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 (PBS).</li>
	<li>Remove the intestines from the PBS and use scissors to gently remove excess fat and connective tissue. Cutting the connective tissue helps the tissue lay flat during subsequent steps.</li>
	<li>Divide the intestine into individual segments using scissors (i.e., duodenum, jejunum, ileum, cecum, and colon). Divide the segments based on anatomical features and/or length, with approximately 13% of the intestinal length being duodenum, 58% being the jejunum, 8% being the ileum, 6% being the cecum, and colon is 15%9. The duodenum follows the stomach, and the ileum is the terminal section of the small intestine immediately before the cecum.</li>
	<li>Free the colon by cutting where it joins the cecum using scissors. Place the duodenum, jejunum, ileum, and colon into the weigh boat or Petri dish of PBS. Discard the cecum or fix it according to other published methods10.</li>
	<li>Attach a P200 pipette tip to a 10 mL syringe by cutting off the large end of the tip with a razor blade. Fill the syringe with PBS.</li>
	<li>Insert the syringe tip into the opening of an intestinal segment using forceps. Use the forceps to hold the intestinal segment on the syringe and gently flush at a rate of ~50 &#181;L/s to remove intestinal contents. Collect the intestinal contents into an empty weigh boat or Petri dish.</li>
	<li>Lay the wet intestinal segment in a straight line on a strip of dry-labeled cellulose filter paper (see Table of Materials). Ensure the intestinal segment is wet enough to properly adhere to the dry filter paper. Label the filter paper with the information for mouse number, genotype, experimental group, etc. using a pencil. 
	NOTE: Using a pen to label the filter paper can result in loss of labels during fixation.</li>
	<li>Use dissection scissors to cut the intestine open longitudinally along the mesenteric line. Cut ~5 mm at a time, using the bottom edge of the scissors or a pair of forceps to lay the cut tissue flat on the filter paper. Continue until the whole section of the intestine is cut and laid flat on the filter paper. Take care to cut the intestine open in as straight a line as possible, as this will make it easier to roll the tissue. 
	NOTE: Ball-tip scissors can be used during this step to avoid tissue damage.</li>
	<li>Place another piece of filter paper on top of the intestinal segment. Ensure the filter paper sandwiches the intestinal segment, preventing the intestine from curling or losing its flattened shape during fixation.</li>
	<li>Staple the edges of the filter paper to secure the tissue in place. Do not staple the tissue. Place enough staples around the tissue to prevent the tissue from becoming detached from the filter paper while fixing.</li>
	<li>Repeat steps 1.5-1.10 for each of the intestinal segments (duodenum, jejunum, ileum, and colon).</li>
	<li>Submerge the tissues in 10% normal buffered formalin (or fixative of choice such as 4% paraformaldehyde, Carnoy&#39;s fixative, etc.) overnight at 4 &#176;C. 
	CAUTION: Ensure correct personal protective equipment is worn as fixatives such as formalin are toxic.</li>
</ol>
2. Intestinal tissue rolling and processing
CAUTION: Perform the steps 2.1-2.6 in a ventilated hood.
<ol>
	<li>Gently remove the top piece of filter paper (the one touching the luminal side of the intestine). Use forceps to carefully peel the intestine off the bottom piece of filter paper. Discard the filter paper.</li>
	<li>Fill three weigh boats with PBS. Wash tissue in PBS 3x to remove formalin from the tissue.</li>
	<li>Using reverse action forceps (see Table of Materials), pick up a piece of intestine along the short edge with the luminal side facing up. Turn the forceps to roll the tissue. Use a pair of regular forceps to help guide the tissue as it is rolled. If the tissue gets folded, unroll it and try again.</li>
	<li>Place the tissue in a dry weigh boat or Petri dish. 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.</li>
	<li>Insert a size 00 dissecting pin or minutien pin into the tissue (see Table of Materials). Use wire cutters to remove the sharp tip of the pin. Place the tissue in a large cassette and then place the cassette in a container of 70% ethanol until ready to process.</li>
	<li>Repeat steps 2.1-2.5 with all intestinal segments (the duodenum, jejunum, ileum, and colon), placing all four tissue rolls into the same large cassette (see Table of Materials). Ensure that the cassette is labeled with a pencil to prevent the label from washing off in the ethanol and xylene (see Table of Materials) solutions.</li>
