Die Autoren haben keine Anerkennung.

Alle experimentellen Verfahren wurden vom Tokyo Metropolitan University Institutional Animal Care and Use Committee (IACUC) genehmigt. Die Tiere wurden in Übereinstimmung mit dem Leitfaden für die Pflege und Verwendung von Labortieren und dem Leitfaden für die Pflege und Verwendung von Versuchstieren des Kanadischen Rates für Tierpflege untergebracht und behandelt . Wir benutzten Tiere, die im Functional Morphology Laboratory, Department of Frontier Health Sciences, Tokyo Metropolitan University, Japan gezüchtet und gepflegt wurden. 1. Tierpflege und Wohnraum <ol><li> Erhalten Sie 7 S. murinus (3 Frauen und 4 Männer mit einem Gewicht von 70-90 g) aus einer geschlossenen Zuchtkolonie. </li><li> Käfig der S. murinus einzeln nach der Entwöhnung (20 d nach der Geburt) in Plastikkäfige ausgestattet mit einem hölzernen Nistkasten mit Papierstreifen. Halten Sie sie in einem konventionell konditionierten Tierraum: 23-27 ° C, keine Feuchtigkeitsregelung und ein 12:12 h Licht: dunkler Zyklus. Versorgung commercPellets und Wasser ad libitum. </li></ol> 2. Gewebevorbereitung <ol><li> Zur Herstellung von 4% (Gew./Vol.) Paraformaldehyd (PFA) werden 40 g PFA in 1000 ml 0,01 M Phosphat-gepufferter Kochsalzlösung (PBS, pH 7,4) gelöst. In einem Abzug, mischen mit einem Wirbel, bis die Lösung klar ist. Die 4% PFA O / N bei 4 ° C aufbewahren. Achtung: Bei der Handhabung von PFA geeignete persönliche Schutzausrüstung (PSA) tragen. </li><li> Um das Tier zu perfundieren und zu fixieren, es mit Äther zu betäuben und dann eine intraperitoneale Injektion von Somnopentyl (Pentobarbital-Natrium, 0,6 ml / kg Körpergewicht) zu geben. </li><li> Nachdem das Tier vollständig narkotisiert ist, öffne die Bauchhöhle mit einem Skalpell und schaffe einen Mittellinien-Bauchschnitt 3 cm lang. Während man die untere Hohlvene zum Exsanguinieren eindringt, führe einen Katheter nachträglich in die Bauchaorta ein, und zwar auf dem Niveau unmittelbar oberhalb der Verzweigung dieser Arterie in die Arteria iliaca-Arterien. </li><li> Injizieren Sie Somnopentyl (Pentobarbital soDium, 1,0 mL / kg Körpergewicht). Nach vollständiger Euthanasie mit 0,01 M PBS (pH 7,4) und dann mit 4% PFA in einem Abzug. </li><li> Nach der Perfusion 2-3 ml weißer Neopren-Latex (verdünnter weißer Neopren-Latex 3: 2 mit destilliertem Wasser (DW)) einspritzen, um die Blutgefäße zu etikettieren. </li><li> Entleeren Sie die Bauchorgane, einschließlich der Leber, Gallenblase, gemeinsame Gallengang, Duodenum und Pankreas en bloc, mit den verwandten Nerven und Gefäßen. </li><li> Injizieren Sie etwa 0,1 ml blauen Neopren-Latex (fügen Sie einen Tropfen blauer Tinte zu 10 ml des verdünnten weißen Neopren-Latex hinzu) an die Gallenblase, um das Gallensystem zu markieren. </li><li> Nach-Fixierung über Nacht mit 4% PFA bei 4 ° C für Vollmontage-Immunfärbung. Achtung: PFA ist giftig. Vermeiden Sie die Handhabung von PFA direkt. PFA sollte in einem Abzug verwendet werden. </li></ol> 3. Vollständige Immunhistochemie Anmerkung: Während des Protokolls, einschließlich während des Waschens, Antikörperinkubation und Färbung, thDie Probe muss auf dem Nutator bleiben, vorsichtig bei RT oder bei 4 ° C schaukeln . <ol><li> Tag 1: Enzymatische Behandlung und Inaktivierung potentieller endogener Peroxidase <ol><li> Die vorbereitete Probe mit DW 4x für jeweils 1 h bei RT in einer entsprechend bemessenen Glasampulle waschen. Führen Sie die Probe sanft auf die Nutator, um die PFA zu entfernen. Vermeiden Sie es, die Probe beim Austausch von Lösungen zu beschädigen. </li><li> Zur Herstellung von 1% (w / v) Orthopiodsäure, löst man 1 g Orthopiodsäure in 100 ml DW auf. </li><li> Inkubieren Sie die Probe in 1% orthoperiodischer Säure für 20 min bei RT, um eine intrinsische Peroxidase-Reaktion zu verhindern. </li><li> 0,004% (w / v) Papain durch Auflösen von 0,004 g Papain in 100 ml 0,025 mol / l