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 C57BL/6 mice carrying the inducible Cre recombinase gene under regulation of the Lgr5 promoter (Lgr5-EGFP/CreERT2 mice).</li>
	<li>To establish Lgr5-EGFP/CreERT2/Klf5fl/fl mice, cross Klf5fl/fl mice with Lgr5-EGFP/CreERT2 mice and then backcross the progeny toKlf5fl/fl mice.</li>
	<li>Treat mice with tamoxifen as per the protocol previously described22. Inject 8-week-old experimental and control mice intraperitoneally (I.P.) with 1 mg of tamoxifen (10 mg/ml), dissolved in sterile corn oil, for 5 consecutive days.</li>
	<li>On day 14 after first tamoxifen injection, sacrifice mice using CO2 asphyxiation by placing them in a chamber connected to CO2 gas source. Allow the gas to flow into the chamber until the mice are unconscious and all movement ceases. To ensure euthanasia perform cervical dislocation.</li>
</ol>
2. Tissue Preparation and Swiss Rolling
<ol>
	<li>Using dissection forceps and scissors, cut an incision in the skin of the abdominal side. With a pair of forceps, hold the skin at the incision and pull gently away from the abdominal muscle tissue. Use a pair of scissors to cut the abdominal skin and expose the abdominal muscles.</li>
	<li>Hold and pull up the peritoneum with the forceps. Make sure not to be holding and pulling at the intestines. Carefully make an incision in the peritoneal tissue and continue cutting away the skin to expose the abdominal muscles. Carefully make an incision in the abdominal muscles and continue cutting them away to expose the intestines.</li>
	<li>Identify the colon and trace to the distal end where it joins the rectum/anus. With a pair of scissors, cut as close to the anal opening as possible.</li>
	<li>Identify the stomach and trace where it joins with the small intestine. With a pair of scissors, cut free the intestine about 1 cm away from the stomach. Transfer the entire length of the freed small intestine and colon to a clean Petri dish containing phosphate-buffered saline (PBS).</li>
	<li>With one hand or a pair of forceps, hold the distal end of the colon and use the other hand to gently unravel the entire length of the colon from any mesenteric connective and/or fat tissue.</li>
	<li>Identify the cecum (small pouch between the small and large intestine) and cut where the cecum and the colon join (the proximal end of the colon) to free the colon.</li>
	<li>With one hand or a pair of forceps, hold the small intestine and use the other hand to gently unravel the entire length of the small intestine from any mesenteric connective and/or fat tissue. Cut away the cecum and discard if there is no need for it. 
	Note: The process can be stopped here by incubating the dissected out intestines in modified Bouin&#39;s fixative (50% ethanol/5% acetic acid in dH2O) overnight and continue the procedure the following day.</li>
	<li>Handling one segment of the intestine (small intestine or colon), fill a 10-ml syringe with modified Bouin&#39;s fixative and attach a gavage needle to it. Insert the needle about half a centimeter in the anterior opening of the intestinal segment.</li>
	<li>Hold the gavage needle inside the intestinal segment by applying firm pressure with an instrument on the intestinal segment. With the other hand holding the syringe, apply gentle but consistent pressure to flush the contents of the intestinal segment using modified Bouin&#39;s fixative. This step allows simultaneous cleaning of the intestinal segment and immediate fixation. Use a Petri dish to collect the flow-through waste. Fixation can be observed by the colon color turning opaque. 
	Note: Make sure not to apply too much pressure during flushing or the intestinal segment might burst open.</li>
	<li>Using scissors, cut open the intestinal segment longitudinally along the mesenteric line. Hold it with a pair of forceps and rinse it briefly in a Petri dish containing PBS.
	<ol>
		<li>Cut the small intestine into 3 equal segments: proximal, mid, and distal. The proximal segment is the one immediately following the stomach, and the distal segment is the one immediately before the colon. The proximal segment is equivalent to the duodenum, the mid to the jejunum, and the distal to the ileum.</li>
		<li>For each segment mark the proximal and the distal end. The proximal end is the one that was originally pointing toward the stomach, and the distal pointed toward the colon.</li>
	</ol>
	</li>
	<li>Use the top of a Petri dish to place the cleaned and opened intestinal segment. Place the intestinal segment with the luminal side facing upward.
	<ol>
		<li>For the colon, identify the luminal side by the variegations/ridges running across its width and are present at the proximal end. The mid and distal regions have some variegations that run longitudinally. For the small intestine, identify the luminal side by the rough-appearing surface, which marks the villi. 
		Note: For the small intestine, there are no external macroscopic anatomical demarcation of proximal, middle and distal regions. However these regions can be can identified in sections under the microscope. The villi are longest in the proximal region and become gradually shorter distally where they are the shortest in length. Microscopically, the colonic crypts are shortest in the proximal region which can also be identified by the presence of ridges. The colonic crypts are tallest in the midsection and shorter in the distal region yet still taller than in the proximal region.</li>
	</ol>
	</li>
	<li>Proceed with Swiss rolling the colon:
	<ol>
		<li>Keep the colon flat open and pull it with forceps from its proximal end toward the edge of the Petri dish.</li>
		<li>Keep luminal side facing up and hold the proximal end with the forceps with one hand and with the other hand hold a toothpick.</li>
