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>All studies involving mice were approved by the Stony Brook University Institutional Animal Care and Use Committee (IACUC). The mice were maintained on a 12:12 hr light-dark cycle.
	<ol>
		<li>Commercially obtain C57BL/6 mice. Obtain C57BL/6 mice carrying Klf5 alleles flanked by loxP sites (Klf5fl/fl). These mice were previously described21 and graciously provided by Dr. Ryozo Nagai.</li>
	</ol>
	</li>
	<li>Purchase C5...

<ol>
	<li>van der Flier, L. G., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells,+self-renewal,+and+differentiation+in+the+intestinal+epithelium.">Stem cells, self-renewal, and differentiation in the intestinal epithelium.</a> Annu Rev Physiol. 71, 241-260 (2009).</li><li>Bjerknes, M., Cheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+isolation+of+intact+epithelium+from+the+mouse+intestine.">Methods for the isolation of intact epithelium from the mouse intestine.</a> Anat Rec. 199, 565-574 (1981).</li><li>Barker, 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=Identification+of+stem+cells+in+small+intestine+and+colon+by+marker+gene+Lgr5.">Identification of stem cells in small intestine and colon by marker gene Lgr5.</a> Nature. 449 (7165), 1003-1007 (2007).</li><li>van der Flier, L. G., Haegebarth, A., Stange, D. E., van de Wetering, M., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OLFM4+is+a+robust+marker+for+stem+cells+in+human+intestine+and+marks+a+subset+of+colorectal+cancer+cells.">OLFM4 is a robust marker for stem cells in human intestine and marks a subset of colorectal cancer cells.</a> Gastroenterology. 137 (1), 15-17 (2009).</li><li>van der Flier, L. G., 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=Transcription+factor+achaete+scute-like+2+controls+intestinal+stem+cell+fate.">Transcription factor achaete scute-like 2 controls intestinal stem cell fate.</a> Cell. 136 (5), 903-912 (2009).</li><li>Sangiorgi, E., Capecchi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmi1+is+expressed+in+vivo+in+intestinal+stem+cells.">Bmi1 is expressed in vivo in intestinal stem cells.</a> Nat Genet. 40 (7), 915-920 (2008).</li><li>Montgomery, R. 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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+Kruppel-like+factors+in+health+and+diseases.">Mammalian Kruppel-like factors in health and diseases.</a> Physiol Rev. 90 (4), 1337-1381 (2010).</li><li>McConnell, B. B., Ghaleb, A. M., Nandan, M. O., Yang, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diverse+functions+of+Kruppel-like+factors+4+and+5+in+epithelial+biology+and+pathobiology.">The diverse functions of Kruppel-like factors 4 and 5 in epithelial biology and pathobiology.</a> Bioessays. 29 (6), 549-557 (2007).</li><li>Nandan, M. O., Ghaleb, A. M., Bialkowska, A. B., Yang, V. W. <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+factor+5+is+essential+for+proliferation+and+survival+of+mouse+intestinal+epithelial+stem+cells.">Kruppel-like factor 5 is essential for proliferation and survival of mouse intestinal epithelial stem cells.</a> Stem Cell Res. 14 (1), 10-19 (2015).