Name: immunostaining to visualize murine enteric nervous system development
Uploaded: 2015-04-29T13:00:00
Description: Watch this Scientific Journal Video about Immunostaining to Visualize Murine Enteric Nervous System Development at JoVE.com

Detta arbete stöddes bythe amerikanska Pediatric kirurgiska Association Foundation Award (AG) och National Institutes of Health K08DK098271 (AG).

University of Wisconsin Djurvård och användning kommittén godkände alla förfaranden. 1. Beredning av lösningar <ol><li> Använd 1x fosfatbuffrad saltlösning (PBS) som dissektion buffert och sköljning lösning. </li><li> Förbered 30% sackaros genom att väga 30 g sackaros och plats i en flaska. Lägg 99 ml 1x PBS och tillsätt 1 ml 10% natriumazid. Blanda omsorgsfullt tills allt av sackarosen har lösts upp. Förvaras vid 4 ° C tills de behövdes. </li><li> Bered Blockering...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+and+polyvalent+antiserum+to+chicken+choline+acetyltransferase.">Monoclonal antibodies and polyvalent antiserum to chicken choline acetyltransferase.</a> J Neurochem. 46 (3), 968-976 (1986).</li><li>Tooyama, I., Kimura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protein+encoded+by+an+alternative+splice+variant+of+choline+acetyltransferase+mRNA+is+localized+preferentially+in+peripheral+nerve+cells+and+fibers.">A protein encoded by an alternative splice variant of choline acetyltransferase mRNA is localized preferentially in peripheral nerve cells and fibers.</a> J Chem Neuroanat. 17 (4), 217-226 (2000).</li><li>Koga, T., Bellier, J. -. P., Kimura, H., Tooyama, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoreactivity+for+Choline+Acetyltransferase+of+Peripheral-Type+(pChAT)+in+the+Trigeminal+Ganglion+Neurons+of+the+Non-Human+Primate+Macaca+fascicularis.">Immunoreactivity for Choline Acetyltransferase of Peripheral-Type (pChAT) in the Trigeminal Ganglion Neurons of the Non-Human Primate Macaca fascicularis.</a> Acta histochemica et cytochemica. 46 (2), 59-64 (2013).</li><li>Sang, Q., Young, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+identification+and+chemical+coding+of+cholinergic+neurons+in+the+small+and+large+intestine+of+the+mouse.">The identification and chemical coding of cholinergic neurons in the small and large intestine of the mouse.</a> Anat Rec. 251 (2), 185-199 (1998).</li><li>Lei, J., Howard, M. 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Vårt laboratorium och andra har visat att tarm defekter i HSCR inte är begränsade till den aganglionic kolon men förlängdes proximalt, även i ganglionated tunntarmen 5,15,16. Dessa förändringar innefattar förändringar i ENS neuronal densitet och signalsubstans fenotyp och kan redogöra för dysmotilitet som har observerats hos patienter med HSCR. Vi har använt ovanstående tekniker i vår strävan att förstå faktorerna för ENS bildning. Specifikt har dessa tekniker använts för att visualisera ...

En fungerande enteriska nervsystemet (ENS), som kontrollerar motilitet, absorptionen av näringsämnen, och lokalt blodflöde, är nödvändigt för liv 1. Den ENS bildas av neurallist celler (NCC) som förökar sig, migrera och kolonisera tarmen, där de differentiera till ganglier innehåller neuroner och gliaceller. Hirschsprungs sjukdom (HSCR, Online Mendelian Inheritance in Man), en multigeneic medfödd sjukdom med en incidens på 1 på 4.000 levande födda, kan anses vara prototypiska sjukdomen för att studera störs ENS bildning. I HSCR NCC inte att migrera till och kolonisera varierande längder av den distala hindgut 2. Dessutom är andra vanliga gastrointestinal (GI) utvecklings defekter i den pediatriska populationen, såsom anorektala missbildningar, tarm atresias och motilitetsstörningar i samband med störningar i grundläggande ENS funktioner, och sannolikt i samband med subtila, underappreciated, anatomiska förändringar och funktionella förändringar iENS 3-6. Därför kan tekniker som gör det möjligt för oss att förstå utvecklings faktorerna för ENS bildning belysa patogenesen och potentiell behandling av pediatriska GI vägssjukdomar. Efter migration och kolonisation, skiljer NCC till nervceller med markörer som är specifika för deras signalsubstans fenotyp. Kolinerga neuroner utgör cirka 60% av enter neuroner 7, och kan detekteras genom färgning för kolinacetyltransferas (chatt), den syntetiserande enzym för den excitatoriska neurotransmittorn acetylkolin. Historiskt, försök att visualisera kolinerga neuron förvirrad av olika antigen specificitet antikroppar riktade mot centrala nervsystemet (CNS) ChAT kontra perifera nervsystemet (PNS) ChAT 8-10. Men antikroppar riktade mot placental ChAT erkänna både centrala och perifera ChAT 11-13, och vi har nyligen beskrivit tekniker som gör det möjligt för videoanläggningsering av ENS kolinerga neuron med hög känslighet tidigare i utvecklingen än vad som uppnåtts med chatt reporter linjer 14. Här presenterar vi en teknik för att dissekera, fastställande och immunfärgning av mus embryonala mag-tarmkanalen för att visualisera ENS signalsubstans uttryck i nervceller. För dessa studier har vi använt ChAT-Cre möss parat sig med R26R: floxSTOP: tdTomato djur att producera ChAT-Cre, R26R: floxSTOP: tdTomato möss (definierade som chatt-Cre tdTomato hela manuskriptet). Dessa djur sedan parades med homozygota ChAT-GFP reporter möss, för att erhålla möss som uttrycker både fluorescerande reportrar att upptäcka ChAT uttryck 14. Dessa två reporter djur är på en C57BL / 6J bakgrund, och är kommersiellt tillgängliga (Jackson Laboratories, Bar Harbor, ME).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Oxoid</td>
			<td>BR0014G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>S2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Fisher</td>
			<td>BP9221</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher</td>
			<td>BP1605</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dishes</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence scope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine science tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tubes</td>
			<td>VWR</td>
			<td>20170-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>SouthernBiotech, Birmingham, AL</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>16004-330</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>Nikon A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Elements</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Vi har tidigare beskrivit generation av möss som uttrycker både GFP och tdTomato fluorescerande reportrar att upptäcka ChAT uttryck 14. I korthet var chat-Cre möss parat sig med R26R: floxSTOP: tdTomato djur för att producera ChAT-Cre, R26R: floxSTOP: tdTomato möss (kallas ChAT-Cre tdTomato). Dessa djur sedan parades med homozygota ChAT-GFP reporter möss. Embryon isolerades och vävnaderna dissekerades innan de fasta och immunofärgades såsom ovan, med de a...

Watch this Scientific Journal Video about Immunostaining to Visualize Murine Enteric Nervous System Development at JoVE.com

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. 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.

Immunofärgning att Visualisera Murin enteriska nervsystemet Utveckling

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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