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. Ratos <ol><li> Todos os estudos envolvendo ratos foram aprovados pelo Comitê Animal Care Institucional e Use o Stony Brook University (IACUC). Os ratinhos foram mantidos num ciclo de luz-escuro 12:12 h. <ol><li> Comercialmente obter ratinhos C57BL / 6. Obter camundongos C57BL / 6 com alelos Klf5 flanqueadas por locais loxP (Klf5 f l / f l). Estes ratos foram previamente descritos 21 e graciosamente cedido pelo Dr. Ryozo Nagai. </li...

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K., 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=Mouse+telomerase+reverse+transcriptase+(mTert)+expression+marks+slowly+cycling+intestinal+stem+cells.">Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells.</a> Proc Natl Acad Sci U S A. 108 (1), 179-184 (2011).</li><li>Takeda, N., 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=Interconversion+between+intestinal+stem+cell+populations+in+distinct+niches.">Interconversion between intestinal stem cell populations in distinct niches.</a> Science. 334 (6061), 1420-1424 (2011).</li><li>May, R., 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=Identification+of+a+novel+putative+gastrointestinal+stem+cell+and+adenoma+stem+cell+marker,+doublecortin+and+CaM+kinase-like-1,+following+radiation+injury+and+in+adenomatous+polyposis+coli/multiple+intestinal+neoplasia+mice.">Identification of a novel putative gastrointestinal stem cell and adenoma stem cell marker, doublecortin and CaM kinase-like-1, following radiation injury and in adenomatous polyposis coli/multiple intestinal neoplasia mice.</a> Stem Cells. 26 (3), 630-637 (2008).</li><li>Powell, A. E., 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=The+pan-ErbB+negative+regulator+Lrig1+is+an+intestinal+stem+cell+marker+that+functions+as+a+tumor+suppressor.">The pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor.</a> Cell. 149 (1), 146-158 (2012).</li><li>Pearson, R., Fleetwood, J., Eaton, S., Crossley, M., Bao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kruppel-like+transcription+factors:+a+functional+family.">Kruppel-like transcription factors: a functional family.</a> Int J Biochem Cell Biol. 40 (10), 1996-2001 (2008).</li><li>McConnell, B. B., Yang, V. 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A técnica de laminação Swiss é um método poderoso para a preparação de tecido intestinal para avaliação histológica e morfológica em grande escala. Em contraste com a técnica Swiss-laminagem anteriormente descrita, que foi originalmente desenvolvido para preparação de secções congeladas 18,19, o processo aqui apresentado permite a preparação do tecido intestinal rápida e a fixação para a fixação de formalina e embebidos em parafina (FFPE). Em comparação com tecido congelado, o tecido ...

