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 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.

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

2.5K 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

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxy&#64258;uorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

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 ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

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 ...