		<li>Wrap the edge of the proximal end around the toothpick using the forceps and slightly pinch the wrapped edge against the toothpick to hold it in place.</li>
		<li>Gently and slowly start rolling the toothpick with fingers to roll the colon around the toothpick to form a Swiss roll.</li>
		<li>Once the entire colon length has been rolled up, use a pair of forceps to carefully slide the colon Swiss roll off the toothpick and into a tissue-processing/-embedding cassette. Place the cassette in 10% buffered formalin overnight at room temperature.</li>
	</ol>
	</li>
	<li>Proceed with Swiss rolling the small intestine:
	<ol>
		<li>Handling one segment at a time, keep the segment flat open with luminal side up and pull it with forceps from its proximal end toward the edge of the Petri dish.</li>
		<li>Proceed with Swiss rolling as described for the colon.</li>
	</ol>
	</li>
	<li>Proceed with processing formalin-fixed tissues for paraffin embedding. Proceed as follows: 
	NOTE: Use an automated processor (see materials and Equipment&#39;s for details).
	<ol>
		<li>While tissue is in the cassette, rinse tissue with PBS until fixative is completely removed.</li>
		<li>Dehydrate tissue using ethanol in the following sequence: 50% ethanol for 10 min, 70% ethanol for 10 min, 80% ethanol for 10 min, 95% ethanol for 10 min, 100% ethanol for 10 min, 100% ethanol for 10 min, and 100% ethanol for 10 min.</li>
		<li>Exchange ethanol with xylene in the following sequence: 2:1 ethanol : xylene for 10 - 15 min, 1:1 ethanol : xylene for 10 - 15 min, 1:2 ethanol : xylene for 10 - 15 min, 100% xylene for 10 - 15 min, 100% xylene for 10 - 15 min, and 100% xylene for 10 - 15 min. CAUTION: Xylene is a health hazard, thus the workplace should be equipped with a local exhaust ventilation with a proper hood and the proper protective equipment should be wore by personnel.</li>
		<li>Exchange xylene with paraffin. Perform the following steps in a vacuum oven set for 54 - 58 &#176;C: 2:1 xylene : paraffin for 30 min, 1:1 xylene : paraffin for 30 min, 1:2 xylene : paraffin for 30 min, 100% paraffin for 1 - 2 hr, and 100% paraffin for 1 - 2 hr or overnight. 
		Note: Do not let the paraffin exceed 60 &#176;C for prolonged periods of time because this will degrade the paraffin polymers and make it hard and brittle.</li>
		<li>Embed in fresh new paraffin and orient tissue as desired before the paraffin hardens. For the Swiss rolls, following tissue paraffinization, it is important to reposition each roll on its side during paraffin embedding. This ensures that during cutting, sections of the entire length of each roll will be produced.</li>
	</ol>
	</li>
	<li>For staining, cut 5-&#956;m thick sections using microtome. Collect the sections on charged slides and dry them (bake) in a 65 &#176;C oven overnight; let cool to room temperature and subsequently used them for staining. 
	Note: The baking time can be shortened to 1 - 2 hr depending on how freshly cut the slides are. If less than a month since sectioning, then it is advisable to bake overnight. If it has been more than a month, then 1 - 2 hr of baking is sufficient to save time. If running out of time, overnight backing can always be used.</li>
</ol>
3. Histological Analysis and Immunofluorescence Staining
Perform histological analysis and immunofluorescence staining as previously described23,24, with modifications. For enhanced green fluorescent protein (EGFP) immunofluorescence:
<ol>
	<li>Bake the slides in a 65 &#176;C oven for 1 hr to inactivate endogenous alkaline phosphatase activity and subsequently deparaffinize in xylene.</li>
	<li>Incubate sections in a bath of 3% hydrogen peroxide in methanol to block endogenous tissue peroxidases; then rehydrate by incubation in a decreasing alcohol bath series (100%, 95%, 70%) followed by antigen retrieval in citrate buffer solution (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) at 125 &#176;C for 10 min using a decloaking chamber.</li>
	<li>Draw a circle around tissue sections using marking pen that provides hydrophobic barrier and incubate sections with blocking buffer (2.5% bovine serum albumin [BSA] in TBS-Tween) for 30 min at 37 &#176;C and then with primary antibody against EGFP (dilution 1:500) at 4 &#176;C overnight in a humidified chamber while shaking gently.</li>
	<li>Wash sections and incubate with fluorescent-tagged secondary antibody at the appropriate concentration for 30 min at 37 &#176;C.</li>
	<li>Wash slides after secondary antibody treatment and stain nuclei with Hoechst and mount with mounting media.
	<ol>
		<li>For Klf5/EGFP/Ki-67 co-staining, process slides as described in steps 3.1 - 3.3 and perform Klf5 staining by incubating with Klf5 antibody (dilution 1:150) overnight.</li>
		<li>Apply rabbit AP polymer detection to slides as per protocol.</li>
		<li>Reveal Klf4 staining using chromogen immunohistochemical staining as per manufacturer&#39;s protocol.</li>
		<li>Perform antibody elution using the previously described protocol25. Incubate slides at 50 &#176;C in Glycine-SDS (pH 2.0) solution with agitation for 1 hr.</li>
		<li>Wash slides thoroughly with distilled water and incubate with blocking buffer (2.5% BSA in TBS-Tween) for 30 min at 37 &#176;C.</li>
		<li>Add Ki-67 (dilution 1:500) and EGFP (dilution 1:500) antibodies simultaneously and incubate at 37 &#176;C for 1 hr.</li>
		<li>Perform fluorescent detection with appropriate secondary antibodies before staining with Hoechst (data not shown) and mount with mounting medium.</li>
	</ol>
	</li>
	<li>Observe histological morphology of the tissues on staining 5-&#956;m sections with hematoxylin and eosin (H&#38;E)26. For fluorescent images, use emission wavelengths of 535, 646, and 700 to visualize EGFP, Klf5, and Ki-67 staining, respectively.</li>
</ol>

