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Tutte le procedure sperimentali sono state approvate dal Comitato istituzionale per la cura e l&#39;uso degli animali di Tokyo Metropolitan University (IACUC). Gli animali sono stati alloggiati e trattati secondo la Guida per la cura e l&#39;uso degli animali da laboratorio e la Guida per la cura e l&#39;uso degli animali sperimentali del Consiglio canadese sulla cura degli animali. Abbiamo usato animali allevati e mantenuti presso il Laboratorio Morfologico Funzionale, Dipartimento di Scienze della Salute Frontiere, Tokyo Metropolitan University, Giappone. 1. Cura degli animali e alloggi <ol><li> Ottenere 7 S. murinus (3 femmine e 4 maschi che pesano 70-90 g) da una colonia di riproduzione chiusa. </li><li> Gabbia il S. murinus individualmente dopo lo svezzamento (20 d dopo la nascita) in gabbie di plastica dotate di una scatola di nido di legno contenente strisce di carta. Tenerli in un ambiente animale convenzionalmente condizionato: 23-27 ° C, nessun controllo dell&#39;umidità e 12:12 h luce: ciclo scuro. Fornitura commercPellicole di trota e acqua ad libitum. </li></ol> 2. Preparazione del tessuto <ol><li> Per preparare 4% (w / v) paraformaldeide (PFA), sciogliere 40 g di PFA in 1.000 ml di salinio fosfato-bufferizzato da 0,01 M (PBS, pH 7,4). In un armadio di fumo, mescolare usando un vortice fino a quando la soluzione è chiara. Conservare il 4% PFA O / N a 4 ° C. Attenzione: indossare adeguate apparecchiature protettive personali (PPE) durante la manipolazione del PFA. </li><li> Per perfezionare e fissare l&#39;animale, anestetizzarlo con l&#39;etere e poi darle un&#39;iniezione intraperitoneale di somnopentil (pentobarbital sodio, 0,6 ml / kg di peso corporeo). </li><li> Dopo che l&#39;animale è completamente narcotizzato, aprire la cavità addominale con un bisturi, creando un&#39;incisione addominale mediana di 3 cm di lunghezza. Mentre incide la vena cava inferiore per exsanguining, inserire un catetere retrogradamente nell&#39;aorta addominale al livello immediatamente superiore alla biforcazione di questa arteria nelle arterie ilieche comuni. </li><li> Iniettare somnopentil (pentobarbital cosìDium, 1,0 mL / kg di peso corporeo). Dopo la completa eutanasia, perfezionare con 0,01 M PBS (pH 7,4) e poi con 4% PFA in un armadio di fumo. </li><li> Dopo la perfusione, iniettare 2-3 mL di lattice in neoprene bianco (diluire il lattice di neoprene bianco 3: 2 con acqua distillata (DW)) per etichettare i vasi sanguigni. </li><li> Estrarre gli organi addominali, compresi il fegato, la cistifellea, il canale biliare comune, il duodeno e il pancreas in blocco, con i nervi e le patologie correlate. </li><li> Iniettare circa 0,1 ml di lattice blu in neoprene (aggiungere una goccia di inchiostro blu a 10 ml del lattice in neoprene bianco diluito) alla cistifellea per etichettare il sistema biliare. </li><li> Post-fix durante la notte con il 4% di PFA a 4 ° C per l&#39;immunostaining completo. Attenzione: il PFA è tossico. Evitare di gestire direttamente PFA. PFA dovrebbe essere utilizzato in un armadio di fumo. </li></ol> 3. Immunohistochemistry intero-mount Nota: Durante il protocollo, incluso durante il lavaggio, l&#39;incubazione degli anticorpi e la colorazione, thIl campione deve rimanere sul nocciolatore, scivolando delicatamente a RT oa 4 ° C. <ol><li> Giorno 1: trattamento enzimatico e inattivazione di potenziali perossidasi endogena <ol><li> Lavare il campione preparato con DW 4x per 1 h ciascuno a RT in un flaconcino di vetro adeguatamente dimensionato. Far scivolare il campione delicatamente sul nocciolatore per rimuovere il PFA. Evitare di danneggiare il campione quando si scambiano soluzioni. </li><li> Per preparare l&#39;acido ortoperiodico al 1% (v / v), sciogliere 1 g di acido orthoperiodic in 100 mL di DW. </li><li> Incubare il campione in 1% di acido orthoperiodic per 20 minuti a RT per evitare qualsiasi reazione perossidasi intrinseca. </li><li> Preparare il papaino 0,004% (w / v) sciogliendo 0,004 