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. Mus <ol><li> Alle undersøgelser med mus blev godkendt af Stony Brook University Institutional Animal Care og brug Udvalg (IACUC). Musene blev holdt på en 12:12 timers lys-mørke-cyklus. <ol><li> Kommercielt få C57BL / 6-mus. Opnå C57BL / 6 mus, der bærer Klf5 alleler flankeret af loxP sites (Klf5 f l / f l). Disse mus blev beskrevet tidligere 21 og nådigt fra Dr. Ryozo Nagai. </li></ol></li><li> Køb C57BL / 6 mus, der bærer ...

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Den schweiziske rullende teknik er en kraftfuld metode til fremstilling af intestinal væv til histologisk og morfologisk vurdering i stor skala. I modsætning til den tidligere beskrevne Swiss-rullende teknik, som oprindeligt blev udviklet til fremstilling af frosne snit 18,19, proceduren præsenteres her muliggør hurtig tarmvæv forberedelse og fiksering til formalin fiksering og paraffinindlejring (FFPE). Sammenlignet med frosset væv, FFPE væv har meget længere holdbarhed og er den foretrukne form for ...

Pattedyrs intestinale epithel omfatter et enkelt lag af søjleformede celler. I tyndtarmen, er de proliferative celler begrænset til krypterne mens differentierede celler besætte villus region. Men fordi der er ingen villi i tyktarmen, er de proliferative celler lokaliseret til bunden af ​​krypterne og differentierede celler optager det øvre område af krypterne. Tarmepitelet undergår hurtig genopfyldning (ca. 3 - 5 dage), der er drevet af kontinuert fordeling af de proliferative celler i krypterne. De proliferative celler i krypter er ikke en homogen befolkning og er yderligere opdelt i stamceller og transit-forstærke (TA) celler 1. Stamcellerne opholde nederst krypten, inden for de første 4 - 5 celler fra selve bunden 2. Den nuværende model understøtter eksistensen af ​​to typer stamceller: krypt basis søjleformede (CBC) stamceller og reserve hvilende stamceller. CBC stem celler er aktivt prolifererende og er markeret med leucin-rige repeat-holdige G-protein koblet receptor 5 (Lgr5) 3, Olfactomedin 4 (Olfm4) 4 og Achaete Scute-lignende 2 (Ascl2) 5. På den anden side er reserve hvilende stamceller mærkes ved B-celle-specifik Moloney leukæmivirus integration site 1 (Bmi1) 6, mus telomerase revers transkriptase (mTERT) 7, HOP homeobox (Hopx) 8, Doublecortin-lignende og CAM kinase -lignende 1 (Dclk1) 9, og leucin-rige gentagelser og immunglobulin-lignende domæner 1 (Lrig1) 10. De aktivt prolifererende stamceller giver anledning til TA celler derefter gennemgå yderligere differentiering i absorberende celler (enterocytter) og sekretoriske celler (enteroendocrine, bæger, Paneth, og Tuft celler). Kontinuerlig celledeling i den proliferative zone resulterer i opadgående bevægelse af epitelceller langs krypten-villus aksen indtil de når toppen af ​​villi, hvor de gennemgår apoptose og erløsnes fra overfladen af ​​epitelet. De forskellige typer af intestinale epitelceller er præget af ekspressionen af distinkte proteiner (f.eks kan intestinale slimceller anerkendes ved farvning med antistof mod MUC2 og Paneth celler med antistof mod lysozym). Vi studerer rolle Krüppel-lignende faktorer (KLFs) i homeostase og patobiologien af tarmepitelet 11-13. Resultaterne præsenteres her støtter muligheden for en modificeret schweizisk-rullende teknik er baseret på tidligere undersøgelser af den rolle, Krüppel-lignende faktor 5 (KLF5) i vedligeholdelse af aktivt prolifererende intestinale epitelceller stamceller 14. KLF5 er en zink-finger transkriptionsfaktor, i høj grad udtrykkes i den aktive intestinal stamme og TA celler 12. Tidligere undersøgelser viste, at KLF5 co-udtrykkes med Ki-67, en kendt proliferativ markør i de intestinale krypter. Den gastrointestinale tr<html> handling er ikke en strukturelt eller funktionelt homogen væv. Tyndtarmen er opdelt i duodenum, jejunum og ileum og tyktarmen i coecum og colon, idet sidstnævnte yderligere opdelt i proksimal, midterste og distale dele. Hver af disse sektioner har unikke histologiske træk og spiller forskellige roller 15. Som sådan kan virkningen af fornærmelser og graden af respons tarmepitelet afhænge regionen studerede væv 16. Derudover forskellige mus stammer demonstrere mangfoldighed af svaret på det histologiske niveau baseret på den type fornærmelse anvendt i undersøgelserne 16. Således sømmer forberedelse væv er nødvendigt at tillade passende histologiske og molekylære analyse af de intestinale væv. Som sådan er den schweizisk-roll teknik tilskud analyse af hele længden af ​​tarmepitelet på én gang og dermed konstaterer velinformerede konklusioner baseret på omfattende information. e_content &quot;&gt; Den schweiziske-roll teknik blev først nævnt af Magnus 17, og beskrevet i detaljer af Moolenbeck og Ruitenberg og Park et al., som en metode til fremstilling af væv og udføre histologiske analyser af gnaver tarmen 18,19 henholdsvis. Protokollen afgrænset i denne publikation præsenterer en forbedret version af den oprindelige metode, der tillader for rettidig og pålidelig forberedelse væv til diagnostiske formål. denne modificerede teknik gør det muligt for effektive samlinger og udarbejdelse af tarmepitelet for universelt anvendte teknikker, såsom immunhistokemi, immunfluorescens, samt in situ hybridisering (fluorescerende og chromogene 20). Endvidere modificerede vævsprøven fremstillingsmetode anvender let tilgængelige og forholdsvis billige reagenser samtidig med en fremgangsmåde til hurtig vævsfiksering og tillader genvinding af protein, DNA og RNA til yderligere evaluering. taget sammen, this teknik er fremragende til omfattende vurdering af histopatologiske, patologiske, og molekylære funktioner i tarmens epitel.

<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.

Den schweiziske rullende teknik i kombination med immunhistokemisk farvning muliggør omfattende analyse af små eller store tarm væv. Eksemplet med H &amp; E-farvning af en tyktarmen af en C57BL / 6 mus (figur 1) er en illustration af gennemførligheden og effektiviteten af denne teknik. Som vist i figur 1, er billedet i stand til at indfange alle dele af tyktarmen: proksimale, midterste og distale. Således giver det mulighed for omfattende histologis...

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

Nøjagtig identificering og lokalisering af epitelceller langs tarmslimhinde foring er afgørende for at definere forskellige cellelinier. Korrekt billeddannelse af tarmvæv er afgørende for identifikation af protein ekspressionsmønstre med maksimal opløsning. Denne undersøgelse har til formål at afgrænse de optimale metoder og betingelser for forarbejdning mus tarm væv.

Forbedret Swiss-rullende Teknik for tarmvæv Forberedelse til immunhistokemisk og Immunofluorescerende Analyser

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

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JoVE Journal

Biology

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