Tris-HCl-Puffer (pH 7,6) vorbereiten. </li><li> Die Probe mit DW für 10 min bei RT waschen. </li><li> Inprobieren Sie die Probe in frisch zubereiteten 0,004% Papain für 2 h bei 37 ° C in einem konstanten Temperaturbad mit sanftem Schütteln. </li><li> Waschen Sie das Exemplar mit DW für 50-60 min bei RT. </li><li> Die Probe in 4% PFA bei 4 ° CO / N aufbewahren. </li></ol></li><li> Tag 2: Enzymatische Behandlung <ol><li> Waschen Sie die gespeicherte Probe mit DW 4 mal für jeweils 1 h bei RT. </li><li> Inprobieren Sie die Probe in frisch zubereiteten 0,004% Papain für 2 h bei 37 ° C in einem konstanten Temperaturbad mit sanftem Schütteln. </li><li> Die Probe mit DW für 50-60 min bei RT waschen. Die Probe in 4% PFA bei 4 ° CO / N aufbewahren. </li></ol></li><li> Tag 3: Einfrieren Behandlung <ol><li> Waschen Sie die gespeicherte Probe mit DW 4x für 1 h jeweils bei RT, wie oben. </li><li> 2% (w / v), 5% (w / v) und 10% (w / v) Saccharose durch Auflösen von 2,5 g, 5 g bzw. 10 g Saccharose in 100 ml 0,01 M PBS ( PH 7,4). </li><li> Tauchen Sie die Probe in 2,5% (w / v), 5% (w / v) und 10% (w / v) Saccharose für jeweils 30 min ein. </li><li> Die Probe bei -20 ° C für 30 - 60 min einfrieren und bei RT vollständig auftauen; Wiederholen Sie den Zyklus 3x. </li><li> Oben2% Triton X-100 (v / v) abgeben, 2 ml Triton X-100 zu 100 ml 0,01 M PBS (pH 7,4) zugeben. </li><li> Die Probe in 2% Triton X-100 bei 4 ° CO / N aufbewahren. </li></ol></li><li> Tag 4: Inkubation des primären Antikörpers <ol><li> Die Verdünnungslösung des primären Antikörpers wird durch Auflösen von 0,2 g Rinderserumalbumin (BSA), 0,3 ml Triton X-100 und 0,1 g Natriumazid in 100 ml 0,01 M PBS (pH 7,4) hergestellt. Achtung: Natriumazid ist akut toxisch. Einatmen und Berühren müssen vermieden werden. Tragen Sie geeignete PPE. </li><li> Verdünnen Sie den primären Antikörper (NFP) 1: 600 im obigen Verdünnungspuffer (Schritt 3.4.1). Inkubieren Sie die Probe mit NFP-Antikörper bei 4 ° C für 3-6 Tage, wobei die Probe vorsichtig auf dem Nuss- Größere Exemplare benötigen erhöhte Inkubationszeiten. </li></ol></li><li> Sekundäre Antikörper-Inkubation <ol><li> Waschen Sie die Probe in PBS 4x für 1 h jeweils bei RT, um ungebundenen primären Antikörper zu entfernen. </lI&gt; <li> Die Verdünnungslösung für den sekundären Antikörper durch Auflösen von 0,2 g BSA und 0,3 ml Triton X-100 in 100 ml 0,01 M PBS (pH 7,4) vorbereiten. </li><li> Verdünnen Sie den sekundären Antikörper (Peroxidase-konjugiertes, affinitätsgereinigtes Schaf-anti-Maus-IgG-HRP) 1: 600 im Verdünnungspuffer (Schritt 3.5.2). Inkubieren Sie die Probe mit dem sekundären Antikörper bei 4 ° C für 3 d und halten Sie die Probe vorsichtig auf den Nutator. </li></ol></li><li> Färbung <ol><li> Vorbereitung der DAB-Farblösung durch Zugabe von 0,002 g 3,3&#39;-Diaminobenzidintetrahydrochlorid (DAB) zu 100 ml 0,05 mol / l Tris-HCl-Puffer (pH 7,6) unter einem Abzug, wobei die Lösung im Dunkeln gehalten wird. </li><li> Waschen Sie die Proben bei RT in PBS 4x für jeweils 1 h. </li><li> 10 μl 30% iges H 2 O 2 in frisch hergestellter DAB-Farblösung zugeben und mit der Lösung bei 4 ° CO / N oder für 3 d inkubieren, während sie vorsichtig auf den Nuss- </li><li> Stoppen Sie die Reaktion von plaCing die Probe in PBS mit 0,04% Natriumazid, wenn die optimale Färbeintensität erreicht ist. HINWEIS: Vermeiden Sie die Verwendung von Natriumazid bis zu diesem Schritt, da es die Meerrettichperoxidase hemmt. </li></ol></li><li> Bildgebung des gefärbten Gewebes <ol><li> Sorgfältig die Probe auf eine Petrischale (5 cm hoch, 10 cm im Durchmesser) mit PBS übertragen. Erfassen Sie ganzheitliche Bilder mit einer Kamera, die auf einem Stereomikroskop montiert ist. HINWEIS: Vollständig gefärbtes Gewebe sollte abgebildet werden, während es vollständig in PBS auf einer transparenten Glasschale eingetaucht wird. </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>