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The authors declare that they have no competing financial interests.

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.

<table><tbody><tr><td>Stainless Steel Dissecting Kits</td><td>VWR</td><td>25640-002</td><td></td></tr><tr><td>Decloaking Chamber</td><td>Biocare Medical</td><td>DC2012</td><td></td></tr><tr><td>Syringe 10 ml</td><td>VWR</td><td>89215-218</td><td></td></tr><tr><td>Swingsette Tissue embedding/processing cassette with lid</td><td>Simport</td><td>M515</td><td></td></tr><tr><td>Superfrost Plus Slides [size: 25 x 75 x 1 mm]</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Manual Slide Staining Set</td><td>Tissue-Tek/Sakura</td><td>4451</td><td></td></tr><tr><td>Staining Dish Green</td><td>Tissue-Tek/Sakura</td><td>4456</td><td></td></tr><tr><td>Staining Dish White</td><td>Tissue-Tek/Sakura</td><td>4457</td><td></td></tr><tr><td>24-Slide Slide Holder with Detachable Handle</td><td>Tissue-Tek/Sakura</td><td>4465</td><td></td></tr><tr><td>Oven</td><td>Thermo Scientific</td><td>6243</td><td>for baking slides at 65 degree</td></tr><tr><td>Dissection microscope</td><td>Zeiss</td><td>Stemi 2000C</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Nikon</td><td>Eclipse 90i</td><td>Bright and fluoerescent light, with objectives: 10X, 20X</td></tr><tr><td>PAP Pen Super-Liquid Blocker Mini</td><td>Fisher Scientific</td><td>DAI-PAP-S-M</td><td></td></tr><tr><td>Ethanol 200 proof</td><td>AAPR</td><td>111000200</td><td></td></tr><tr><td>Methanol</td><td>VWR</td><td>BDH1135-4LP</td><td></td></tr><tr><td>Glacial acetic acid</td><td>AAPR</td><td>281000ACS</td><td></td></tr><tr><td>Xylene</td><td>Fisher Scientific</td><td>X5P-1GAL</td><td></td></tr><tr><td>Hydrogen peroxide 25% solution in water</td><td>ACROS</td><td>202465000</td><td></td></tr><tr><td>10% bufered formalin</td><td>Fisher Scientific</td><td>22-026-213</td><td></td></tr><tr><td>Bovine serum fraction V, heat shock</td><td>Roche</td><td>3116956001</td><td></td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P7949</td><td></td></tr><tr><td>Sodium citrate</td><td>Fisher Scientific</td><td>S279</td><td></td></tr><tr><td>Gavage needle</td><td>VWR</td><td>20068-624</td><td></td></tr><tr><td>Rabbit anti Klf5 antibody</td><td>Santa Cruz Biotechnology</td><td>sc-22797</td><td>Dilution 1:150</td></tr><tr><td>Chicken anti EGFP antibody</td><td>Millipore</td><td>AB16901</td><td>Dilution 1:500</td></tr><tr><td>Rabbit anti Ki67 antibody</td><td>Biocare Medical</td><td>CRM325B</td><td>Dilution 1:500</td></tr><tr><td>Mach3 rabbit AP polymer detection kit</td><td>Biocare Medical</td><td>M3R533L</td><td></td></tr><tr><td>Warp red chromogen kit</td><td>Biocare Medical</td><td>WR806 H</td><td></td></tr><tr><td>Lgr5-EGFP/CreER&lt;sup&gt;T2&lt;/sup&gt; mice&amp;nbsp;</td><td>Jackson labs</td><td>008875&amp;nbsp;</td><td></td></tr><tr><td>Automated processor</td><td>Leica</td><td>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.

