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 ...

optimized-protocol-for-intestinal-swiss-rolls-immunofluorescent

Research

JoVE Journal

Biology

1.2K vistas.  Medical University of South Carolina. Nuestra investigación se centra en comprender cómo funciona el tracto gastrointestinal y cómo estas funciones se ven afectadas por diferentes estados patológicos. Estamos interesados en conocer la respuesta de diferentes células epiteliales intestinales a diversas afecciones, como infección e inflamación, obesidad y mutaciones genéticas. La importancia de la especificidad de los anticuerpos es cada vez más evidente.Sin embargo, el impacto de l...