This study was supported by the National Institutes of Health (NIH) grants K01 DK121869 to ACE and this publication was supported in part by T32 GM132055 (RME), F31 DK139736 (SAD), T32 DK124191 (SAD), TL1 TR001451 (RS), UL1 TR001450 (RS) and the HCS cornerstone grants to SAD &#38; RS. This work was supported by startup funds from the Medical University of South Carolina (MUSC) to ACE and was supported by the MUSC Digestive Disease Research Core Center (P30 DK123704) and the COBRE in Digestive and Liver Disease (P20 GM120475). Imaging was performed using the cell and molecular imaging core at MUSC.

The Institutional Animal Care and Use Committee of the Medical University of South Carolina approved all animal care, maintenance, and treatment. Intestinal tissue was collected from adult C57BL/6J mice (males and females 3-5 months old, weighing about 30 g) for use in the present study.
1. Intestinal tissue fixation
<ol>
	<li>Carefully dissect out the entire intestine from an euthanized mouse and place it in a weigh boat or Petri dish containing phosphate-buffered saline (P...

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

<ol>
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The authors have nothing to disclose.

Here, we present an optimized method for tissue fixation using the Swiss roll technique to preserve intestinal architecture and promote accurate immunostaining. Once mastered, this technique can be used to investigate a wide variety of research questions involving intestinal physiology and cell biology19. Several optimized Swiss rolling methods have been published and are very useful20,21. An advantage of this technique is the ease of accu...

The cellular architecture of the intestine poses a unique challenge in maintaining its structural integrity when intestinal tissue is being preserved for immunostaining. The small intestine is made up of elongated fingerlike structures known as villi1. These villi often become malformed during embedding processes. Ensuring that researchers have techniques for properly embedding intestines to achieve cross sections, allowing visualization of all regions of the intestine, as well as the layers that make up the intestine (i.e., muscularis propria, mucosa, and the serosa), is crucial for robust experimental analysis2. Inadequate fixation, excessive fixation, and improper tissue handling will compromise tissue integrity, resulting in inadvertent damage to the intestinal epithelium3,4. Damaging the intestinal epithelium during these steps can significantly diminish the quality of subsequent analyses, like immunofluorescence, irrespective of the efficacy of immunohistochemistry protocols and antibodies employed.
Immunostaining, like proper tissue fixation, is an important part of biomedical research. When done well, immunostaining can illuminate previously unknown aspects of cellular structure and function. Immunofluorescence staining of paraffin sections can be challenging due to physicochemical modifications resulting from the fixation and paraffin embedding process5. Fixation and paraffin embedding results in antigen masking that can interfere with immunofluorescence detection of epitopes of interest6. Delayed fixation can induce proteolytic degradation, which results in weakened or absent staining of critical epitopes7. Additionally, antibodies are often inaccurate with high levels of background. Immunostaining protocols that promote consistent and specific antibody binding and a high signal-to-noise ratio can provide valuable information for researchers. 
Here, we provide a comprehensive protocol designed to obtain high-quality immunofluorescence images through intestinal tissue fixation, Swiss roll preparation8, and immunostaining. Emphasizing guidelines to preserve the integrity of the intestine, the protocol aims to provide researchers with a robust methodology to enhance the quality and reliability of immunofluorescence imaging studies. We have also sought to use cost-effective resources, including filter paper and homemade antigen retrieval, blocking solutions, and antibody diluents to make the protocol more accessible to labs that may have restricted funds. As for all experimental protocols, researchers should optimize the current protocol based on their experimental approach and areas of interest.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#946;-CATENIN</td>
			<td>GeneTex</td>
			<td>GTX101435</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulose filter paper</td>
			<td>Cytiva</td>
			<td>10427804</td>
			<td>Thick Whatman paper</td>
		</tr>
		<tr>
			<td>Charged glass slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>23888114</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Epredia</td>
			<td>152440</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting pins size 00</td>
			<td>Phusis</td>
			<td>B082DH4TZF</td>
			<td></td>
		</tr>
		<tr>
			<td>E-CADHERIN</td>
			<td>R&#38;D Systems</td>
			<td>AF748</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer gloves</td>
			<td>Tempshield</td>
			<td>UX-09113-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating block</td>
			<td>Premiere</td>
			<td>XH-2001</td>
			<td>Slide Warmer</td>
		</tr>
		<tr>
			<td>Histo-Clear II</td>
			<td>Electron Microscopy Sciences</td>
			<td>64111-04</td>
			<td>Clearing reagent</td>
		</tr>
		<tr>
			<td>Hoescht</td>
			<td>Thermo Fisher Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma Aldrich</td>
			<td>320331</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic pen</td>
			<td>Millipore</td>
			<td>402176</td>
			<td></td>
		</tr>
		<tr>
			<td>LAMININ</td>
			<td>GeneTex</td>
			<td>GTX27463</td>
			<td></td>
		</tr>
		<tr>
			<td>LAMP1</td>
			<td>Santa Cruz</td>
			<td>SC-19992</td>
			<td></td>
		</tr>
		<tr>
			<td>Large cassettes</td>
			<td>Tissue-Tek</td>
			<td>4173</td>
			<td></td>
		</tr>
		<tr>
			<td>Minutien pins</td>
			<td>Fine Science Tools</td>
			<td>NC9679721</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse-on-mouse blocking reagent</td>
			<td>Vector Laboratories</td>
			<td>MKB-2213</td>
			<td>Mouse-on-mouse block</td>
		</tr>
		<tr>
			<td>MUC2</td>
			<td>GeneTex</td>
			<td>GTX100664</td>
			<td></td>
		</tr>
		<tr>
			<td>PCNA</td>
			<td>Cell Signaling Technology</td>
			<td>2586S</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Cooker</td>
			<td>Cuisinart</td>
			<td>B000MPA044</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong gold antifade</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36934</td>
			<td>Mounting medium</td>
		</tr>
		<tr>
			<td>Reverse action forceps</td>
			<td>Dumont</td>
			<td>5748</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Rack</td>
			<td>Tissue-Tek</td>
			<td>62543-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Staining Set</td>
			<td>Tissue-Tek</td>
			<td>62540-01</td>
			<td>Solvent Resistant Dishes and Metal Frame</td>
		</tr>
		<tr>
			<td>Small cassettes</td>
			<td>Fisherbrand</td>
			<td>15-200-403B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate dihydrate</td>
			<td>Fisher Bioreagents</td>
			<td>BP327-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Teleostein Gelatin</td>
			<td>Sigma</td>
			<td>G7765</td>
			<td>Blocking buffer</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Thermo Fisher Scientific</td>
			<td>A16046</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Thermo Fisher Scientific</td>
			<td>J20605-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Wipes</td>
			<td>KimTech</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes</td>
			<td>Fisher Chemical</td>
			<td>1330-20-7</td>
			<td></td>
		</tr>
		<tr>
			<td>&#947;-ACTIN</td>
			<td>Santa Cruz</td>
			<td>SC-65638</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