</li><li>Gelberg, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+anatomy,+physiology,+and+mechanisms+of+disease+production+of+the+esophagus,+stomach,+and+small+intestine.">Comparative anatomy, physiology, and mechanisms of disease production of the esophagus, stomach, and small intestine.</a> Toxicol Pathol. 42 (1), 54-66 (2014).</li><li>De Robertis, M., 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+AOM/DSS+murine+model+for+the+study+of+colon+carcinogenesis:+From+pathways+to+diagnosis+and+therapy+studies.">The AOM/DSS murine model for the study of colon carcinogenesis: From pathways to diagnosis and therapy studies.</a> J Carcinog. 10 (9), (2011).</li><li>Magnus, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+presence+of+intestinal+epithelium+in+the+gastric+mucosa.">Observations on the presence of intestinal epithelium in the gastric mucosa.</a> The Journal of Pathology and Bacteriology. 44 (2), 389-398 (1937).</li><li>Moolenbeek, C., Ruitenberg, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+"Swiss+roll":+a+simple+technique+for+histological+studies+of+the+rodent+intestine.">The "Swiss roll": a simple technique for histological studies of the rodent intestine.</a> Lab Anim. 15 (1), 57-59 (1981).</li><li>Park, C. M., Reid, P. E., Walker, D. C., MacPherson, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+practical+'swiss+roll'+method+of+preparing+tissues+for+paraffin+or+methacrylate+embedding.">A simple, practical 'swiss roll' method of preparing tissues for paraffin or methacrylate embedding.</a> J Microsc. 145, 115-120 (1987).</li><li>Summersgill, B., Clark, J., Shipley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+and+chromogenic+in+situ+hybridization+to+detect+genetic+aberrations+in+formalin-fixed+paraffin+embedded+material,+including+tissue+microarrays.">Fluorescence and chromogenic in situ hybridization to detect genetic aberrations in formalin-fixed paraffin embedded material, including tissue microarrays.</a> Nat Protoc. 3 (2), 220-234 (2008).</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=Cardiac+fibroblasts+are+essential+for+the+adaptive+response+of+the+murine+heart+to+pressure+overload.">Cardiac fibroblasts are essential for the adaptive response of the murine heart to pressure overload.</a> J Clin Invest. 120 (1), 254-265 (2010).</li><li>el Marjou, F., 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=Tissue-specific+and+inducible+Cre-mediated+recombination+in+the+gut+epithelium.">Tissue-specific and inducible Cre-mediated recombination in the gut epithelium.</a> Genesis. 39 (3), 186-193 (2004).</li><li>McConnell, B. 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The Swiss rolling technique is a powerful method for preparing intestinal tissue for histological and morphological assessment on a large scale. In contrast to the previously described Swiss-rolling technique, which was originally developed for preparation of frozen sections18,19, the procedure presented here allows prompt intestinal tissue preparation and fixation for formalin fixation and paraffin embedding (FFPE). Compared to frozen tissue, FFPE tissue has much longer shelf life and is the preferred type of...

