Name: improved swiss-rolling technique for intestinal tissue preparation for immunohistochemical and immunofluorescent analyses
Uploaded: 2016-07-13T14:00:00
Description: Watch this Scientific Journal Video about Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses at JoVE.com

We would like to thank Ainara Ruiz de Sabando for providing H&amp;E images. This work was supported by grants from the National Institutes of Health (DK052230, DK093680 and CA172113) awarded to Dr. Vincent W. Yang.

1. Mice <ol><li> Alle studier med mus ble godkjent av Stony Brook University Institutional Animal Care og bruk Committee (IACUC). Musene ble opprettholdt på en 12:12 timers lys-mørke-syklusen. <ol><li> Kommersielt oppnå C57BL / 6 mus. Skaff C57BL / 6 mus som bærer Klf5 alleler flankert av loxP nettsteder (Klf5 f l / f l). Disse musene ble tidligere beskrevet 21 og graciously levert av Dr. Ryozo Nagai. </li></ol></li><li> Kjøp C57B...

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Den sveitsiske rullende teknikken er en kraftig metode for fremstilling av tarmvevet for histologisk og morfologisk vurdering på en stor skala. I motsetning til den tidligere beskrevne Swiss-valseteknikk, som opprinnelig ble utviklet for fremstilling av frosne seksjoner 18,19, fremgangsmåten som presenteres her muliggjør rask tarmvevet forberedelse og fiksering for formalin fiksering og forankring parafin (FFPE). Sammenlignet med frossent vev, har FFPE vev mye lengre holdbarhet og er den foretrukne type ve...

Pattedyr tarmepitelet omfatter et enkelt lag av søyleceller. I tynntarmen, blir de proliferative cellene begrenset til krypter mens differensierte cellene opptar villus-regionen. Imidlertid, fordi det ikke er noen villi i tykktarmen, er de proliferative cellene lokalisert til bunnen av kryptene og differensierte celler opptar det øvre område av kryptene. Tarmepitelet gjennomgår hurtig etterfylling (ca 3 - 5 dager) som er drevet ved kontinuerlig oppdeling av proliferative celler i kryptene. De proliferative celler av krypter er ikke en homogen populasjon, og er videre delt inn i stamceller og transitt-amplifisering (TA) celler 1. Stamcellene ligge på bunnen av krypten, i løpet av de første 4 - 5 celler fra bunnen 2. Den nåværende modellen støtter eksistensen av to typer stamceller: krypten basen søyle (CBC) stamceller og reserve stille stamceller. CBC stem celler er aktivt prolifererende og er merket med leucine-rik gjenta holdige G-proteinkoblet reseptor 5 (Lgr5) 3, Olfactomedin 4 (Olfm4) 4 og Achaete scute lignende 2 (Ascl2) 5. På den annen side, er reserve stille stamceller merket med B-celle-spesifikke Moloney murine leukemi virus integrering for 1. (Bmi1) 6, mus telomerase revers transkriptase (mTert) 7, HOP Homeobox (Hopx) 8, Doublecortin-aktig og CAM Kinase -lignende 1 (Dclk1) 9, og leucin-rich gjentar og immunoglobulin-lignende domener 1 (Lrig1) 10. De aktivt prolifererende stamceller gir opphav til TA cellene deretter gjennomgå ytterligere differensiering i absorberende celler (entero) og sekretoriske celler (enteroendocrine, beger, Paneth, og tuft celler). Kontinuerlig celledelingen i den proliferative sonen resulterer i oppadgående bevegelse av epitelceller langs krypten-villus akse inntil de når toppen av villi, hvor de gjennomgår apoptose og ersloughed av fra overflaten av epitelet. De ulike typene av intestinale epitelceller er preget av uttrykket av ulike proteiner (f.eks, kan tarmslimceller bli gjenkjent av farging med antistoff mot Muc2 og Paneth celler med antistoff mot lysozym). Vi studere rollen til Krüppel-lignende faktorer (KLFs) i homeostase og patobiologi av tarmepitelet 11-13. Resultatene som presenteres her støtter gjennomførbarheten av en modifisert Swiss-valseteknikk er basert på tidligere undersøkelser av rollen til Krüppel-lignende faktor 5 (KLF5) i vedlikehold av aktivt prolifererende intestinale epitelceller stamceller 14. KLF5 er en sink-finger transkripsjonsfaktor som er sterkt uttrykt i aktiv tarm stammen og TA celler 12. Tidligere studier har vist at KLF5 er co-uttrykkes med Ki-67, en kjent proliferativ markør i tarm krypter. Mage-tr<html> handling er ikke et strukturelt eller funksjonelt homogen vev. Tynntarmen er delt inn i duodenum, jejunum og ileum og tykktarmen inn i cecum og tykktarm, med den sistnevnte videre delt inn proksimale, midtre og distale deler. Hver av disse seksjonene har unike histologiske funksjoner og spiller forskjellige roller 15. Som sådan, kan virkningene av fornærmelser og graden av responsen til tarmepitelet er avhengig av området av undersøkt vev 16. I tillegg er forskjellige musestammer demonstrere mangfold av responsen på det histologiske nivå basert på den type av fornærmelse anvendt i studiene 16. Således som gjenspeiler dette vevspreparatet er nødvendig for å tillate passende histologiske og molekylær analyse av tarmvevet. Som sådan, Swiss-rull teknikk tilskudd analyse av hele lengden av tarmepitelet på en gang og dermed konstaterer velinformerte konklusjoner basert på omfattende informasjon. e_content &quot;&gt; The Swiss-roll teknikken ble først nevnt av Magnus 17, og beskrevet i detalj av Moolenbeck og Ruitenberg og Park et al. som en metode for å forberede vev og utføre histologisk analyser av gnager tarmen 18,19, henholdsvis. Protokollen skissert i denne publikasjon viser en forbedret versjon av den opprinnelige metode som tillater for presis og pålitelig vev forberedelse for diagnostiske formål. denne modifiserte teknikken gjør det mulig for effektiv samlinger og fremstillingen av tarmepitelet for universelt brukte teknikker, slik som immunhistokjemi, immunfluorescens, såvel som in situ hybridisering (fluorescerende og kromogen 20). Videre er det modifiserte vev prøven fremstillingsmetode utnytter lett tilgjengelige og relativt rimelige reagenser samtidig som de gir en fremgangsmåte for hurtig vev fiksering og gjør det mulig for utvinning av protein, DNA og RNA for ytterligere evaluering. Tatt sammen, this teknikken er utmerket for helhetlig vurdering av histopatologiske, patologiske og molekylære funksjoner i tarmepitelet.