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

Our research focuses on understanding how the gastrointestinal tract functions, and how these functions are impacted by different pathological states. We are interested in learning the response of different intestinal epithelial cells to various conditions, such as infection and inflammation, obesity and genetic mutations. The importance of antibody specificity is becoming increasingly apparent.However, the impact of tissue fixation, preservation, and standardized staining methods are underappreciated in the field. Consistent and robust immunofluorescent staining greatly depends on the quality of tissue preservation, processing, and orientation that occurs in the initial steps of data collection. This protocol provides a simple and effective method for investigating various questions regarding intestinal cell biology and physiology.Our lab is currently using these protocols to better our understanding of the roles of different intestinal cell types in maintaining homeostasis and limiting inflammation. This protocol allows detailed visualization of all regions of a mouse intestine in a single paraffin embedded block. This saves money on paraffin, and paraffin embedding services, the amount of time required for sectioning, and reagents.It's also highly reproducible using diverse antibodies.

Hematoxylin and eosin (H&#38;E) staining was performed, as previously described12. Using the optimized method, intestinal Swiss rolls included all three segments of the small intestine and the large intestine on a single slide. Having the entire intestine accommodated on a slide allows researchers to analyze changes throughout all portions of the intestine and saves costs on sectioning and staining reagents (Figure 1). Also, exposing all intestinal segments to the sam...

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 ...