The mammalian intestinal epithelium comprises a single layer of columnar cells. In the small intestine, the proliferative cells are confined to the crypts while differentiated cells occupy the villus region. However, because there are no villi in the large bowel, the proliferative cells are localized to the bottom of the crypts and differentiated cells occupy the upper region of the crypts. The intestinal epithelium undergoes rapid replenishment (about 3 - 5 days) that is driven by continuous division of the proliferative cells within the crypts. The proliferative cells of the crypts are not a homogeneous population and are further subdivided into stem cells and transit-amplifying (TA) cells1. The stem cells reside at the bottom of the crypt, within the first 4 - 5 cells from the very bottom2. The current model supports the existence of two types of stem cells: crypt base columnar (CBC) stem cells and reserve quiescent stem cells. The CBC stem cells are actively proliferating and are marked by Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5)3, Olfactomedin 4 (Olfm4)4 and Achaete scute-like 2 (Ascl2)5. On the other hand, reserve quiescent stem cells are labeled by B cell-specific Moloney murine leukemia virus integration site 1 (Bmi1)6, mouse telomerase reverse transcriptase (mTert)7, HOP Homeobox (Hopx)8, Doublecortin-Like And CAM Kinase-Like 1 (Dclk1)9, and Leucine-Rich Repeats And Immunoglobulin-Like Domains 1 (Lrig1)10. The actively proliferating stem cells give rise to TA cells then undergo further differentiation into absorptive cells (enterocytes) and secretory cells (enteroendocrine, goblet, Paneth, and Tuft cells). Continuous cell division in the proliferative zone results in upward movement of epithelial cells along the crypt-villus axis until they reach top of the villi, where they undergo apoptosis and are sloughed off from the surface of the epithelium. The different types of intestinal epithelial cells are marked by the expression of distinct proteins (e.g., intestinal goblet cells can be recognized by staining with antibody against Muc2 and Paneth cells with antibody against lysozyme). We study the role of Kr&#252;ppel-like factors (KLFs) in the homeostasis and pathobiology of the intestinal epithelium11-13. The results presented here supporting the feasibility of a modified Swiss-rolling technique are based on previous studies of the role of Kr&#252;ppel-like factor 5 (KLF5) in the maintenance of the actively proliferating intestinal epithelial stem cells14. KLF5 is a zinc-finger transcription factor that is highly expressed in the active intestinal stem and TA cells12. Previous studies demonstrated that KLF5 is co-expressed with Ki-67, a known proliferative marker in the intestinal crypts.
The gastrointestinal tract is not a structurally or functionally homogeneous tissue. The small intestine is divided into duodenum, jejunum, and ileum and the large intestine into cecum and colon, with the latter further divided into proximal, middle, and distal portions. Each of these sections has unique histological features and plays distinct roles15. As such, the effects of insults and the degree of the response of the intestinal epithelium may depend on the region of studied tissue16. Additionally, various mice strains demonstrate diversity of the response at the histological level based on the type of insult used in the studies16. Thus, befitting tissue preparation is necessary to permit appropriate histological and molecular analysis of the intestinal tissues. As such, the Swiss-roll technique grants analysis of the complete length of the intestinal epithelium at one time and thus ascertains well-informed conclusions based on comprehensive information.
The Swiss-roll technique was first mentioned by Magnus17, and described in detail by Moolenbeck and Ruitenberg and Park et al. as a method for preparing tissues and performing histological analyses of the rodent intestine18,19, respectively. The protocol delineated in this publication presents an improved version of the original method that permits for timely and reliable tissue preparation for diagnostic purposes. This modified technique allows for efficient collections and preparation of the intestinal epithelium for universally used techniques, such as immunohistochemistry, immunofluorescence, as well as in situ hybridization (fluorescent and chromogenic20). Furthermore, the modified tissue specimen preparation method utilizes readily available and relatively inexpensive reagents while offering a method of rapid tissue fixation and allows for recovery of protein, DNA, and RNA for additional evaluation. Taken together, this technique is excellent for comprehensive assessment of histopathological, pathological, and molecular features of the intestinal epithelium.