	<li>Once all samples have been rolled, put samples into a tissue processor. Use the following settings for intestinal tissue: 70% ethanol for 35 min; 90% ethanol for 35 min; 95% ethanol for 35 min; 100% ethanol for 35 min, 3x; xylene for 35 min, 3x; paraffin for 60 min, 3x. Cassettes can be left in melted paraffin wax for extended time periods. 
	CAUTION: Ensure proper protective equipment is worn as xylene is a toxic chemical.</li>
</ol>
3. Embedding intestinal tissue
<ol>
	<li>Preheat large embedding molds to keep the paraffin melted.</li>
	<li>Remove cassettes from the tissue processor and place them in a beaker of melted paraffin.</li>
	<li>At an embedding station, place a small amount of paraffin into a mold. Remove the four Swiss rolls from the cassette and place them all in one mold, laying them as flat as possible. Add more paraffin to fill the mold.</li>
	<li>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 (see Table of Materials) on the mold and add more paraffin if needed.</li>
	<li>Once the wax has solidified, remove the block from the mold. The tissue is now ready to be cut into 5 &#181;m slices and floated onto charged glass slides for immunostaining11.</li>
</ol>
4. Tissue adherence and slide preparation
<ol>
	<li>To ensure the prepared 5 &#181;m paraffin tissue section adheres to the slide, heat glass slides on a slide warmer or heat block set to 60 &#176;C for 15-30 min. Slides can also be left heating for longer; this step is not time sensitive.</li>
	<li>Let slides cool to room temperature (~15 min).</li>
</ol>
5. Deparaffinization
<ol>
	<li>Upon beginning the de-paraffinization of slides, ensure that the tissue remains in a solution and does not dry out for the entire course of immunostaining.</li>
	<li>Use a slide rack (see Table of Materials) to hold the glass slides and place them inside a solvent-resistant dish (see Table of Materials) that is filled with a clearing/de-paraffinization reagent (see Table of Materials). Ensure the reagent completely covers the tissue on each slide. Before completely immersing the slides in the clearing reagent, dip or jostle the slide rack several times in the clearing solution. Leave slides submerged for 10 min or longer. 
	NOTE: This step may need to be performed in a ventilated hood due to hazardous fumes, depending on the chosen clearing agent. Deparaffinization of the tissue in this step is not time sensitive, if using a non-toxic clearing agent.</li>
	<li>Remove and agitate the slide rack to remove excess clearing reagent. In a new solvent-resistant dish, repeat step 5.2.</li>
	<li>In a new solvent-resistant dish, repeat step 5.3. Leave slides submerged for 15 min or longer. This step is not time sensitive.</li>
</ol>
6. Rehydration
<ol>
	<li>Remove and agitate the slide rack to remove excess clearing reagent. In a new solvent-resistant dish filled with 100% ethanol, dip or jostle the slide rack several times and leave the slides submerged for 5 min. This step is time sensitive. Repeat 2x.</li>
	<li>Remove and agitate the slide rack to remove excess 100% ethanol. In a new solvent-resistant dish filled with 95% ethanol, dip or jostle the slide rack several times and leave the slides submerged for 5 min. This step is time sensitive. Repeat 1x.</li>
	<li>Remove and agitate the slide rack to remove excess 95% ethanol. In a new solvent-resistant dish filled with 70% ethanol, dip or jostle the slide rack several times and leave the slides submerged for 5 min. This step is time sensitive.</li>
	<li>Remove and agitate the slide rack to remove excess 70% ethanol. In a new solvent-resistant dish filled with 50% ethanol, dip or jostle the slide rack several times and leave the slides submerged for 5 min. This step is time sensitive.</li>
	<li>Remove and agitate the slide rack to remove excess 50% ethanol. In a new solvent-resistant dish filled with deionized (DI) water, dip or jostle the slide rack several times and leave the slides submerged for 5 min. This step is time sensitive.
	<ol>
		<li>To pause here, place slides in a container with PBS until immunostaining can be resumed after the 5 min water rehydration step.</li>
	</ol>
	</li>
</ol>
7. Antigen retrieval
<ol>
	<li>Fill a new solvent-resistant dish with an appropriate antigen retrieval buffer. Directly transfer the slide rack into this solution. Ensure that the slides are fully submerged in the antigen retrieval solution, covering the tissue completely. 
