Name: using a whole-mount immunohistochemical method to study the innervation of the biliary tract in suncus murinus
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Alle eksperimentelle procedurer blev godkendt af Tokyo Metropolitan University Institutional Animal Care and Use Committee (IACUC). Dyrene blev indkvarteret og håndteret i overensstemmelse med vejledningen til pleje og brug af laboratoriedyr og vejledningen til pleje og brug af eksperimentelle dyr i det canadiske råd om dyrepleje . Vi brugte dyr opdrættet og vedligeholdt på Functional Morphology Laboratory, Department of Frontier Health Sciences, Tokyo Metropolitan University, Jap...

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Dette værk beskriver den eksperimentelle procedure til visualisering af det ekstrahepatiske galdevejs indervering ved anvendelse af et antistof mod neurofilamentprotein. Denne protokol blev tilpasset fra protokollen beskrevet af Kuratani og Tanaka 1 , 2 , 3 . For dette eksperiment skal du planlægge tidslinjen for hver af eksperimenterne, da hele monteringsfarvningen går videre i 2-3 uger. Skibet,...

Husets muskeskrue , Suncus murinus , tilhører ordren Insectivora og familien Soricidae. Dette lille pattedyr er fordelt i hele Sydøstasien og beboer huse og græsarealer, der ligger tæt på menneskelige boliger eller kvægpinde 4 . Denne art udviser generelle morfologiske egenskaber mere ligner mennesker end andre laboratoriedyr, såsom musen, rotten og kaninen 5 . Tidligere blev helbredsimmunfarvning med en perifer neuronmarkør for S. murinus neurofilamentprotein (NFP) brugt til at mærke perifere nerver i bugspytkirtlen 6 , major duodenal papilla 7 , pylorus 8 , galdeblære 5 og ekstrahepatisk galdeveje 9 . NFP er et makromolekylært kompleks, som er en del af det modne neuronale cytoskelet. Neurofilament-relateret proteinS formidle interaktioner mellem NFP og zygosomet. Både enzymfunktionen og strukturen af ​​linkerproteiner reguleres af NFP 10 . NFP-komplekset er fremstillet af tre polypeptider: NF-L (70 kDa), NF-M (150-160 kDa) og NF-H (200 kDa). NFP kan findes i neuronale axoner i både det centrale og perifere nervesystem. Anti-humant NFP antistof har vist sig at mærke axoner i det centrale og perifere nervesystem. Anatomiske og elektrofysiologiske undersøgelser har vist, at sphincter, galdeblære og proksimal mave-tarmkanalen er forbundet 11 , 12 , 13 , 14 . Imidlertid er de morfologiske egenskaber ved deres neuronale forbindelser endnu ikke defineret. Dette arbejde demonstrerer anvendelsen af ​​en helmonteret immunohistokemisk farvningsmetode til at mærke innerveringen af S. murinus bilIarytisk kanal med et NFP antistof.

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	<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 arbejde beskriver detaljeret immunhistokemisk farvningsmetode i detaljer ved anvendelse af neurofilamentproteinantistof til at mærke innervation af galdevejen i Suncus murinus ( S. murinus &#160; ). Først blev prøven dissekeret fra S. murinus og fikseret i 4% paraformaldehyd (PFA). For det andet blev en enzymatisk behandling og potentiel endogen peroxidase inaktivering udført. Prøven blev derefter udsat for det primære antistof, anti-neurofilament protein antistof i 3-6 dage. Det blev derefter inkuberet med det sekundære antistof konjugeret med peberrodperoxidase. Farvereaktionen blev afsløret ved at reagere prøven med et 3,3&#39;-diaminobenzidin (DAB) substrat. Denne metode kan anvendes til at analysere innerveringen af ​​alle organer. Desuden kan denne protokol også tilpasses til at teste andre neuronale antistoffer, men optimering af antibodiesDet skal først gøres. Denne metode blev oprindeligt indført af Kuratani og Tanaka 1 , 2 , 3 .

Figur 1 - 4 viser typiske resultater for NFP-positive nervefibre i den ekstrahepatiske galde i S. murinus . Antistoffet mod NFP reproducerbart mærket innervationen i hele billedet af den ekstrahepatiske galde ( figur 1 ), galdeblæren ( figur 2 ), den øvre galdekanal ( figur 3 ) og duodenal papillaområdet ( figur 4 ) med hø...

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 helmonteret immunohistokemisk tilgang til at visualisere neurofilamentproteinekspression i det ekstrahepatiske galde i Suncus murinus. Er præsenteret her. Denne protokol kan bruges til at analysere innerveringen af ​​alle viscerale organer i S. murinus eller andre arter.

Brug af en helmonteret immunhistokemisk metode til at studere indervation af galdevejen 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 ...

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

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