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			<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>
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		<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>
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		<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>
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			<td height="21" style="height:21px;width:316px;">Tween 20</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td>P7949</td>
			<td></td>
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		<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>
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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.

The Swiss rolling technique in combination with immunohistochemical staining allows for comprehensive analysis of small or large intestinal tissue. The example of H&#38;E staining of a large bowel of a C57BL/6 mouse (Figure 1) is an illustration of the feasibility and the effectiveness of this technique. As shown in Figure 1, the image is able to capture all portions of the colon: proximal, middle, and distal. Thus, it allows for comprehensive histologica...

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

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

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

The overall goal of this technique is to efficiently process long sections of mouse intestinal tissue for uniform histological staining along its length. To get the intestinal sections there can be variations in staining along the entire length of the intestine. I tried utilizing the standard Swiss rolling technique but found it yields intestinal tissue that is very stiff and not easy to open.With some experimentation, I came up with a quick fixation method that uses less toxic chemicals and that allows much better Swiss rolling of the intestinal tissue. The modified fixative can be used to quickly fix other thick tissues. In preparation, euthanize the test animal according to the text protocol and ready the surgical setting.To begin, use dissection forceps and scissors to make a lateral incision into the middle of the abdomen about one to two centimeters below the ribcage depending on the size of the mouse. Then gently pull apart and cut away the skin to expose the abdominal muscles. Next, grip and pull up on the muscles without holding or pulling at the intestines.Then, carefully make an initial incision in the abdominal muscles and carefully cut through them to expose the intestines. Now identify the colon and trace it to its distal end where it joins the rectum. Then using scissors transect the colon as close to the anal opening as possible.Next find where the stomach joins the small intestines and using scissors cut free the intestines about one centimeter from the stomach. Now transfer the entire length of the free small intestine and colon to a clean petri dish containing PBS. Next secure the distal end of the colon and gently unravel the entire length of the colon removing any attached mesenteric tissues.Then identify the cecum, and transect the intestine where the cecum joins the colon, thus isolating the colon. Similarly unravel the rest of the small intestine from the attached mesenteric connective and fatty tissues. Then transect the small intestine where it joins the cecum.The cecum may then be removed and discarded. Now fill a 10 milliliter syringe with modified Bouin's fixative and attach a gavage needle. Insert the needle about half a centimeter into the anterior opening of an intestinal segment.While firmly securing the gavage needle inside the segment, slowly flush the contents of the tissue with fixative. Be careful, as too much pressure will burst the segment. If the small intestine looks too long to handle, you might want to cut it into three equal segments first and flush each segment separately.The fixation will cause the intestinal segment's color to become opaque. Next using scissors cut the segment longitudinally along the mesenteric line and then briefly rinse the segment in a dish of PBS while holding it open with forceps. Now cut the small intestine into three equal segments, proximal, mid and distal, which correspond roughly to the duodenum, the jejunum and the ileum.Macroscopically, there are no discerning features to each section. Be sure to make a mark to identify the proximal from the distal end on each segment. Then place a segment with the lumenal side facing upward on the lid of a petri dish.On the colon segment, identify the lumenal side by the ridges running across its width at the proximal end. The mid and distal regions have some variegations that run longitudinally. On the small intestine, identify the lumenal side by the rougher surface which marks the villi.Microscopic examination shows that the villi are longest in the proximal regions of each segment, whereas the colonic crypts are shortest in the proximal regions and are at their tallest in the mid section. Proceed with Swiss rolling of the colon. With the colon flat open and the lumenal side up, pull it with forceps from its proximal end toward the edge of the petri dish.Keeping the lumenal side up, hold the proximal end with the forceps and wrap up the edge of the proximal end round a toothpick and tightly pinch the wrapped edge against the toothpick. Then gently and slowly roll the colon around the toothpick to form a Swiss roll. Once the entire colon length has been rolled up, use another pair of forceps to carefully slide the roll into a tissue processing cassette.Then transfer the cassette into 10 percent buffered formalin and allow it to incubate overnight at room temperature. To Swiss roll a segment of small intestine, open it lumenal side up and pull it with forceps from its proximal end toward the edge of a dish. Then use the same technique as shown with the colon to make the Swiss roll and fix it in formalin.Using the described methods, HND staining for the large bowel of a C57 black 6 mouse revealed all portions of the colon for a comprehensive histological assessment. Next, the preparation was used for mice expressing EGFP driven by the LGR5 promoter which is active only in peripherating intestinal crypts. This EGFP transgene only has about 5 to 10 percent penetrance, but regions of interest could be easily found.Using the same mice with EGFP LGR5 expression, KLF5 and KI67 were stained. First control mice with normal LGR5 expression were studied. EGFP positive cells are marked with blue arrows and EGFP negative cells are marked by white arrows.Then the same transgenes were expressed in a KLF5 knockout mouse. Co-staining of KLF5 and KI67 is missing in EGFP positive stem cells, but remains present in EGFP negative crypts. Clearly the quality of the preparation for multi fluoro force staining is very good.Once mastered, this technique can be done in about 15 minutes per mouse if you are collecting both the small intestine and the colon. Don't forget that working with Bouin's fixative can be extremely hazardous to the eyes. Precautions such as proper eye protection should always be taken while performing this procedure.Thank you for watching and good look with your experiments.

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