	NOTE: Optimal antigen retrieval solutions can vary depending on the particular antibody being used. The antigen retrieval required for individual antibodies may need to be determined by the researcher.
	<ol>
		<li>To make 1x citrate antigen retrieval buffer, add 2.94 g of sodium citrate dihydrate (see Table of Materials) per 1 L of deionized water. Once the sodium citrate dihydrate is dissolved, add hydrogen chloride (see Table of Materials) until the solution reaches a pH of 6. Lastly, add 500 &#181;L of Tween20 (see Table of Materials) per 1 L of deionized water.</li>
		<li>To make 1x Tris-EDTA antigen retrieval buffer, add 1.211 g Tris Base and 0.292 g EDTA in 1 L of deionized water. Once dissolved, adjust to pH 9.</li>
	</ol>
	</li>
	<li>Cover the solvent-resistant dish containing the antigen retrieval solution and slides with a lid and secure the lid with rubber bands.</li>
	<li>Place the secure solvent-resistant dish in a pressure cooker (see Table of Materials) on top of the metal rack or trivet. Ensure there is enough water in the pressure cooker to reach the metal rack or trivet so pressure can build.</li>
	<li>Place the lid on the pressure cooker and twist clockwise to lock the lid into place. Twist the pressure limit valve that is on the lid to the pressure setting.</li>
	<li>Under the menu setting, select high pressure on the pressure cooker, then under the time setting, set the pressure cooker to 30 min. Select Start.</li>
	<li>After 30 min the pressure cooker will beep and automatically set to the keep warm setting. Wait until the pressure has released naturally and the lid unscrews freely.</li>
	<li>Use heat-resistant gloves (see Table of Materials) to remove the solvent-resistant dish from the pressure cooker and remove the rubber band and lid. Place the dish in an ice bath for ~30 min to allow the slides to cool to room temperature. This step is not time sensitive.</li>
	<li>Once cooled to room temperature, remove the slide rack from the solvent-resistant dish and place it in a new solvent-resistant dish filled with DI water to remove residual antigen retrieval solution. Ensure that the slides are fully submerged.</li>
	<li>Remove the slide rack from the DI water and place it in a new solvent-resistant dish filled with PBS for 5 min. Ensure that the slides are fully submerged. This step is not time sensitive.</li>
</ol>
8. Blocking nonspecific background staining
<ol>
	<li>Prepare a humidified chamber by taking a large slide box and removing the cardboard cover adhered to the lid. At the base of the slide box, place damp paper towels vertically down. Ensure the damp paper towels lay flat.</li>
	<li>Remove a slide one at a time from the slide rack submerged in PBS. Carefully wipe away excess PBS on the glass slide, avoiding the tissue.</li>
	<li>With a hydrophobic pen (see Table of Materials) form a barrier that surrounds the tissue by drawing a box around the tissue with the hydrophobic pen. Be careful to avoid the tissue and not use the hydrophobic pen too close to the tissue.</li>
	<li>Place the outlined slide horizontally over the damp paper towels. Add blocking buffer to cover the tissue (~100 &#181;L).
	<ol>
		<li>To make a blocking buffer, add 100 &#181;L of cold water fish skin gelatin (Teleostein Gelatin; see Table of Materials) to 9.85 mL of PBS. Finally, add 50 &#181;L of 20% Triton X-100 (see Table of Materials) and mix until dissolved.</li>
	</ol>
	</li>
	<li>Repeat steps 8.2-8.4 for each slide. Close the humidified chamber and incubate slides at room temperature for 90 min.</li>
</ol>
9. Mouse on mouse blocking
<ol>
	<li>When using a primary mouse antibody for immunostaining mouse tissue, perform an additional blocking step. Mouse-on-mouse blocking reagents (mouse-on-mouse block; see Table of Materials) are commercially available; follow the commercial instructions when preparing the mouse-on-mouse block.</li>
	<li>Gently tap off the blocking solution and immediately add the prepared mouse-on-mouse block to cover the tissue (~100 &#181;L). Repeat for each slide containing mouse tissue, which will be stained using a primary antibody raised in mice.</li>
	<li>Incubate tissue sections in the humidified chamber box at room temperature for 15 min.</li>
	<li>Remove slides from the humidified chamber and place them directly into a slide rack submerged in PBS. Leave slides in PBS for 5 min to wash off the mouse-on-mouse block.</li>
</ol>