Die Autoren erklären keine Interessenkonflikte.

Diese Arbeit beschreibt das experimentelle Verfahren zur Visualisierung der Innervation des extrahepatischen Gallenweges unter Verwendung eines Antikörpers gegen Neurofilamentprotein. Dieses Protokoll wurde aus dem von Kuratani und Tanaka 1 , 2 , 3 beschriebenen Protokoll angepasst. Für dieses Experiment planen Sie die Timeline für jedes der Experimente, da der Voll-Mount-Färbe-Ansatz für 2-3 W...

Das Haus Moschusspitzmaus , Suncus murinus , gehört zur Ordnung Insektivora und die Familie Soricidae. Dieses kleine Säugetier ist in Südostasien verteilt und bewohnt Häuser und Rasenflächen, die in der Nähe von menschlichen Wohnräumen oder Viehstiften liegen 4 . Diese Spezies weist allgemeine morphologische Merkmale auf, die den Menschen ähnlicher sind als andere Labortiere wie Maus, Ratte und Kaninchen 5 . Bisher wurde eine Vollmontage-Immunfärbung mit einem peripheren Neuron-Marker für S. murinus- Neurofilament-Protein (NFP) verwendet, um die peripheren Nerven in der Bauchspeicheldrüse 6 , der großen Duodenalpapille 7 , dem Pylorus 8 , der Gallenblase 5 und dem extrahepatischen Gallengang 9 zu markieren. NFP ist ein makromolekularer Komplex, der Teil des reifen neuronalen Zytoskeletts ist. Neurofilament-verwandtes ProteinS Zwischenwechsel zwischen NFP und dem Zygosom. Sowohl die Enzymfunktion als auch die Struktur der Linkerproteine ​​werden durch NFP 10 reguliert. Der NFP-Komplex besteht aus drei Polypeptiden: NF-L (70 kDa), NF-M (150-160 kDa) und NF-H (200 kDa). NFP kann in neuronalen Axonen sowohl im zentralen als auch im peripheren Nervensystem gefunden werden. Anti-Human-NFP-Antikörper wurde gezeigt, um Axone des zentralen und peripheren Nervensystems zu etikettieren. Anatomische und elektrophysiologische Untersuchungen haben gezeigt, dass der Sphinkter, die Gallenblase und der proximale Magen-Darm-Trakt 11 , 12 , 13 , 14 verbunden sind. Allerdings sind die morphologischen Merkmale ihrer neuronalen Verbindungen noch nicht definiert. Diese Arbeit zeigt die Verwendung einer ganz-montierten immunhistochemischen Färbemethode, um die Innervation des S. murinus bil zu etikettierenIary Trakt mit einem NFP-Antikörper.