g di papava in 100 ml di tampone Tris-HCl 0,025 mol / l (pH 7,6). </li><li> Lavare il campione con DW per 10 minuti a RT. </li><li> Incubare il campione in papato di 0,004% preparato fresco per 2 ore a 37 ° C in un bagno a temperatura costante con un leggero dondolo. </li><li> Lavare il campione con DW per 50-60 minuti a RT. </li><li> Conservare il campione in 4% PFA a 4 ° CO / N. </li></ol></li><li> 2 ° giorno: trattamento enzimatico <ol><li> Lavare il campione memorizzato con DW 4 volte per 1 h ciascuno a RT. </li><li> Incubare il campione in papato di 0,004% preparato fresco per 2 ore a 37 ° C in un bagno a temperatura costante con un leggero dondolo. </li><li> Lavare il campione con DW per 50-60 minuti a RT. Conservare il campione in 4% PFA a 4 ° CO / N. </li></ol></li><li> Giorno 3: trattamento congelante <ol><li> Lavare il campione memorizzato con DW 4x per 1 h ciascuno a RT, come sopra. </li><li> Preparare rispettivamente 2,5 g, 5 g e 10 g di saccarosio in 100 ml di PBS di 0,01 M (si raccomanda il 2,5% (w / v), il 5% (w / v) e il 10% PH 7,4). </li><li> Immergere il campione in 2,5% (w / v), 5% (w / v) e 10% (w / v) di saccarosio per 30 minuti ciascuno. </li><li> Fermare il campione a -20 ° C per 30-60 minuti e poi scollegarlo completamente a RT; Ripetere il ciclo 3x. </li><li> SuperioreRicaricare il 2% di Triton X-100 (v / v), aggiungere 2 mL di Triton X-100 a 100 mL di 0,01 M PBS (pH 7,4). </li><li> Conservare il campione in 2% di Triton X-100 a 4 ° CO / N. </li></ol></li><li> 4 ° giorno: Incubazione primaria dell&#39;anticorpo <ol><li> Preparare la soluzione di diluizione dell&#39;anticorpo primario sciogliendo 0,2 g di albumina Bovine Serum (BSA), 0,3 ml di Triton X-100 e 0,1 g di sodio azide in 100 ml di PBS da 0,01 M (pH 7,4). Attenzione: l&#39;azido di sodio è acutamente tossico. Inalazione e tocco devono essere evitati. Indossare un adeguato DPI. </li><li> Diluire l&#39;anticorpo primario (NFP) 1: 600 nel precedente tampone di diluizione (punto 3.4.1). Incubare il campione con l&#39;anticorpo NFP a 4 ° C per 3-6 giorni, mantenendo il campione delicatamente dondolando sul nutatore. I campioni più grandi richiedono tempi di incubazione aumentati. </li></ol></li><li> Incubazione secondaria anticorpale <ol><li> Lavare il campione in PBS 4x per 1 h ciascuno a RT per rimuovere l&#39;anticorpo primario non legato. </li&gt; <li> Preparare la soluzione di diluizione per l&#39;anticorpo secondario sciogliendo 0,2 g di BSA e 0,3 ml di Triton X-100 in 100 ml di PBS di pH 0,01 (pH 7,4). </li><li> Diluire l&#39;anticorpo secondario (perossidasi-coniugato, affinità-pecore anti-mouse IgG-HRP) 1: 600 nel tampone di diluizione (punto 3.5.2). Incubare il campione con l&#39;anticorpo secondario a 4 ° C per 3 d, e mantenere il campione dondolando delicatamente sul nocciolo. </li></ol></li><li> Colorazione <ol><li> Preparare la soluzione di colorazione DAB con l&#39;aggiunta di 0,002 g di 3,3&#39;-diaminobenzidin-tetraidloruro (DAB) a 100 ml di tampone Tris-HCl (pH 7,6) di 0,05 mol / l sotto un armadio di fumo mantenendo la soluzione al buio. </li><li> Lavare gli esemplari a RT in PBS 4x per 1 h ciascuno. </li><li> Aggiungere 10 μl di 30% di H 2 O 2 in soluzione di colorazione DAB preparata di fresco e incubare con la soluzione a 4 ° CO / N o per 3 giorni mentre dondolando delicatamente sul nutatore. </li><li> Arrestare la reazione con plaIl campione in PBS con azoto sodico al 0,04% quando viene raggiunta l&#39;intensità di colorazione ottimale. NOTA: Evitare l&#39;uso di sodio azide fino a questo passaggio, in quanto inibisce la perossidasi di rafano. </li></ol></li><li> Imaging del tessuto macchiato <ol><li> Trasferire con cura il campione su un piatto di vetro di Petri (5 cm di altezza, 10 cm di diametro) contenente PBS. Cattura immagini integrali utilizzando una fotocamera montata su uno stereomicroscopio. NOTA: Il tessuto macchiato interamente montato dovrebbe essere imaged mentre è immerso completamente in PBS su un piatto di vetro trasparente. </li></ol></li></ol>

