Name: sivq-lcm protocol for the arcturusxt instrument
Uploaded: 2014-07-23T14:00:00
Description: Watch this Scientific Journal Video about SIVQ-LCM Protocol for the ArcturusXT Instrument at JoVE.com

Studien ble støttet i en del av egenutført Research Program av National Institutes of Health, National Cancer Institute, Center for Cancer Research.

Den beskrevne protokollen ble ansatt i henhold til NIH regler om bruk av menneskelige vevsprøver. En. Vevpreparerina <ol><li> Før begynnelsen, få menneskelige vev prøver i henhold til Institutional Review Board (IRB)-protokoller. </li><li> Velg den type vev / celleblokk og tilhørende prosesseringsmetode [formalinfiksert parafin-embedded (FFPE), frossen, eller etanol-fast parafininnleiret (EFPE)]. Formalin fiksering gir optimal histologi, etterfulgt av etanol fiksering og flash-frosset...

<ol>
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W., 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=Impact+of+sample+acquisition+and+linear+amplification+on+gene+expression+profiling+of+lung+adenocarcinoma:+laser+capture+micro-dissection+cell-sampling+versus+bulk+tissue-sampling.">Impact of sample acquisition and linear amplification on gene expression profiling of lung adenocarcinoma: laser capture micro-dissection cell-sampling versus bulk tissue-sampling.</a> BMC medical genomics. 2, 13 (2009).</li><li>Zheng, J., Garg, S., Wang, J., Loose, D. S., Hauer-Jensen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissected+mucosa+versus+whole+tissue+specimens+for+assessment+of+radiation-induced+dynamic+molecular+and+pathway+changes+in+the+small+intestine.">Laser capture microdissected mucosa versus whole tissue specimens for assessment of radiation-induced dynamic molecular and pathway changes in the small intestine.</a> PloS one. 8, e53711 (2013).</li><li>Boone, D. R., Sell, S. L., Hellmich, H. 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(39), (2010).</li><li>Hipp, J., 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=SIVQ-aided+laser+capture+microdissection:+A+tool+for+high-throughput+expression+profiling.">SIVQ-aided laser capture microdissection: A tool for high-throughput expression profiling.</a> Journal of pathology informatics. 2, 19 (2011).</li><li>Hipp, J. D., Cheng, J. Y., Toner, M., Tompkins, R. G., Balis, U. 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Vi presenterer en protokoll for anvendelse av SIVQ-LCM å microdissect immunfargete epitelceller fra FFPE menneskelig brystvev. Bruken av en bildeanalyse-algoritmen, slik som SIVQ, reduserer mengden av praktisk nødvendige tid for mikrodisseksjon prosessen. Dette er en potensielt viktig fremskritt i feltet siden operatøren tid og anstrengelse er typisk det hastighetsbegrensende trinnet for nøyaktig disseksjon av celler av interesse. I den foreliggende protokoll, vi spesielt tilpasset for vår fremgangsmåte til Arctur...

Opprinnelig utviklet på midten av 1990-tallet, laser capture microdissection (LCM) gjør det mulig for brukeren å presist fange spesifikke celler eller cellulære regioner fra en histologisk vevsnitt.F.eks via mikroskopisk visualisering 1, 2. Mange studier som sammenligner molekylær analyse av LCM versus vev skraper illustrere verdien av metoden 3-12. I tillegg er det tre videoprotokoller publikasjoner på den teknologi som er tilgjengelig for visning 13, 14.. Men til tross for sin bevist verdien, kan LCM være kjedelig og arbeidskrevende når målet om interesse er en spredt cellepopulasjon i et heterogent vev delen, eller når et stort antall celler er nødvendig for spesifikke nedstrøms applikasjoner som proteomikk. Den belastningen på den menneskelige operatøren ledet oss til å utvikle en semi-automatisert disseksjon tilnærming for LCM ved å kombinere en kraftig bildeanalyse algoritme for å veilede LCM prosess 15. &lt;p class = &quot;jove_content&quot;&gt; I samarbeid med Universitetet i Michigan, vårt laboratorium ved NIH utvidet tidligere utviklet og rapportert Romlig invariant vektor kvantisering (SIVQ) algoritme på en måte å la den semi-automat vevet utvelgelsesprosessen egenverdi for guidet microdissection, og dermed gjør tilgjengelig et verktøy med patologen eller livsvitenskapsmann i tankene. Romlig invariant vektor kvantisering (SIVQ) er en algoritme som gjør at brukeren kan ganske enkelt &quot;klikk&quot; på en histologisk funksjon av interesse å lage en ring vektor (predikat bildefunksjonen) som kan brukes til å søke i hele histologiske bildet, justere statistisk terskel som trengs 16-21. Den resulterende varmekartet viser kvaliteten på kampene til den opprinnelige predikatet bildefunksjon og er senere omgjort til en enkelt farge (rød) merknad kart som kan importeres inn i LCM instrument. Den automatiserte utvalg programvare, AutoScanXT, blir så brukt til å tegne et kart basertpå SIVQ sin merknad guiding fangst av målcellene fra vevsprøve. Den detaljerte protokollen nedenfor beskriver gjennomføringen av SIVQ inn i microdissection arbeidsflyten.