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Research

JoVE Journal

Biology

Intestinal Tissue Fixation and Processing Using the Swiss Roll Technique

To begin, attach a large P200 cut pipette tip to a 10-milliliter syringe and fill it with PBS. Use forceps to insert the syringe tip into the opening of an intestinal segment isolated from the euthanized mouse. Hold the intestinal segment on the syringe with the forceps and gently flush at a rate of approximately 50 microliters per second to remove the contents.Collect the intestinal contents into an empty weigh boat. Then lay the wet intestinal segment in a straight line on a strip of dry labeled cellulose filter paper. Using dissection scissors, cut the intestine open longitudinally along the mesenteric line, approximately five millimeters at a time.Use the bottom edge of the scissors or a pair of forceps to lay the cut tissue flat on the filter paper. Now place another piece of filter paper on top of the intestinal segment to prevent it from curling or losing its shape during fixation. To secure the tissue in place, staple the edges of the filter paper while avoiding stapling the tissue.Submerge the tissues in 10%normal buffered formalin. Gently remove the top piece of filter paper touching the luminal side of the intestine. Use forceps to carefully peel the intestine off the bottom piece of filter paper before discarding it.Then wash the tissue in PBS three times to remove formalin from the tissue. Using reverse action forceps, pick up a piece of intestine along the short edge with the luminal side facing up and turn the forceps to roll the tissue. Place the tissue in a dry weigh boat.Hold the tissue in place with forceps in one hand. Carefully open the reverse action forceps and release the tissue, using the other forceps to dislodge the tissue from the reverse action forceps. Insert a size 00 dissecting pin into the tissue.Use wire cutters to remove the sharp tip of the pin. Once all samples have been rolled, place all four tissue rolls into the same cassette and insert the cassette into a tissue processor. At an embedding station, place a small amount of paraffin into a mold.Remove the four Swiss rolls from the cassette and place them into an individual mold, laying them as flat as possible. Add more paraffin to fill the mold. Move the mold to the cold plate and ensure that all Swiss rolls are flat on the bottom of the mold.Place a labeled small cassette top on the mold. Once the wax has solidified, remove the block from the mold.

Optimized Immunofluorescence Staining of Intestinal Tissue and Microscopic Preparation for Imaging

To begin, prepare a humidified chamber by taking a large slide box and removing the cardboard cover adhered to the lid. Place damp paper towels vertically at the base of the slide box, ensuring they lay flat. Prepare the slide for staining by deparaffinization, rehydration, antigen retrieval, and washing.Remove the slide from PBS and wipe off excess PBS while avoiding the tissue. With a hydrophobic pen, draw a box around the tissue to form a barrier. Place the outline slide horizontally over the damp paper towels.Add approximately 100 microliters of blocking buffer to cover the tissue. Close the humidified chamber and incubate slides at room temperature for 90 minutes. Then, gently tap off the blocking solution.Immediately add the prepared mouse-on-mouse block to cover the tissue and incubate at room temperature for 15 minutes. At the end of the incubation, remove the slides from the humidified chamber and place them directly into a slide rack submerged in PBS. Then, remove the slides from PBS.Gently tap off the excess PBS and place them horizontally back in the humidified chamber. Add appropriately diluted primary antibodies of interest to cover the tissue. Once the slides are removed from the humidified chamber, gently tap off the primary antibodies and place the slides in a slide rack submerged in PBS for five minutes.After removing slides from PBS and tapping off excess PBS, place them horizontally back in the humidified chamber. Add appropriately diluted secondary antibody fluorophores or conjugated primary antibodies of interest to cover the tissue. Then, add appropriately diluted Huist directly on the tissue before incubating at room temperature in the dark.After that, place the slides in a new solvent-resistant dish filled with PBS for five minutes in the dark. Remove one slide at a time, keeping the remaining slides submerged in PBS in the dark. After removing excess PBS, add one to two drops of antifade mounting medium to the center of the tissue.Hold a clean cover slip by the edges and slowly lower it onto the slide at a 45 degree angle. Starting at the center of the tissue, gently press down on the cover slip with two fingers. Attach a cut, non-filtered P200 pipette to the tip of a serological pipette connected to a vacuum.Following the edges of the cover slip, use the vacuum to remove the excess mounting medium. In a new slide box, horizontally lay the slide flat before letting it dry in the dark at room temperature. Microscopic images showed the morphology of different intestinal segments and staining for goblet cells.The apical membrane, the lateral membrane of epithelial cells, and nuclei. Immunofluorescent staining of the intestine highlighted many different cellular compartments, including proliferative cells, interstitium, the lysosomal domain, and the epithelium.

1.1K Views.  Medical University of South Carolina.  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.

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

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