Name: using a whole-mount immunohistochemical method to study the innervation of the biliary tract in suncus murinus
Uploaded: 2017-06-15T12:30:00.000+00:00
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Description: 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

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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>

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	<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>

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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 ...

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><tbody><tr><td>orthoperiodic acid</td><td>Wako</td><td>162-00732</td><td></td></tr><tr><td>papain</td><td>Wako</td><td>164-00172</td><td></td></tr><tr><td>bovine serum albumin (BSA)</td><td>Wako</td><td>010-15131</td><td></td></tr><tr><td>sodium azide</td><td>Nacalai Tesque</td><td>312-33</td><td></td></tr><tr><td>neurofilament protein (NFP) antibody</td><td>Dako</td><td>M0762, lot 089, clone: 2F11</td><td></td></tr><tr><td>peroxidase-conjugated affinity-purified sheep anti-mouse IgG-HRP</td><td>MBL</td><td>Code 330</td><td></td></tr><tr><td>3,3&#039;-diaminobenzidine tetrahydrochloride (DAB)</td><td>Wako</td><td>349-00903</td><td></td></tr><tr><td>imidazole</td><td>Sigma</td><td>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 del...

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 ...

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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

Immunostaining to Visualize Murine Enteric Nervous System Development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Immunostaining Per visualizzare Murine enterico nervoso Sistemi Di Sviluppo

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must ...

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Biologia dello sviluppo

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

Localizzazione dell'immunofluorescenza in vivo per la valutazione della biodistribuzione terapeutica e diagnostica degli anticorpi nella ricerca sul cancro

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

Ricerca sul cancro

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

Determinazione della densità del dotto biliare nel fegato del topo

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

Bile Duct Ligation in topi: Induzione infiammatoria epatica Lesioni e fibrosi da colestasi ostruttiva

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Medicina

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

In Situ Ca 2+ Imaging del sistema nervoso enterico

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Neuroscienze

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

Visualizzazione delle cellule interstiziali di Cajal (ICC) di rete in topi

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Biologia

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Imaging intravitale dei linfociti intraettali a Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Immunologia e infezioni

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian countries, with a reported frequency of 1 in 5,000 in Taiwan. Although much is known about the management of biliary atresia, its pathogenesis is still elusive. The sea lamprey (Petromyzon marinus) provides a unique opportunity to examine the mechanism and progression of biliary degeneration. Sea lamprey develop through three distinct life stages: larval, parasitic, and adult. During the transition from larvae to parasitic juvenile, sea lamprey undergo metamorphosis with dramatic reorganization and remodeling in external morphology and internal organs. In the liver, the entire biliary system is lost, including the gall bladder and the biliary tree. A newly-developed method called &#8220;CLARITY&#8221; was modified to clarify the entire liver and the junction with the intestine in metamorphic sea lamprey. The process of biliary degeneration was visualized and discerned during sea lamprey metamorphosis by using laser scanning confocal microscopy. This method provides a powerful tool to study biliary atresia in a unique animal model.

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus

Sea lamprey lose the gall bladder and bile ducts during metamorphosis, a process similar to human biliary atresia. A new fixation and clarification method (CLARITY) was modified to visualize the entire biliary tree using laser scanning confocal microscopy. This method provides a powerful tool to study biliary degeneration.

Un metodo Chiarimento Nuovo per visualizzare biliare degenerazione Durante fegato Metamorfosi in mare Lamprey ( Petromyzon marinus)

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Murine Precision-Cut Liver Slices come un modello Ex Vivo di biologia del fegato

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Location, Dissection, and Analysis of the Murine Stellate Ganglion

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter of investigation in the pathophysiology of ventricular arrhythmias (VA). Neurons in the stellate ganglia (SG)&#160;&#8211;&#160;bilateral star-shaped structures of the sympathetic chain &#8211;&#160;are an important component of the sympathetic infrastructure. The SG are a recognized target for&#160;treatment via cardiac sympathetic denervation in patients with therapy-refractory VA. While neuronal remodeling and glial activation in the SG have been described in patients with VA, the underlying cellular and molecular processes that potentially precede the onset of arrhythmia are only insufficiently understood and should be elucidated to improve autonomic modulation. Mouse models allow us to study sympathetic neuronal remodeling, but identification of the murine SG is challenging for the inexperienced investigator. Thus, in-depth cellular and molecular biological studies of the murine SG are lacking for many common cardiac diseases. Here, we describe a basic repertoire for dissecting and studying the SG in adult mice for analyses at RNA level (RNA isolation for gene expression analyses, in situ hybridization), protein level (immunofluorescent whole mount staining), and cellular level (basic morphology, cell size measurement). We present potential solutions to overcome challenges in the preparation technique, and how to improve staining via quenching of autofluorescence. This allows for the visualization of neurons as well as glial cells via established markers in order to determine cell composition and remodeling processes. The methods presented here allow characterizing the SG to gain further information on autonomic dysfunction in mice prone to VA and can be complemented by additional techniques investigating neuronal and glial components of the autonomic nervous system in the heart.

Pathophysiological changes in the cardiac autonomic nervous system, especially in its sympathetic branch, contribute to the onset and maintenance of ventricular arrhythmias. In the present protocol, we show how to characterize murine stellate ganglia to improve the understanding of the underlying molecular and cellular processes.

Posizione, dissezione e analisi del Ganglio Stellato Murine

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter ...

Utilizzando un metodo immunohistochemico integrale per studiare l'innervazione del tratto bipolare in

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