10. Primary antibodies
<ol>
	<li>For each slide, select primary antibodies of interest that are raised in different species. Dilute primary antibodies in antibody diluent as per the manufacturer&#39;s instruction. The following primary antibodies were used: E-CADHERIN (1:100), MUC2 (1:200), PCNA (1:500), LAMININ (1:200), &#946;-CATENIN (1:200), and LAMP1 (1:50). 
	NOTE: If the manufacturer does not provide recommended dilutions, 1:100 dilution is a good starting point. If the primary antibody of interest is conjugated to a fluorophore see step 12.2.
	<ol>
		<li>To make the antibody diluent, add blocking buffer 1:20 to PBS, if using fish gelatin blocking buffer described above.</li>
	</ol>
	</li>
	<li>Remove slides from PBS or from the humidified chamber, gently tap off the excess blocking solution or PBS, and place them horizontally back in the humidified chamber. Ensure the barrier created with the hydrophobic pen in step 8.3 remains; outline the tissue again if necessary.</li>
	<li>Add appropriately diluted primary antibodies of interest to cover the tissue (~100 &#181;L).</li>
	<li>Close the humidified chamber and incubate slides on a flat surface at 4 &#176;C in the dark overnight.</li>
</ol>
11. Washing the slides
<ol>
	<li>Remove slides from the humidified chamber, gently tap off the primary antibodies, and place the slides in a slide rack submerged in PBS for 5 min.</li>
	<li>Remove the slide rack and place it in a new solvent-resistant dish filled with PBS for 5 min. Repeat step 1x.</li>
</ol>
12. Secondary antibodies and nuclei counterstaining
<ol>
	<li>For each slide, select secondary antibody fluorophores designed to bind to the primary antibody species. Dilute secondary antibody fluorophores or conjugated primary antibodies in secondary antibody diluent. The secondary antibodies used were donkey anti-goat 488 (1:200), donkey anti-rabbit cy3 (1:200), donkey anti-mouse 647 (1:200), donkey anti-rabbit 647 (1:200), and donkey anti-rat cy3 (1:200). The primary antibody &#947;-ACTIN conjugated to fluorophore 647 was used at a 1:100 dilution.
	<ol>
		<li>To make secondary antibody diluent, add blocking buffer 1:100 to PBS, if using fish gelatin blocking buffer as described above.</li>
	</ol>
	</li>
	<li>Remove slides from PBS, gently tap off excess PBS, and place them horizontally back in the humidified chamber. Ensure the barrier created with the hydrophobic pen in step 8.3 remains; outline the tissue again if necessary.</li>
	<li>Add appropriately diluted secondary antibody fluorophores or conjugated primary antibodies of interest to cover the tissue (~100 &#181;L).</li>
	<li>Close the humidified chamber and incubate slides at room temperature in the dark for 1 h.</li>
	<li>Dilute 10 mg/mL Hoechst (see Table of Materials) or DAPI using PBS for a final concentration of 1 &#181;g/mL Hoechst&#160;or DAPI using PBS and add directly to the tissue (~100 &#181;L). Incubate for 5 min at room temperature in the dark.</li>
	<li>Remove slides from the humidified chamber and place them in a slide rack that is submerged in PBS for 5 min in the dark.</li>
	<li>Remove the slide rack and place in a new solvent resistant dish filled with PBS for 5 min in the dark. Repeat step 1x.</li>
</ol>
13. Mounting and preparation for microscopy
<ol>
	<li>Remove one slide at a time from the slide rack submerged in PBS, keeping the remaining slides submerged in PBS in the dark. Gently tap off excess PBS, and if needed, use a lint-free wipe to carefully wipe away excess PBS on the glass slide, avoiding the tissue.</li>
	<li>Add one to two drops of antifade mounting medium (see Table of Materials) to the center of the tissue.</li>
	<li>Hold a clean coverslip (see Table of Materials ) by the edges and slowly lower it onto the slide at a 45&#176; angle. Ensure the mounting medium spreads evenly over the tissue.</li>
	<li>Starting at the center of the tissue, gently press down on the coverslip with two fingers to help remove air bubbles and excess mounting medium. If needed, continue pressing toward the edges and maintain gentle pressure throughout.</li>
	<li>Cut off the small end of a non-filtered P200 pipette and attach it to the tip of a serological pipette connected to a vacuum.</li>
	<li>Following the edges of the coverslip, use the vacuum to remove the excess mounting medium.</li>
	<li>In a new slide box, horizontally lay the slide flat and allow the slide to dry in the dark at room temperature.</li>
	<li>Repeat this process for each slide.</li>
</ol>

