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

Undersøgelsen blev støttet delvist af Intramural Research Program af National Institutes of Health, National Cancer Institute, Center for Cancer Research.

Den beskrevne protokol blev ansat i overensstemmelse med NIH regler om anvendelse af menneskelige vævsprøver. 1. Vævspræparat <ol><li> Forud for starten, få menneskelige vævsprøver ifølge Institutional Review Board (IRB) protokoller. </li><li> Vælg den type væv / celler blok og tilsvarende forarbejdningsmetode [formalin paraffinindlejret (FFPE), frosne, eller ethanol-paraffinindlejret (EFPE)]. Formalinfiksering giver optimal histologi, efterfulgt af ethanol fiksering og lynfrosse...

<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. 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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 præsenterer en protokol for anvendelse af SIVQ-LCM til microdissect immunofarvet epitelceller fra FFPE human brystvæv. Brugen af ​​et billede analyse algoritme, som SIVQ, reducerer mængden af ​​hands-on tid, der kræves for mikrodissektion processen. Dette er et potentielt vigtigt fremskridt for området siden operatøren tid og kræfter er typisk hastighedsbegrænsende trin for den præcise dissektion af celler af interesse. I den nuværende protokol, vi specifikt tilpasset vores procedure til ArcturusXT ...

Oprindeligt udviklet i midten af 1990&#39;erne, laser capture mikrodissektion (LCM) gør det muligt for brugeren at netop fange specifikke celler eller cellulære regioner fra en histologisk vævssnit via mikroskopisk visualisering 1, 2. Mange studier, der sammenligner molekylær analyse af LCM versus væv skrammer illustrere værdien af metoden 3-12. Desuden er der tre video protokol publikationer om den teknologi, der er til rådighed for visning 13, 14. Men på trods af dens dokumenterede værdi, kan LCM være trættende og besværlig, når målet af interesse er en spredt celle population i en heterogen vævssnit, eller når et stort antal celler er nødvendige for bestemte downstream-applikationer såsom proteomics. Byrden på den menneskelige operatør førte os til at udvikle en halvautomatisk dissektion tilgang til LCM ved at kombinere et kraftfuldt billede analyse algoritme til at styre LCM processen 15. &lt;p class = &quot;jove_content&quot;&gt; I samarbejde med University of Michigan, vores laboratorium på NIH forlængede tidligere udviklet og rapporteret Rumligt invariant vektorkvantisering (SIVQ) algoritme på en måde at gøre det muligt for semi-automatisere udvælgelsen væv proces iboende guidede mikrodissektion, hvorved fås et værktøj med patologen eller liv videnskabsmand i tankerne. Rumligt invariant vektorkvantisering (SIVQ) er en algoritme, der giver brugeren mulighed for at blot &quot;klikke&quot; på en histologisk træk af interesse at skabe en ring vektor (prædikat billede-funktion), der kan bruges til at søge i hele histologiske billede, justere den statistiske tærskel efter behov 16-21. Den resulterende zonekort viser kvaliteten af ​​kampe til den oprindelige prædikat billedet funktionen og efterfølgende omdannet til en enkelt farve (rød) anmærkning kort, der kan importeres til LCM instrumentet. Den automatiserede udvælgelse software, AutoScanXT, derefter bruges til at tegne et kort baseretpå SIVQ anmærkningssredskaber vejlede indfangning af target celler fra vævsprøve. Den detaljerede protokol nedenfor beskriver implementeringen af ​​SIVQ i mikrodissektion arbejdsgang.

<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, der automatiserer og strømliner de mere traditionelle, bruger-afhængig laser dissektion proces. Det sigter mod at skabe en avanceret, hurtigt kan tilpasses laser dissektion platform-teknologi. I denne rapport beskriver vi integrationen af ​​billedet analyse software Rumligt Invariant Vector Quantization (SIVQ) på ArcturusXT instrumentet. Det ArcturusXT-systemet indeholder både en infrarød (IR) og ultraviolet (UV)-laser, der giver mulighed for specifik celle eller store areal dissektioner. Det primære mål er at forbedre den hastighed, præcision og reproducerbarhed af laseren dissektion for at øge prøve gennemløb. Denne nye fremgangsmåde letter mikrodissektion af både dyr og mennesker væv i forskning og kliniske arbejdsgange.

En FFPE humant brystvæv afsnit blev immunfarvet for cytokeratin AE1/AE3 ved hjælp af en standard IHC-protokol 23. Efter farvning blev vævet slide placeret på ArcturusXT scenen og SIVQ-LCM-protokollen blev indledt som beskrevet ovenfor. Da væv ikke kan dækglas til mikrodissektion kan IHC + farvede celler være vanskelige at skelne visuelt (figur 1A). Således, for at give bedre indeks-svarende og et forbedret billede blev xylener hvormed vævssnittet for at skabe en pseudo-dækglas ...

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 om ArcturusXT Instrument

SIVQ-LCM er en innovativ tilgang, der udnytter en computer algoritme, Rumligt Invariant Vector Quantization (SIVQ), til at køre laser capture mikrodissektion (LCM) proces. Den SIVQ-LCM workflow i høj grad forbedrer hastigheden og nøjagtigheden af ​​mikrodissektion med anvendelser i både forskning og kliniske indstillinger.

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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