<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

Diese Arbeit beschreibt die Vollständige Immunhistochemie Färbung Methode im Detail, mit Neurofilament Protein-Antikörper, um die Innervation der Gallenwege in Suncus murinus ( S. murinus &#160; ). Zuerst wurde das Exemplar von S. murinus seziert und in 4% Paraformaldehyd (PFA) fixiert. Zweitens wurde eine enzymatische Behandlung und potentielle endogene Peroxidase-Inaktivierung durchgeführt. Die Probe wurde dann dem primären Antikörper, Anti-Neurofilament-Protein-Antikörper, für 3-6 Tage ausgesetzt. Es wurde dann mit dem sekundären Antikörper inkubiert, der mit Meerrettichperoxidase konjugiert war. Die Farbreaktion wurde durch Umsetzen der Probe mit einem 3,3&#39;-Diaminobenzidin (DAB) -Substrat aufgedeckt. Diese Methode kann angewendet werden, um die Innervation aller viszeralen Organe zu analysieren. Darüber hinaus kann dieses Protokoll auch angepasst werden, um andere neuronale Antikörper zu testen, aber die Optimierung der antibodiEs sollte zuerst getan werden. Diese Methode wurde ursprünglich von Kuratani und Tanaka 1 , 2 , 3 eingeführt.

Die Abbildungen 1 - 4 zeigen typische Ergebnisse für NFP-positive Nervenfasern im extrahepatischen Gallengang in S. murinus . Der Antikörper gegen NFP markierte reproduzierbar die Innervation im gesamten Bild des extrahepatischen Gallenganges (Abbildung 1 ), Gallenblase (Abbildung 2 ), oberer Gallengang (Abbildung 3 ) und Duodenalpapillen...

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

Ein ganzheitlicher immunhistochemischer Ansatz, um die Neurofilament-Protein-Expression im extrahepatischen Gallengang in Suncus murinus zu visualisieren. Wird hier vorgestellt. Dieses Protokoll kann verwendet werden, um die Innervation aller viszeralen Organe in S. murinus oder anderen Spezies zu analysieren.

Mit einer vollständigen immunhistochemischen Methode, um die Innervation der Gallenwege zu studieren<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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Biology

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

6.7K Aufrufe.  Tokyo Metropolitan University.  Ein ganzheitlicher immunhistochemischer Ansatz, um die Neurofilament-Protein-Expression im extrahepatischen Gallengang in Suncus murinus zu visualisieren. Wird hier vorgestellt. Dieses Protokoll kann verwendet werden, um die Innervation aller viszeralen Organe in S. murinus oder anderen Spezies zu analysieren. 

Serie: 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.

Testen visueller Sensibilität für die Geschwindigkeit und Richtung der Bewegung in Lizards

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

Forschung

Biologie

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.

Doppel Whole Mount in situ Hybridisierung von Anfang Hühnerembryonen

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.

Verfahren zur Whole Mount Antikörper-Färbung in Küken

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.

Zebrafisch Whole Mount High-Resolution Doppel Fluorescent In-situ Hybridisierung

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.

Hallo-C: eine Methode, um die dreidimensionale Architektur des Genoms Study.

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.

Mit Whole Berg In situ Hybridisierung zu Molecular Biology und Organismal Link-

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.

Whole-mount Immunhistochemische Analyse für Embryonale Limb Haut Gefäßsystem: ein Modellsystem zur Vascular Branching Morphogenese in Embryo-Studie

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.

Ganze Berge In-situ Hybridisierung von E8.5 bis E11.5 Maus Embryonen

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.

Ein hoher Durchsatz In situ Hybridisierung Methode zur mRNA Expression Patterns in der Fetal-Maus unteren Urogenitaltrakts Charakterisieren

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 ...