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

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+techniques+for+a+comprehensive+gut+histopathological+analysis:+Swiss+roll+versus+intestine+strips.">Comparison of two techniques for a comprehensive gut histopathological analysis: Swiss roll versus intestine strips.</a> Exp Mol Pathol. 111, 104302 (2019).</li><li>Williams, J. M., Duckworth, C. A., Vowell, K., Burkitt, M. D., Pritchard, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+preparation+techniques+for+histological+analysis+in+the+mouse.">Intestinal preparation techniques for histological analysis in the mouse.</a> Curr Prot Mouse Biol. 6 (2), 148-168 (2016).</li><li>Boenisch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+heat-induced+antigen+retrieval+following+inconsistent+formalin+fixation.">Effect of heat-induced antigen retrieval following inconsistent formalin fixation.</a> Appl Immunohistochem Mol Morphol. 13 (3), 283-286 (2005).</li><li>Hasegawa, Y., Mark Welch, J. L., Rossetti, B. J., Borisy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+three-dimensional+spatial+structure+in+the+gut+microbiome.">Preservation of three-dimensional spatial structure in the gut microbiome.</a> PLoS One. 12 (11), e0188257 (2017).</li><li>Garabedian, E. M., Roberts, L. J., Mcnevin, M. S., Gordon, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+the+role+of+paneth+cells+in+the+small+intestine+by+lineage+ablation+in+transgenic+mice.">Examining the role of paneth cells in the small intestine by lineage ablation in transgenic mice.</a> J Biol Chem. 272 (38), 23729-23740 (1997).</li><li>Moshi, J. M., Ummelen, M., Broers, J. L. V., Ramaekers, F. C. S., Hopman, A. H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+antigen+retrieval+protocols+on+the+immunohistochemical+detection+of+epigenetic+DNA+modifications.">Impact of antigen retrieval protocols on the immunohistochemical detection of epigenetic DNA modifications.</a> Histochem Cell Biol. 159 (6), 513-526 (2023).</li><li>Krenacs, L., Krenacs, T., Stelkovics, E., Raffeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-induced+antigen+retrieval+for+immunohistochemical+reactions+in+routinely+processed+paraffin+sections.">Heat-induced antigen retrieval for immunohistochemical reactions in routinely processed paraffin sections.</a> Methods Mol Biol. 588, 103-119 (2010).</li><li>Pereira, E. S. A., Lourenco, A. L., Marmello, B. O., Bitteti, M., Teixeira, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+techniques+for+a+comprehensive+gut+histopathological+analysis:+Swiss+roll+versus+intestine+strips.">Comparison of two techniques for a comprehensive gut histopathological analysis: Swiss roll versus intestine strips.</a> Exp Mol Pathol. 111, 104302 (2019).</li><li>Bolognesi, M. a. O., 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=Antibodies+validated+for+routinely+processed+tissues+stain+frozen+sections+unpredictably.">Antibodies validated for routinely processed tissues stain frozen sections unpredictably.</a> Bio Techniques. 3 (3), 137-148 (2021).</li></ol>

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><tbody><tr><td>&amp;beta;-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&amp;amp;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>&amp;gamma;-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.

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...

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.

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 ...

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

Research

JoVE Journal

Biology

2.8K 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.

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

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the &#8216;&#8216;stemness&#8217;&#8217; of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia.

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

Developmental Biology

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

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

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 ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Murine Fecal Isolation and Microbiota Transplantation

Gut microbiota dysbiosis plays a role in the pathophysiology of cardiovascular and metabolic disorders, but the mechanisms are not well understood. Fecal microbiota transplantation (FMT) is a valuable approach to delineating a direct role of the total microbiota or isolated species in disease pathophysiology. It is a safe treatment option for patients with recurrent Clostridium difficile infection. Preclinical studies demonstrate that manipulating the gut microbiota is a useful tool to study the mechanistic link between dysbiosis and disease. Fecal microbiota transplantation may help elucidate novel gut microbiota-targeted therapeutics for the management and treatment of cardiometabolic disease. Despite a high success rate in rodents, there remains translational changes associated with the transplantation. The goal here is to provide guidance in studying the effects of gut microbiome in experimental cardiovascular disease. In this study, a detailed protocol for the collection, handling, processing, and transplantation of fecal microbiota in murine studies is described. The collection and processing steps are described for both human and rodent donors. Lastly, we describe using a combination of the Swiss-rolling and immunostaining techniques to assess gut-specific morphology and integrity changes in cardiovascular disease and related gut microbiota mechanisms.

The goal here is to outline a protocol to investigate the mechanisms of dysbiosis in cardiovascular disease. This paper discusses how to aseptically collect and transplant murine fecal samples, isolate intestines, and use the "Swiss-roll" method, followed by immunostaining techniques to interrogate changes in the gastrointestinal tract.

Gut microbiota dysbiosis plays a role in the pathophysiology of cardiovascular and metabolic disorders, but the mechanisms are not well understood. Fecal ...

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.

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 ...

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 ...

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

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