O epitélio intestinal de mamífero compreende uma única camada de células colunares. No intestino delgado, as células proliferativas são confinados às criptas enquanto que as células diferenciadas ocupar a região da vilosidade. No entanto, porque não há nenhum vilosidades no intestino grosso, as células proliferativas são localizadas na parte inferior das criptas e células diferenciadas ocupar a região superior das criptas. O epitélio intestinal sofre reposição rápida (cerca de 3-5 dias) que é accionado por divisão contínua das células proliferativas nas criptas. As células proliferativas das criptas não são uma população homogénea e são ainda subdivididas em células estaminais e células-amplificação de trânsito (TA) 1. As células estaminais reside na parte inferior da cripta, dentro das primeiras 4 - 5 células a partir da parte inferior 2. O modelo atual suporta a existência de dois tipos de células-tronco: as células-tronco da cripta colunar base (CBC) e de reserva de células estaminais quiescentes. A CBC scélulas dez estão se proliferando de forma activa e são marcados por rica em leucina contendo repetição G-proteína receptor acoplado 5 (Lgr5) 3, Olfactomedin 4 (Olfm4) 4 e Achaete scute-like 2 (Ascl2) 5. Por outro lado, as células estaminais quiescentes de reserva são rotulados por Moloney site B específico da célula vírus da leucemia murina de integração 1 (Bmi1) 6, a telomerase rato transcriptase reversa (mTert) 7, HOP homeobox (Hopx) 8, duplacortina-like e CAM Kinase -como 1 (Dclk1) 9, e leucina-ricos repete e do tipo imunoglobulina domínios 1 (Lrig1) 10. As células estaminais que proliferam activamente dar origem a células TA, em seguida, submetido a posterior diferenciação em células absortivas (enterócitos) e células secretoras (enteroendócrinas, caliciformes, células de Paneth e flocos). divisão celular contínua na zona proliferativa resulta em movimento ascendente de células epiteliais ao longo do eixo de cripta-vilosidade até atingirem a parte superior da vilosidade, onde eles sofrem apoptose e sãodescartadas a partir da superfície do epitélio. Os diferentes tipos de células epiteliais intestinais estão marcados pela expressão de proteínas diferentes (por exemplo, células caliciformes intestinais podem ser reconhecidos através de coloração com anticorpo contra células de Paneth MUC2 e com anticorpo contra lisozima). Nós estudar o papel de factores como Krüppel-(KLFs) na homeostase e patobiologia do epitélio intestinal 11-13. Os resultados aqui apresentados apoiar a viabilidade de uma técnica de laminagem Swiss modificado baseiam-se em estudos anteriores sobre o papel do factor-Krüppel como 5 (KLF5) na manutenção das proliferam activamente células estaminais epiteliais intestinais 14. KLF5 é um factor de transcrição dedo de zinco que é altamente expresso na haste intestinal activa e 12 alvéolos Ta. Estudos anteriores demonstraram que KLF5 é co-expressa com Ki-67, um marcador conhecido proliferativa nas criptas intestinais. O tr gastrointestinal<html> ato não é um tecido estruturalmente ou funcionalmente homogénea. O intestino delgado é dividido em duodeno, jejuno e íleo e o intestino grosso no ceco e no cólon, com esta última ainda divididas em segmentos proximal, médio e distal. Cada uma destas secções tem características histológicas únicas e desempenha papéis distintos 15. Como tal, os efeitos de insultos e o grau de resposta do epitélio intestinal pode depender da região de tecido estudado 16. Além disso, várias estirpes de ratos demonstrar a diversidade da resposta ao nível histológico com base no tipo de insulto utilizado nos estudos 16. Assim, condizente com a preparação do tecido é necessário para permitir histológico apropriado e análise molecular dos tecidos intestinais. Como tal, a-rolo suíço análise bolsas técnica de todo o comprimento do epitélio intestinal de uma só vez e, assim, verifica conclusões bem informadas com base numa informação global. e_content &quot;&gt; A técnica Swiss-rolo foi mencionado pela primeira vez por Magnus 17, e descrito em detalhe por Moolenbeck e Ruitenberg e Park et al., como um método para a preparação de tecidos e realizando análises histológicas do intestino roedor 18,19, respectivamente. O protocolo delineada na presente publicação apresenta uma versão melhorada do método original que permite a preparação do tecido oportuna e confiável para fins de diagnóstico. esta técnica modificada permite coleções e preparação do epitélio intestinal de técnicas universalmente utilizados, tais como imuno-histoquímica, imunofluorescência, bem eficientes como hibridação in situ (fluorescente e cromogénico 20). Além disso, o tecido modificado espécime método de preparação utiliza reagentes facilmente disponíveis e relativamente pouco dispendiosos, enquanto que oferecem um método de fixação rápida dos tecidos e permite a recuperação de proteína, ADN e ARN para a avaliação adicional. Tomados juntos, this técnica é excelente para avaliação abrangente dos aspectos histopatológicos, patológicas e moleculares do epitélio intestinal.

<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.

A técnica de rolamento suíça em combinação com coloração imuno-histoquímica permite a análise abrangente do tecido intestinal pequeno ou grande. O exemplo de coloração de H &amp; E de um intestino grosso de um C57BL / 6 de ratinho (Figura 1) é uma ilustração da exequibilidade e a eficácia desta técnica. Como mostrado na Figura 1, a imagem é capaz de capturar todas as porções do cólon: proximal, médio e distal. Assim,...

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

A identificação precisa e localização de células epiteliais ao longo da mucosa intestinal são essenciais para definir diferentes linhagens celulares. imagiologia adequado dos tecidos intestinais é crucial para a identificação de padrões de expressão de proteínas com uma resolução máxima. Este estudo visa delinear os métodos ótimos e as condições de processamento do mouse tecidos intestinais.

Técnica Swiss-rolamento melhorado para preparo intestinal tecido por imuno-histoquímica e análises de imunofluorescência

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

Research

JoVE Journal

Biology

Pressure-polishing Pipettes for Improved Patch-clamp Recording

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Biologia

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

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