<ol>
	<li>Kuratani, S., Tanaka, S., Ishikawa, Y., Zukeran, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+development+of+the+hypoglossal+nerve+in+the+chick+embryo+as+observed+by+the+whole-mount+nerve+staining+method.">Early development of the hypoglossal nerve in the chick embryo as observed by the whole-mount nerve staining method.</a> Am. J. Anat. 182 (2), 155-168 (1988).</li><li>Kuratani, S., Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+development+of+the+avian+vagus+nerve+with+special+reference+to+the+morphological+innervation+of+heart+and+lung.">Peripheral development of the avian vagus nerve with special reference to the morphological innervation of heart and lung.</a> Anat. Embryol. (Berl). 182 (5), 435-445 (1990).</li><li>Kuratani, S., Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+development+of+avian+trigeminal+nerves.">Peripheral development of avian trigeminal nerves.</a> Am. J. Anat. 187 (1), 65-80 (1990).</li><li>Yi, S. Q., 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=House+musk+shrew,+Suncus+murinus:+a+novel+and+natural+obesity-resistant+animal+model.">House musk shrew, Suncus murinus: a novel and natural obesity-resistant animal model.</a> Obes. Metab. 6, 22-28 (2010).</li><li>Yi, S. Q., 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=Surgical+anatomy+of+innervation+of+the+gallbladder+in+humans+and+Suncus+murinus+with+special+reference+to+morphological+understanding+of+gallstone+formation+after+gastrectomy.">Surgical anatomy of innervation of the gallbladder in humans and Suncus murinus with special reference to morphological understanding of gallstone formation after gastrectomy.</a> World J. Gastroenterol. 13 (14), 2066-2071 (2007).</li><li>Yi, S. Q., 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=Anatomical+study+of+the+pancreas+in+the+house+musk+shrew,+Suncus+murinus,+with+special+reference+to+the+blood+supply+and+innervation.">Anatomical study of the pancreas in the house musk shrew, Suncus murinus, with special reference to the blood supply and innervation.</a> Anat. Rec. A Discov. Mol. Cell Evol. Biol. 273 (1), 630-635 (2003).</li><li>Yi, S. Q., 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=Surgical+anatomy+of+the+innervation+of+the+major+duodenal+papilla+in+humans+and+Suncus+murinus,+from+the+perspective+of+preserving+innervation+in+organ-saving+procedures.">Surgical anatomy of the innervation of the major duodenal papilla in humans and Suncus murinus, from the perspective of preserving innervation in organ-saving procedures.</a> Pancreas. 30 (3), 211-217 (2005).</li><li>Yi, S. Q., 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=Surgical+anatomy+of+the+innervation+of+the+pylorus+in+humans+and+Suncus+murinus,+in+relation+to+the+surgical+technique+for+pylorus-preserving+pancreatoduodenectomy.">Surgical anatomy of the innervation of the pylorus in humans and Suncus murinus, in relation to the surgical technique for pylorus-preserving pancreatoduodenectomy.</a> World J. Gastroenterol. 12 (14), 2209-2216 (2006).</li><li>Yi, S. Q., Ren, K., Kinoshita, M., Takano, N., Itoh, M., Ozaki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innervation+of+Extrahepatic+Biliary+Tract,+With+Special+Reference+to+the+Direct+Bidirectional+Neural+Connections+of+the+Gall+Bladder,+Sphincter+of+Oddi+and+Duodenum+in+Suncus+murinus,+in+Whole-Mount+Immunohistochemical+Study.">Innervation of Extrahepatic Biliary Tract, With Special Reference to the Direct Bidirectional Neural Connections of the Gall Bladder, Sphincter of Oddi and Duodenum in Suncus murinus, in Whole-Mount Immunohistochemical Study.</a> Anat. Histol. Embryol. 45 (3), 184-188 (2016).</li><li>Wang, H., 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=Neurofilament+proteins+in+axonal+regeneration+and+neurodegenerative+diseases.">Neurofilament proteins in axonal regeneration and neurodegenerative diseases.</a> Neural Regen. Res. 7 (8), 620-626 (2012).</li><li>Wyatt, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+of+the+sphincter+of+Oddi+to+the+stomach,+duodenum+and+gall-bladder.">The relationship of the sphincter of Oddi to the stomach, duodenum and gall-bladder.</a> J. Physiol. 193 (2), 225-243 (1967).</li><li>Mawe, G. M., Kennedy, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duodenal+neurons+provide+nicotinic+fast+synaptic+input+to+sphincter+of+Oddi+neurons+in+guinea+pig.">Duodenal neurons provide nicotinic fast synaptic input to sphincter of Oddi neurons in guinea pig.</a> Am. J. Physiol. 277, G226-G234 (1999).</li><li>Seo, J. H., Cho, S. S., Lee, I. S., Lee, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+and+neuropeptidergic+properties+of+the+duodenal+neurons+projecting+to+the+gallbladder+in+the+golden+hamster.">Anatomical and neuropeptidergic properties of the duodenal neurons projecting to the gallbladder in the golden hamster.</a> Arch. Histol. Cytol. 65 (4), 317-321 (2002).</li><li>Padbury, R. T., Furness, J. B., Baker, R. A., Toouli, J., Messenger, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+of+nerve+cells+from+the+duodenum+to+the+sphincter+of+Oddi+and+gallbladder+of+the+Australian+possum.">Projections of nerve cells from the duodenum to the sphincter of Oddi and gallbladder of the Australian possum.</a> Gastroenterology. 104 (1), 130-136 (1993).</li><li>White, J. J., Reeber, S. L., Hawkes, R., Sillitoe, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wholemount+immunohistochemistry+for+revealing+complex+brain+topography.">Wholemount immunohistochemistry for revealing complex brain topography.</a> J. Vis. Exp. (62), e4042 (2012).</li></ol>