The Swiss rolling technique in combination with immunohistochemical staining allows for comprehensive analysis of small or large intestinal tissue. The example of H&amp;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 ...

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Research

JoVE Journal

Biology

68.6K Views.  Stony Brook University School of Medicine.  Nauwkeurige identificatie en locatie van epitheelcellen langs de intestinale slijmvlies essentieel verschillende cellijnen definiëren. Een goede beeldvorming van darmweefsel is van cruciaal belang voor de identificatie van eiwit expressie patronen met maximale resolutie. Dit onderzoek heeft als doel om de optimale methoden en voorwaarden voor de verwerking van de muis intestinale weefsels af te bakenen. 

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

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of Crohn&rsquo;s Disease - Crohn&rsquo;s Colitis. Acute colitis may present with abdominal pain and distention, malabsorption, diarrhea, hematochezia and mucus in the stool. We are beginning to understand the complex interactions between the environment, genetics, and epithelial barrier dysfunction in Inflammatory Bowel Disease and animal models of colitis have been essential in advancing our understanding of this disease. One popular model involves supplementing the drinking water of mice with low-molecular weight Dextran Sodium Sulfate (DSS), resulting in epithelial damage and a robust inflammatory response in the colon lasting several days 1.Variations of this approach can be used to model acute injury, acute injury followed by repair, and repeated cycles of DSS interspersed with recovery modeling chronic inflammatory diseases 2. After a single four-day treatment of 3% DSS in drinking water, mice show signs of acute colitis including weight loss, bloody stools, and diarrhea. Mice are euthanized at the conclusion of the treatment course and at necropsy dissected colons are processed and can be 'Swiss rolled" 3 to allow microscopic analysis of the entire colon or infused with formalin as "sausages" to allow macroscopic analysis. Tissue is then embedded in paraffin, sectioned, and stained for histologic review.

Murine Colitis Modeling using Dextran Sulfate Sodium DSS

Dextran sulfate sodium (DSS) administered in the drinking water is an established murine inflammatory injury model of acute colitis. This protocol outlines the method for DSS treatment and the preparation of tissues.

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

Medicine

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Neuroscience

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. ...

Immunology and Infection

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

Use of Anti-phospho-girdin Antibodies to Visualize Intestinal Tuft Cells in Free-Floating Mouse Jejunum Cryosections

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is phosphorylated by both receptor and non-receptor tyrosine kinases at tyrosine 1798. Omori et al. developed site- and phosphorylation status-specific antibodies against human girdin at tyrosine-1798 (pY1798), which specifically bind to phosphorylated tyrosine-1798, but not to unphosphorylated tyrosine-1798. pY1798 antibodies have been used to specifically label tuft cells (TCs) that are present in mammalian gastrointestinal tissues, but the function of these cells is unclear. This protocol allows the robust visualization of TCs in the jejunum using pY1798 antibodies and immunofluorescence. To ensure successful and simple TC visualization, this protocol includes two histological techniques: production of free-floating cryosections from gelatin-filled jejunum tissue, and low-temperature antigen retrieval at 50 &#176;C for 3 h. Filling the jejunum with gelatin maintains the shape of free-floating sections throughout the staining procedure, whereas low-temperature antigen retrieval ensures robust signals from TCs. Successful use of this protocol results in pY1798 staining of TCs distributed from villus tip to crypt. Stained TCs have a spool-shaped soma and fluorescent signals condense at the lumenal tip, which corresponds to the protruding &#39;tuft.&#39;&#160;Phalloidin staining colocalized with pY1798-positive TCs at the thickened brush border, and corresponds to a rootlet mass extending from the TC tuft. This protocol could be used to examine TCs in human biopsy samples collected with gastrointestinal endoscopes. Furthermore, TCs were recently reported to accumulate following parasite infection in mice, suggesting that this protocol could have applications for diagnosis of parasite infections in the human gut.

