
			<td>102032</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides ~76x26x1, 1/2-w. ground plain</td>
			<td>nordicbiolabs</td>
			<td>1030418</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Razor Blades</td>
			<td>VWR</td>
			<td>55411-055</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430828</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430055</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman prepleated qualitative filter paper, Grade 113V, creped</td>
			<td>Sigma-Aldrich</td>
			<td>WHA1213125</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed stage research mircoscope</td>
			<td>Olympus</td>
			<td>BX61WI</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 6 inbred mice, male, C57BL/6NTac</td>
			<td>Taconic</td>
			<td>B6-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 6 inbred mice,female, C57BL/6NTac</td>
			<td>Taconic</td>
			<td>B6-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Greenough Stereo Microscope</td>
			<td>Leica</td>
			<td>Leica S6 E</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection and culture of mouse embryonic kidney

L&#39;obiettivo di questo protocollo è quello di descrivere un metodo per la dissezione, l&#39;isolamento e la cultura dei rudimenti metanofili del mouse. Durante lo sviluppo del rene dei mammiferi, i due tessuti progenitrici, il germoglio ureterico e il mesenchim metanufrico, comunicano e reciprocamente inducono meccanismi cellulari per formare eventualmente il sistema di raccolta e i nefroni del rene. Poiché gli embrioni di mammiferi crescono intrauterini e pertanto sono inaccessibili all&#39;osservatore, è stata sviluppata una cultura dell&#39;organo. Con questo metodo è possibile studiare le interazioni epiteliali-mesenchimali e il comportamento cellulare durante l&#39;organogenesi renale. Inoltre, l&#39;origine di malformazioni del rene congenito e delle vie urogenitali può essere studiata. Dopo un&#39;attenta dissezione, i rudimenti metanufricani vengono trasferiti su un filtro che galleggia sul mezzo di coltura e può essere mantenuto in un incubatore di colture di cellule per diversi giorni. Tuttavia, bisogna essere consapevoli che le condizioni sonoArtificiale e potrebbe influenzare il metabolismo nel tessuto. Inoltre, la penetrazione delle sostanze di prova potrebbe essere limitata a causa della matrice extracellulare e della membrana basale presente nell&#39;esplosivo. Un vantaggio principale della cultura dell&#39;organo è che lo sperimentatore può ottenere l&#39;accesso diretto all&#39;organo. Questa tecnologia è economica, semplice e consente un gran numero di modifiche, come l&#39;aggiunta di sostanze biologicamente attive, lo studio delle varianti genetiche e l&#39;applicazione di tecniche di imaging avanzate.

L&#39;anlagen renale metanico è stato derivati ​​da topi in gravidanza Black-6 in E11.5 e sono stati coltivati. Dopo 3 giorni, il germoglio ureterico era ramificato fino a 5 volte, con conseguente ramificazione del germoglio uretrico inizialmente a forma di T. Ogni esplante è stato fotografato e il numero di segmenti e endpoint è stato quantificato per determinare le generazioni di ramificazione e per calcolare il numero di endpoint per ramo ( Figura 1 ). ImageJ (...

Watch this Scientific Journal Video about Dissection and Culture of Mouse Embryonic Kidney at JoVE.com

Dissection and Culture of Mouse Embryonic Kidney

Questo protocollo descrive un metodo per isolare e coltivare rudimenti metanufricani da embrioni di topo.

Dissezione e cultura del rene embrionale del mouse

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

dissection-and-culture-of-mouse-embryonic-kidney

Research

JoVE Journal

Developmental Biology

11.8K Views.  Uppsala University.  Questo protocollo descrive un metodo per isolare e coltivare rudimenti metanufricani da embrioni di topo. 

Video: Dissection and Culture of Mouse Embryonic Kidney

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

Su larga scala Zebrafish embrionali Cuore dissezione per l&#39;analisi trascrizionale

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

Ricerca

Biologia dello sviluppo

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Cultura della embrionali di topo cocleari espianti e Gene Transfer con elettroporazione

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

Cultura e co-coltura di mouse ovaie e follicoli ovarici

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

Differenziamento neurale delle cellule staminali embrionali di topo in siero privo monostrato Cultura

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Trascrittoma Profiling di<em&gt; In-Vivo</em&gt; Embrioni bovini pre-impianto prodotto con piattaforma microarray a due colori

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

Cultura della murini embrionali Metatarso: Un modello fisiologico di endocondrale Ossificazione

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

Dissezione e preparazione coronale fetta di sviluppare la ghiandola pituitaria del Mouse

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

Dissezione e colorazione delle ovaie Pupal Drosophila

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

Metodo di coltura organotypic per studiare lo sviluppo dei tessuti embrionali di pollo

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

Dissezione, cultura e analisi delle cellule staminali primarie dal mouse per lo studio della delaminazione e della migrazione delle cellule creste neurali

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...