Gli autori non dichiarano conflitti di interesse.

Questo lavoro descrive la procedura sperimentale per la visualizzazione dell&#39;innervazione del tratto biliare extraepatico usando un anticorpo contro la proteina del neurofilamento. Questo protocollo è stato adattato dal protocollo descritto da Kuratani e Tanaka 1 , 2 , 3 . Per questo esperimento, pianificare la timeline per ciascuno degli esperimenti, in quanto l&#39;approccio di colorazione a tutto tondo continua per 2-3 settimane. La vasca contenente il tessuto deve essere ruotata in un nutatore in qualsiasi momento, tranne che durante il processo di congelamento / scongelamento. Particolarmente durante l&#39;incubazione con gli anticorpi primari e secondari, il campione è stato impostato in una camera di stoccaggio fredda mentre ruota su un nutatore per garantire l&#39;esposizione uniforme del campione alle soluzioni di reazione. Diverse soluzioni - 1% (w / v) acido ortoperiodico, 0,004% (w / v) papain e il tampone di diluizione per l&#39;antibo primario / secondarioMuore - deve essere fresco per ogni esperimento. Durante il protocollo, per evitare di toccare e danneggiare il campione quando si cambiano le soluzioni, le soluzioni devono essere versate invece di essere rimosse con le pinze 15 . Dopo il fissaggio con PFA, i campioni devono essere lavati sufficientemente con DW o PBS. Inoltre, il trattamento enzimatico con l&#39;incubazione papaina è importante. Il calore e l&#39;enzima usato per il recupero di antigeni aiutano a preparare il tessuto per la penetrazione di anticorpi. Per migliorare la penetrazione delle soluzioni, la membrana esterna del tessuto deve essere interrotta con un trattamento di congelamento a -20 ° C per 30 minuti o -80 ° CO / N 15 . È importante sommare completamente il campione sperimentale in adeguati volumi di soluzione tampone per assicurare che la soluzione possa incubare l&#39;intero campione. Il tempo di incubazione dell&#39;anticorpo primario deve essere determinato empiricamenteD per diversi tessuti e dimensioni del campione. Di solito, deve essere di 3 giorni per un campione di 2-3 cm. La diluizione ottimale deve essere determinata empiricamente per ciascun anticorpo. Si raccomanda una diluizione a 1: 600 sia per gli anticorpi primari che per quelli secondari quando si utilizza il appropriato tampone anticorpale. Questo lavoro descrive in dettaglio il protocollo di un approccio immunohistochemico versatile a tutto sesto per rivelare l&#39;espressione proteica del neurofilamento nel tratto biliare di S. murinus . Questo metodo può essere utilizzato per analizzare l&#39;innervazione di tutti gli organi viscerali in molte specie. Inoltre, questo protocollo può anche essere adattato per testare altri anticorpi neuronali, ma deve essere eseguita l&#39;ottimizzazione degli anticorpi. Tuttavia, la tecnica è stata limitata nell&#39;etichettatura dei tessuti nervosi superficiali e nella costruzione di un modello tridimensionale di innervazione. Inoltre, non ha potuto identificare le caratteristiche delle fibre nervose ( cioè simpatiche o parAsintomatici, motori o sensoriali, ecc. ).

