Name: improving the application of high molecular weight biotinylated dextran amine for thalamocortical projection tracing in the rat
Uploaded: 2018-04-12T12:30:00
Description: Watch this Scientific Journal Video about Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat at JoVE.com

Denne studien ble finansiert av National Natural Science Foundation of China (Project kode nr. 81373557, nr. 81403327).

Denne studien ble godkjent av den etiske komiteen ved den Kina kinesisk medisinsk vitenskapsakademi (referansenummer 20160014). Alle prosedyrer ble utført i samsvar med den nasjonale institutter for helse Guide og bruk av forsøksdyr (National Academy Press, Washington, DC, 1996). Fire voksen mannlig rotter (vekt 250-280 g) ble brukt i denne studien. Alle dyrene ble plassert i en 12t lys/mørke syklus med kontrollert temperatur og fuktighet, og gratis tilgang til mat og vann. Instrumentene og materialer som brukes i stu...

<ol>
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Velge en riktig tracer er et kritisk punkt for en vellykket nevrale sporing eksperiment. I familien av BDA, høy molekylvekt BDA (10.000 molekylvekt) ble anbefalt transporteres fortrinnsvis gjennom anterograd nevrale sti i motsetning til lav molekylvekt BDA (3000 molekylvekt)2,3 , 11 , 12 , 13. imidlertid mange studier også antydet at høy molekylvekt BDA kan...

Høy molekylvekt BDA (10.000 molekylvekt), en svært følsom tracer, har blitt brukt for å spore nervebaner i sentralnervesystemet for over 20 år1. Selv om bruken av BDA er en vanlig nevrale traktat sporing teknikk, kan kvaliteten på BDA merking påvirkes i dyr av ulike faktorer1,2,3. Våre siste studie viste at den optimale strukturen av BDA merking er ikke bare forbundet med riktig etter injeksjon overlevelse tid, men også korrelert med flekker metode4. Inntil nå, konvensjonelle avidin-biotin-peroxidase kompleks (ABC), streptavidin-fluorescein isothiocyanate og streptavidin-AF594 ble flekker metodene brukt for å avsløre BDA merkingen i tidligere studier2,3, 4,5. Sammenligning kan fluorescerende flekker for BDA enkelt utføres.
For å utvide bruken av høy molekylvekt BDA, ble en raffinert protokoll introdusert i studien. Etter injeksjon av BDA i VPM i thalamus med rotte hjernen ble BDA merking avslørt den vanlige metoden for standard ABC flekker og dobbel fluorescerende flekker, som ble gjennomført for å observere korrelasjon av BDA merking og grunnleggende nevrale elementer eller interneurons i kortikale målet med streptavidin-AF594 og fluorescerende Nissl histochemistry eller PV-immunochemistry, henholdsvis. Gjennom de gjensidige nervebaner mellom VPM og den primære somatosensory cortex (S1)6,7,8fokuserer vi vår observasjon på BDA merking i thalamocortical anslått axons og corticothalamic anslått celle somas i S1. Ved denne prosessen forventet vi å gir ikke bare en detaljert protokoll for å få den høye kvaliteten på neural merking med høy molekylvekt BDA, men også en raffinert protokoll på kombinasjonen av fluorescerende BDA merking og andre fluorescerende nevrale markører med Histochemistry eller immunochemistry. Denne tilnærmingen er å foretrekke å studere lokal nevrale kretser og deres kjemiske egenskaper under en AC confocal mikroskopi for laserskanning.

<table>
	<tbody>
		<tr>
			<td>Biotinylated dextran amine (BDA)</td>
			<td>Molecular Probes</td>
			<td>D1956</td>
			<td>10,000 molecular weight</td>
		</tr>
		<tr>
			<td>Streptavidin-Alexa Fluor 594</td>
			<td>Molecular Probes</td>
			<td>S32356</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>500/525 green fluorescent Nissl stain</td>
			<td>Molecular Probes</td>
			<td>N21480</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Brain stereotaxis instrument</td>
			<td>Narishige</td>
			<td>SR-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing microtome</td>
			<td>Thermo</td>
			<td>Microm International GmbH</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal imaging</td>
			<td>Olympus</td>
			<td>FV1200</td>
			<td></td>
		</tr>
		<tr>
			<td>system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Drill</td>
			<td>Saeyang Microtech</td>
			<td>Marathon-N7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley</td>
			<td>Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences</td>
			<td>SCKX (JUN) 2012-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain ABC Kit</td>
			<td>Vector Laboratories</td>
			<td>PK-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>superfrost plus microscope slides</td>
			<td>Thermo</td>
			<td>#4951PLUS-001</td>
			<td>25x75x1mm</td>
		</tr>
		<tr>
			<td>Photoshop and Illustration</td>
			<td>Adobe</td>
			<td>CS5</td>
			<td></td>
		</tr>
	</tbody>
</table>

improving the application of high molecular weight biotinylated dextran amine for thalamocortical projection tracing in the rat

Høy molekylvekt biotinylated dekstran Amin (BDA) har blitt brukt som en svært følsom nevroanatomi tracer i mange tiår. Siden kvaliteten på sin merking ble påvirket av ulike faktorer, her gir vi en raffinert protokoll for anvendelse av høy molekylvekt BDA for å studere optimal nevrale merking i sentralnervesystemet. Etter stereotactic injeksjon av BDA i ventrale posteromedial kjernen (VPM) i thalamus med rotta gjennom en delikat glass pipette, var BDA farget med fluorescerende streptavidin-Alexa (AF) 594 og counterstained med fluorescerende Nissl flekk AF500/525. På bakgrunn av grønne Nissl flekker, ble den røde BDA merking, inkludert neuronal cellen legemer og axonal terminaler, mer tydelig demonstrert i somatosensory cortex. Videre doble fluorescerende flekker for BDA og kalsium-bindende protein parvalbumin (PV) ble utført for å observere korrelasjon av BDA merking og PV-positive interneurons i kortikale målet, gir mulighet til å studere lokalt nevrale kretser og deres kjemiske egenskaper. Dermed dette raffinert metoden er ikke bare egnet for å visualisere høykvalitets nevrale merking med høy molekylvekt BDA gjennom gjensidige nervebaner mellom thalamus og hjernebarken, men også tillater samtidig demonstrasjon av andre nevrale markører med fluorescerende histochemistry eller immunochemistry.

Overlevelse av 10 dager innlegget injeksjon av BDA i VPM var tilstrekkelig for å produsere intens nevrale merking på tilsvarende kortikale områder ipsilateral til injeksjon siden (figur 2). Både konvensjonell ABC og fluorescerende flekker prosedyrer for BDA avslørt lignende mønster av nevrale merking på S1, inkludert anterogradely merket thalamocortical axons og retrogradely corticothalamic neurons (figur 2C, D </s...

Watch this Scientific Journal Video about Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat at JoVE.com

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

Her presenterer vi en raffinert protokoll for å effektivt avsløre biotinylated dekstran Amin (BDA) merking med et fluorescerende flekker metoden ved hjelp av en gjensidig nevrale sti. Det er egnet for analyse av fine strukturen av BDA merking og skille den fra andre nevrale elementer under en AC confocal laserskanning mikroskop.

Forbedre anvendelsen av høy molekylvekt Biotinylated dekstran Amin for Thalamocortical projeksjon sporing i rotte

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of ...

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