Kuga et al. discovered that phosphorylation-status specific antibodies against the actin binding protein girdin phosphorylated at tyrosine 1798 (pY1798) can be used to label tuft cells (TCs). This protocol allows robust visualization of TCs using immunofluorescent staining of free-floating jejunum cryosections with pY1798 antibodies.

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is ...

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier impairment has been associated with the development of various pathologies such as inflammatory bowel diseases (IBD). In the inflamed mucosa, superficial erosions or micro-erosions that corrupt epithelial monolayers correspond to sites of high permeability. Several mechanisms have been implicated in the formation of micro-erosions including cell shedding and apoptosis. These micro-erosions often represent microscopic epithelial gaps randomly distributed in the colon. Visualization and quantification of those epithelial gaps has emerged as an important tool to investigate intestinal epithelial barrier function. Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa. Additional use of cell death markers revealed that HPR encompass apoptotic foci where epithelial extrusion/shedding occurs. The protocol described here provides a simple but effective approach to visualize and quantify micro-erosions in the intestine, which is a very useful tool in disease models, in which the intestinal epithelial barrier is compromised.

Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa.

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier ...

Efficient Swiss Roll Method for High-Quality Immunostaining of Intestinal Tissue

Optimized Protocol for Intestinal Swiss Rolls and Immunofluorescent Staining of Paraffin Embedded Tissue

The intestine is a complex organ composed of the small and the large intestines. The small intestine can be further divided into duodenum, jejunum, and ileum. Each anatomical region of the intestine has a unique function that is reflected by differences in cellular structure. Investigating changes in the intestine requires an in-depth analysis of different tissue regions and cellular alterations. To study the intestine and visualize large pieces of tissue, researchers commonly use a technique known as intestinal Swiss rolls. In this technique, the intestine is divided into each anatomical region and fixed in a flat orientation. Then, the tissue is carefully rolled and processed for paraffin embedding. Proper tissue fixation and orientation is an often-overlooked laboratory technique but is critically important for downstream analysis. Additionally, improper Swiss rolling of intestinal tissue can damage the fragile intestinal epithelium, leading to poor tissue quality for immunostaining. Ensuring well-fixed and properly oriented tissue with intact cellular structures is a crucial step that ensures optimal visualization of intestinal cells. We present a cost-effective and simple method for making Swiss rolls to include all sections of the intestine in a single paraffin-embedded block. We also describe optimized immunofluorescence staining of intestinal tissue to study various aspects of the intestinal epithelium. The following protocol provides researchers with a comprehensive guide to obtaining high-quality immunofluorescence images through intestinal tissue fixation, Swiss-roll technique, and immunostaining. Employing these refined approaches preserves the intricate morphology of the intestinal epithelium and fosters a deeper understanding of intestinal physiology and pathobiology.

Author Spotlight: Effective Immunofluorescent Staining Protocol for Intestinal Cell Biology Research

The intestine is vital for digestion and absorption. Each region-duodenum, jejunum, ileum, colon-serves distinct functions due to unique cellular structures. Studying intestinal physiology demands meticulous tissue analysis. This protocol outlines tissue fixation and processing using the Swiss roll technique, ensuring accurate immunostaining through proper tissue preservation and orientation.

The intestine is a complex organ composed of the small and the large intestines. The small intestine can be further divided into duodenum, jejunum, and ...

An Immunofluorescence-Based Method for Visualizing Tuft Cells in Jejunum Cryosections

Source: Mizutani, Y. et al., <a href="https://app.jove.com/v/57475">Use of Anti-phospho-girdin Antibodies to Visualize Intestinal Tuft Cells in Free-Floating Mouse Jejunum Cryosections.</a>&#160;J. Vis. Exp. (2018)
This video illustrates an immunofluorescence-based method for visualizing intestinal tuft cells in free-floating mouse jejunum cryosections. Within the intestinal villi, the co-localization of green fluorescence at the luminal tip with an extended red rootlet mass indicates the presence of tuft cells.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

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