Il muschio della casa, Suncus murinus , appartiene all&#39;ordine Insectivora e alla famiglia Soricidae. Questo piccolo mammifero è distribuito in tutto il Sud-Est asiatico e abita case e aree erbose che si trovano vicino a abitazioni umane o penne di bestiame 4 . Questa specie presenta caratteristiche morfologiche generali più simili agli esseri umani rispetto ad altri animali da laboratorio, come il topo, il topo e il coniglio 5 . In precedenza, l&#39;immunostaining con un marker di neurone periferico per la proteina del neurofilamento di S. murinus (NFP) è stato usato per etichettare i nervi periferici nel pancreas 6 , maggiore papilla duodenale 7 , pylorus 8 , cistifellea 5 e tratto biliare extraepatico 9 . NFP è un complesso macromolecolare che fa parte del citoscheletro neuronale maturo. Proteina legata al neurofilamentoLe interazioni mediate tra NFP e zigosoma. La funzione enzimatica e la struttura delle proteine ​​di collegamento sono regolate da NFP 10 . Il complesso NFP è costituito da tre polipeptidi: NF-L (70 kDa), NF-M (150-160 kDa) e NF-H (200 kDa). NFP può essere trovato in assoni neuronali sia nel sistema nervoso centrale che nei periferici. È stato dimostrato che l&#39;anticorpo anti-umano del NFP ha etichettato assoni del sistema nervoso centrale e periferico. Le indagini anatomiche ed elettrofisiologiche hanno dimostrato che lo sfintere, la cistifellea e il tratto gastrointestinale prossimale sono collegati 11 , 12 , 13 , 14 . Tuttavia, le caratteristiche morfologiche delle loro connessioni neuronali non sono ancora state definite. Questo lavoro dimostra l&#39;utilizzo di un metodo di colorazione immunohistochemico a tutto campo per etichettare l&#39;innervazione del S. murinus bilCon un anticorpo NFP.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">orthoperiodic acid</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">162-00732</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">papain</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">164-00172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bovine serum albumin (BSA)</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">010-15131</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sodium azide</td>
			<td style="width:71px;">Nacalai Tesque</td>
			<td style="width:119px;">312-33</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">neurofilament protein (NFP) antibody</td>
			<td style="width:71px;">Dako</td>
			<td style="width:119px;">M0762, lot 089, clone: 2F11</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">peroxidase-conjugated affinity-purified sheep anti-mouse IgG-HRP</td>
			<td style="width:71px;">MBL</td>
			<td style="width:119px;">Code 330</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3,3&#39;-diaminobenzidine tetrahydrochloride (DAB)</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">349-00903</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">imidazole</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">I-0125</td>
			<td></td>
		</tr>
	</tbody>
</table>