<table border="0" cellpadding="0" cellspacing="0" width="586">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Positive Charged Glass Slides</td>
			<td style="width:84px;">Thermo Scientific</td>
			<td style="width:119px;">4951Plus-001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Xylenes, ACS reagent, &#8805;98.5% xylenes + ethylbenzene basis&#160;</td>
			<td style="width:84px;">Sigma Aldrich</td>
			<td style="width:119px;">247642</td>
			<td style="width:167px;">CAUTION: PLEASE USE PROPER SAFETY PROCEDURES.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Ethyl Alcohol, U.S.P. 200 Proof, Anhydrous</td>
			<td style="width:84px;">The Warner-Graham Company</td>
			<td style="width:119px;">6.505E+12</td>
			<td style="width:167px;">CAUTION: PLEASE USE PROPER SAFETY PROCEDURES.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Arcturus CapSure Macro LCM Caps</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;">LCM0211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">ArcturusXT Laser Microdissection Instrument</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;">ARCTURUSXT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:216px;">AutoScanXT Software</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">An optional image analysis program for the ArcturusXT Laser Microdissection Device. This is software is required for SIVQ-LCM.</td>
		</tr>
		<tr height="169">
			<td height="169" style="height:169px;width:216px;">Spatially Invariant Vector Quantization (SIVQ)</td>
			<td style="width:84px;">University of Michigan</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">This tool suite is publicly available for academic collaborations. For access to the SIVQ algorithm, please contact Dr. Ulysses Balis [Ulysses@med.umich.edu]</td>
		</tr>
	</tbody>
</table>

sivq-lcm protocol for the arcturusxt instrument

SIVQ-LCM er en ny metode som automatiserer og effektiviserer mer tradisjonelle, brukeravhengig laser disseksjon prosess. Det har som mål å skape et avansert, raskt tilpasses laser disseksjon plattformteknologi. I denne rapporten beskriver vi integrering av bildeanalyse programvare Romlig Invariant Vector Quantization (SIVQ) på ArcturusXT instrument. Den ArcturusXT Systemet inneholder både en infrarød (IR) og ultrafiolett (UV) laser, noe som åpner for bestemt celle eller stort område disseksjoner. Hovedmålet er å forbedre hastighet, nøyaktighet og reproduserbarhet av laser disseksjon å øke prøvekapasitet. Denne romanen tilnærming forenkler microdissection av både dyr og menneskelige vev i forskning og kliniske arbeidsflyter.

En FFPE menneskelig brystvev delen ble immunostained for cytokeratin AE1/AE3 ved hjelp av en standard IHC protokoll 23. Etter farging ble vevet glide plassert på ArcturusXT scenen og SIVQ-LCM-protokollen ble initiert som beskrevet ovenfor. Ettersom vevet ikke kan coverslipped for mikrodisseksjon, kan IHC + fargede celler er vanskelig å skjelne visuelt (figur 1A). Derfor, for å gi bedre indekstilpasning og en forbedret bilde, ble xylen tilsatt til vev-delen for å skape et pseudo-dekkglass ...

Watch this Scientific Journal Video about SIVQ-LCM Protocol for the ArcturusXT Instrument at JoVE.com

SIVQ-LCM Protocol for the ArcturusXT Instrument

SIVQ-LCM-protokollen for ArcturusXT Instrument

SIVQ-LCM er en innovativ tilnærming som utnytter en datamaskin algoritme, Romlig Invariant Vector Quantization (SIVQ), å drive laser-fangst microdissection (LCM) prosess. Den SIVQ-LCM arbeidsflyt forbedrer hastigheten og nøyaktigheten av microdissection, med anvendelser i både forskning og klinisk innstillinger.

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an ...

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