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

Forfatterne har ingen bekreftelser.

Alle eksperimentelle prosedyrer ble godkjent av Tokyo Metropolitan University Institutional Animal Care and Use Committee (IACUC). Dyrene ble innkapslet og håndtert i samsvar med veiledningen for pleie og bruk av laboratoriedyr og veiledningen for pleie og bruk av eksperimentelle dyr i det kanadiske råd om dyrepleie . Vi brukte dyr oppdrettet og vedlikeholdt på Funksjons Morfologi Laboratory, Department of Frontier Health Sciences, Tokyo Metropolitan University, Japan. </p...

<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>

Dette arbeidet beskriver den eksperimentelle prosedyren for visualisering av innerveringen av den ekstrahepatiske gallveien ved bruk av et antistoff mot nevrofilamentprotein. Denne protokollen ble tilpasset fra protokollen beskrevet av Kuratani og Tanaka 1 , 2 , 3 . For dette eksperimentet planlegger du tidslinjen for hvert av forsøkene, ettersom den fullstendige fargingsmetoden fortsetter i 2-3 uk...

Huset muskeskrue , Suncus murinus , tilhører ordren Insectivora og familien Soricidae. Dette lille pattedyret er fordelt over hele Sørøst-Asia og beboer hus og grøntområder som ligger i nærheten av menneskelige beboer eller storfepenner 4 . Denne arten viser generelle morfologiske egenskaper mer lik mennesker enn andre laboratoriedyr, som mus, rotte og kanin 5 . Tidligere ble helmontert immunfarging med en perifer neuronmarkør for S. murinus nevrofilamentprotein (NFP) brukt til å merke perifere nerver i bukspyttkjertelen 6 , major duodenal papilla 7 , pylorus 8 , galleblæren 5 og ekstrahepatisk biliary 9 . NFP er et makromolekylært kompleks som er en del av det modne neuronale cytoskeletet. Neurofilament-relatert proteinS formidler interaksjoner mellom NFP og zygosomet. Både enzymfunksjonen og strukturen av linkerproteiner reguleres av NFP 10 . NFP-komplekset er laget av tre polypeptider: NF-L (70 kDa), NF-M (150-160 kDa) og NF-H (200 kDa). NFP kan bli funnet i neuronal axon i både sentrale og perifere nervesystemet. Anti-humant NFP antistoff har vist seg å markere aksoner i sentral- og perifert nervesystem. Anatomiske og elektrofysiologiske undersøkelser har vist at sphincter, galleblæren og proksimal gastrointestinalt kanal er forbundet 11 , 12 , 13 , 14 . Imidlertid er de morfologiske egenskapene til deres nevronforbindelser ennå ikke definert. Dette arbeidet demonstrerer bruk av en helmontert immunhistokemisk fargemetode for å merke innerveringen av S. murinus bilIary-kanalen med et NFP-antistoff.

<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

Dette arbeidet beskriver den fullstendige immunhistokjemiske fargemetoden i detalj, ved bruk av nevrofilament-antistoff til å merke innerveringen av galdevegen i Suncus murinus ( S. murinus &#160; ). Først ble prøven dissekert fra S. murinus og fiksert i 4% paraformaldehyd (PFA). For det andre ble en enzymatisk behandling og potensiell endogen peroksidaseinaktivering utført. Prøven ble deretter utsatt for det primære antistoff, anti-nevrofilamentproteinantistoff i 3-6 dager. Den ble deretter inkubert med det sekundære antistoffet konjugert med pepperrotperoksidase. Fargereaksjonen ble avslørt ved å reagere prøven med et 3,3&#39;-diaminobenzidin (DAB) substrat. Denne metoden kan brukes for å analysere innervaringen av alle organer. Videre kan denne protokollen også tilpasses for å teste andre neuronale antistoffer, men optimalisering av antibodiesDet bør gjøres først. Denne metoden ble opprinnelig innført av Kuratani og Tanaka 1 , 2 , 3 .

Figurene 1 - 4 viser typiske resultater for NFP-positive nervefibre i den ekstrahepatiske galdeveien i S. murinus . Antistoffet mot NFP reproduserbart merket innerveringen i hele bildet av den ekstrahepatiske gallveien ( figur 1 ), galleblæren ( figur 2 ), den øvre gallekanalen ( figur 3 ) og duodenal papillatområdet ( figur 4</stron...

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

En helmontert immunohistokemisk tilnærming, for å visualisere nevrofilamentproteinuttrykk i den ekstrahepatiske galdevegen i Suncus murinus. Presenteres her. Denne protokollen kan brukes til å analysere innerveringen av alle viskoser i S. murinus eller andre arter.

Bruke en helmontert immunhistokjemisk metode for å studere innerveringen av galdevegen i<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 ...

Research

JoVE Journal

Biology

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 ...

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 ...

Method for Whole Mount Antibody Staining in Chick

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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 ...

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, ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

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 ...

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 ...

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

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 ...

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

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 ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

Høy oppløsning Whole Mount<em&gt; In Situ</em&gt; Hybridisering innenfor sebrafisk embryoer å studere Gene Expression og funksjon

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...