using a whole-mount immunohistochemical method to study the innervation of the biliary tract in suncus murinus

Questo lavoro descrive dettagliatamente il metodo di colorazione immunohistochemica a tutto campo, utilizzando l&#39;anticorpo proteico neurofilamento per etichettare l&#39;innervazione del tratto biliare in Suncus murinus ( S. murinus &#160; ). In primo luogo, il campione è stato disciolto da S. murinus e fissato in 4% di paraformaldeide (PFA). In secondo luogo, sono stati eseguiti un trattamento enzimatico e una potenziale inattivazione endogena della perossidasi. Il campione è stato poi esposto all&#39;anticorpo primario, anticorpo anti-neurofilamento proteico, per 3-6 giorni. Fu quindi incubato con l&#39;anticorpo secondario coniugato con perossidasi di rafano. La reazione a colori è stata rivelata reagendo il campione con un substrato 3,3&#39;-diaminobenzidina (DAB). Questo metodo può essere applicato per analizzare l&#39;innervazione di tutti gli organi viscerali. Inoltre, questo protocollo può anche essere adattato per testare altri anticorpi neuronali, ma l&#39;ottimizzazione degli antibodiDovrebbe essere fatto prima. Questo metodo è stato originariamente introdotto da Kuratani e Tanaka 1 , 2 , 3 .