<table border="0" cellpadding="0" cellspacing="0" width="1016">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Stainless Steel Dissecting Kits</td>
			<td style="width:179px;">VWR</td>
			<td style="width:119px;">25640-002</td>
			<td style="width:403px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Decloaking Chamber</td>
			<td style="width:179px;">Biocare Medical</td>
			<td>DC2012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Syringe 10ml</td>
			<td style="width:179px;">VWR</td>
			<td>89215-218</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:316px;">Swingsette Tissue embedding/processing cassette with lid</td>
			<td style="width:179px;">Simport</td>
			<td>M515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Superfrost Plus Slides [size: 25x75x1mm]</td>
			<td style="width:179px;">VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Manual Slide Staining Set</td>
			<td style="width:179px;">Tissue-Tek/Sakura</td>
			<td>4451</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Staining Dish Green</td>
			<td style="width:179px;">Tissue-Tek/Sakura</td>
			<td>4456</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Staining Dish White</td>
			<td style="width:179px;">Tissue-Tek/Sakura</td>
			<td>4457</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">24-Slide Slide Holder with Detachable Handle</td>
			<td style="width:179px;">Tissue-Tek/Sakura</td>
			<td>4465</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Oven</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td>6243</td>
			<td>for baking slides at 65 degree</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Dissection microscope</td>
			<td style="width:179px;">Zeiss</td>
			<td>Stemi 2000C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence Microscope</td>
			<td style="width:179px;">Nikon</td>
			<td>Eclipse 90i</td>
			<td>Bright and fluoerescent light, with objectives: 10x, 20x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">PAP Pen Super-Liquid Blocker Mini</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td>DAI-PAP-S-M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Ethanol 200 proof</td>
			<td style="width:179px;">AAPR</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Methanol</td>
			<td style="width:179px;">VWR</td>
			<td>BDH1135-4LP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Glacial acetic acid</td>
			<td style="width:179px;">AAPR</td>
			<td>281000ACS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Xylene</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td>X5P-1GAL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Hydrogen peroxide 25% solution in water</td>
			<td style="width:179px;">ACROS</td>
			<td>202465000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">10% bufered formalin</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td>22-026-213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Bovine serum fraction V, heat shock</td>
			<td style="width:179px;">Roche</td>
			<td>3116956001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Tween 20</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Sodium citrate</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">S279</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Gavage needle</td>
			<td style="width:179px;">VWR</td>
			<td style="width:119px;">20068-624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Rabbit anti Klf5 antibody</td>
			<td style="width:179px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-22797</td>
			<td>Dilution 1: 150</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Chicken anti EGFP antibody</td>
			<td style="width:179px;">Millipore</td>
			<td style="width:119px;">AB16901</td>
			<td>Dilution 1: 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Rabbit anti Ki67 antibody</td>
			<td style="width:179px;">Biocare Medical</td>
			<td style="width:119px;">CRM325B</td>
			<td>Dilution 1: 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Mach3 rabbit AP polymer detection kit</td>
			<td style="width:179px;">Biocare Medical</td>
			<td style="width:119px;">M3R533L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Warp red chromogen kit</td>
			<td style="width:179px;">Biocare Medical</td>
			<td style="width:119px;">WR806 H</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Lgr5-EGFP/CreERT2 mice&#160;</td>
			<td>Jackson labs</td>
			<td>008875&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Automated processor</td>
			<td style="width:179px;">Leica</td>
			<td style="width:119px;">Leica TP1020</td>
			<td></td>
		</tr>
	</tbody>
</table>

improved swiss-rolling technique for intestinal tissue preparation for immunohistochemical and immunofluorescent analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Det sveitsiske valseteknikk i kombinasjon med immunhistokjemisk farging gir mulighet for fullstendig analyse av små eller store tarmvevet. Eksemplet med H &amp; E farging av et stort tarm av en C57BL / 6 mus (figur 1) er en illustrasjon av gjennomførbarheten og effektiviteten av denne teknikken. Som vist i figur 1, er bildet i stand til å fange opp alle partier av tykktarmen: proksimale, midtre og distale. Dermed gir det mulighet for omfattende histol...

Watch this Scientific Journal Video about Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses at JoVE.com

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Nøyaktig identifikasjon og plassering av epitelceller langs intestinal mucosal fôr er viktig å definere ulike celle linjene. Riktig avbildning av intestinal vev er avgjørende for identifisering av protein uttrykk mønstre med maksimal oppløsning. Denne studien tar sikte på å avgrense de optimale metoder og forhold for prosessering musetarmvevet.

Forbedret Swiss-rullende teknikk for tarmvevet Forberedelse til Immunhistokjemisk og Immunofluorescent Analyser

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

Research

JoVE Journal

Biology

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