Le figure da 1 a 4 mostrano risultati tipici per le fibre nervose NFP-positive nel tratto biliare extraepatico di S. murinus . L&#39;anticorpo contro NFP riproduce etichettato l&#39;innervazione nell&#39;intera immagine del tratto biliare extraepatico ( Figura 1 ), della cistifellea ( Figura 2 ), del canale biliare superiore ( Figura 3 ) e dell&#39;area della papilla duodenale ( Figura 4 ), con elevata specificità e minima sfondo. Per tutti i tessuti del campione, indipendentemente dalla forma e dalla dimensione, i nervi tegmentali possono essere tinti contemporaneamente. Oltre all&#39;innervazione del tratto biliare extraepatico, la densità di funzionamento e distribuzione del vagus dell&#39;esofago e dello stomaco è stata esposta in modo non ambiguo ( Figura 1 ). <p clMentre i vasi sanguigni della cistifellea erano etichettati con lattice in neoprene bianco, le fibrose nervose sottili sono state chiaramente dimostrate con un elevato contrasto. La densità dell&#39;innervazione era più alta nel collo che nel fondo della cistifellea ( Figura 2 ). Nel condotto biliare superiore sono stati etichettati due tipi di fasci nervosi. I fasci nervosi fini formavano una rete irregolare e densa di nervi, corse ad adesivo e risiedevano nel tratto biliare; I fasci neurali più spessi sono stati distribuiti paralleli alla superficie del tratto biliare ( Figura 3 ). Il condotto biliare comune è stato etichettato con lattice blu neoprene e le vasi con lattice in neoprene bianco. Le fibre nervose sottili alla fine del canale biliare comune e l&#39;area papilla duodenale sono state chiaramente dimostrate con c<html> Ontrast ( Figura 4 ). <img alt="Figura 1" class="xfigimg" src="/files/ftp_upload/55483/55483fig1.jpg" /> Figura 1: Fibre nervose NFP-positive nel tratto biliare, l&#39;esofago e lo stomaco. Le frecce mostrano il vagus che corre lungo l&#39;esofago allo stomaco. CBD, Common Bile Duct; Es, esofago; St, stomaco. Barra di scala = 1.300 μm. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55483/55483fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a> <img alt="figura 2" class="xfigimg" src="/files/ftp_upload/55483/55483fig2.jpg" /> Figura 2: NFP-positive fibre nervose nella cistifellea. I vasi sanguigni della cistifellea erano etichettati con lattice bianco. L&#39;innervazione abbondante si è verificata nel collo della colecisti (frecce). Barra di scala = 1000 μm.Ref = &quot;https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55483/55483fig2large.jpg&quot; target = &quot;_ blank&quot;&gt; Fare clic qui per visualizzare una versione più grande di questa figura. <img alt="Figura 3" class="xfigimg" src="/files/ftp_upload/55483/55483fig3.jpg" /> Figura 3: Fibre nervose NFP-positive nel tubo superiore. Sono stati osservati due tipi di fasci nervosi. Un tipo era i fasci nervosi fini che correvano adesivamente e si trovavano nel / nel tratto biliare extraepatico (frecce); L&#39;altro tipo era fasci neurali più spessi distribuiti parallelamente alla superficie del tratto biliare extraepatico e correva tra la colecisti e il duodeno (triangoli). PV, Portal Vein. Barra di scala = 650 μm. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55483/55483fig3large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a> <img alt="Figura 4" class= &quot;Xfigimg&quot; src = &quot;/ files / ftp_upload / 55483 / 55483fig4.jpg&quot; /&gt; Figura 4: Fibre nervose NFP-positive nell&#39;area papilla duodenale. Il canale biliare comune è stato etichettato con lattice blu e le vasi con lattice bianco (*). Le frecce mostrano l&#39;innervazione dell&#39;estremità del condotto biliare comune e i triangoli mostrano l&#39;innervazione dell&#39;area della papilla duodenale (P), proveniente dal canale biliare comune e dai vasi. Barra di scala = 1000 μm. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55483/55483fig4large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus at JoVE.com

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

Un approccio immunoistochimico a tutto tondo, per visualizzare l&#39;espressione di proteine ​​del neurofilamento nel tratto biliare extraepatico in Suncus murinus. È qui presentato. Questo protocollo può essere utilizzato per analizzare l&#39;innervazione di tutti gli organi viscerali di S. murinus o di altre specie.

Utilizzando un metodo immunohistochemico integrale per studiare l&#39;innervazione del tratto bipolare in<em&gt; Suncus murinus</em

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the ...

using-whole-mount-immunohistochemical-method-to-study-innervation

Nanyang Technological University

Research

JoVE Journal

Biology

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

6.7K Views.  Tokyo Metropolitan University.  Un approccio immunoistochimico a tutto tondo, per visualizzare l'espressione di proteine ​​del neurofilamento nel tratto biliare extraepatico in Suncus murinus. È qui presentato. Questo protocollo può essere utilizzato per analizzare l'innervazione di tutti gli organi viscerali di S. murinus o di altre specie. 

Video: Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test di sensibilità visiva alla velocità e direzione del movimento in lucertole

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Ricerca

Biologia

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 ...

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 ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hi-C: un metodo per studiare l&#39;architettura tridimensionale dei genomi.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

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

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

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 ...

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 ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

A High Throughput In situ Metodo di ibridazione di caratterizzare gli schemi di espressione di mRNA del mouse fetale basso tratto urogenitale

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

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 ...