Name: diagnosis of hirschsprung's disease by immunostaining rectal suction biopsies for calretinin, s100 protein and protein gene product 9.5
Uploaded: 2019-04-26T12:30:00.000+00:00
Duration: 5 min 45 s
Description: Watch this Scientific Journal Video about Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5 at JoVE.com

The authors thank Weibing Tang for his great help in providing the filming lab. This article is supported by the Public Welfare Research, and special funds were received from the National Health and Family Planning of China (Grant No.201402007).

This protocol was approved by the Research Ethics Board of the Union Hospital of Huazhong University of Science and Technology.
1. Rectal Suction Biopsy
<ol>
	<li>Perform the rectal suction biopsy by a well-trained pediatric surgeon and an assistant using a Rbi2 suction rectal biopsy system after obtaining informed consent from the guardian.
	<ol>
		<li>Perform RSB on patients that have the following indications: inability to pass meconium in infants or long-term bloating with or without constipation in children; children with long-term constipation who need paraffin oil to complete defecation; intestinal obstruction or intestinal perforation with unknown reasons in children undergoing an enterostomy. 
		NOTE: The contraindications for RSB are as follows: children who have severe symptoms, who are in poor physical condition, or who cannot tolerant the RSB; children with acute-stage enterocolitis; children undergoing intestinal anastomosis without complete healing.</li>
	</ol>
	</li>
	<li>Perform a normal saline retention enema as bowel preparation 36 h before the procedure to avoid loose stools and excessive edema of the mucosa. Add magnesium sulfate solution if the child has severe constipation. Administer vitamin K (1 mg/kg) to neonates. 
	NOTE: Do not perform preoperative blood tests or administer antibiotics, as they are not required.</li>
	<li>Put patient in the lithotomy position. Coat the surface of the tube with paraffin oil. Insert the blunt-ended tube 4-7 cm into the rectum with the side hole facing the posterior or lateral walls.</li>
	<li>Apply gentle pressure on the tube to facilitate adequate adhesion of the side hole to the rectal wall. Press the trigger and withdraw the tool immediately.</li>
	<li>Use the biopsy instrument to obtain 4 suction biopsies with a diameter of 2 mm at 3 cm and 6 cm above the dental line, anteriorly and posteriorly. Use a needle to place the specimen on gauze moistened with saline.</li>
	<li>Observe the patient until 2 h after the procedure to rule out rectal bleeding. Avoid further rectal and anal manipulations in the first 24 h after RSB.</li>
</ol>
2. Preparation of the RSB Sections
<ol>
	<li>Place the rectal suction biopsies in 4% paraformaldehyde for 6 h to fix the tissue. Use a fixative volume 5 times more than the tissue volume.</li>
	<li>Dehydrate the tissue using the pathological tissue dehydrator for 11 h. The whole programmed process consists of the following 5 steps:
	<ol>
		<li>Place the tissue in formaldehyde and fix for 1 h.</li>
		<li>Place the tissue in 75% ethanol for 4 h.</li>
		<li>Place the tissue in absolute ethanol (two changes, 1 h each).</li>
		<li>Place the tissue in absolute ethanol (two changes, 30 min each).</li>
		<li>Place the tissue in xylene (three changes, 1 h each).</li>
	</ol>
	</li>
	<li>Place the tissue in paraffin wax (58-60 &#176;C) (three changes, 1 h each). Embed the RSB tissues in paraffin blocks.</li>
	<li>Trim the paraffin blocks using a microtome until the tissue is entirely exposed with a complete section plane.</li>
	<li>Cut the blocks into 0.4 &#956;m sections with a microtome.</li>
	<li>Place the sections into 45-50 &#176;C water for a few seconds each to allow the sections to spread into a flat plate.&#160;</li>
	<li>Transfer the paraffin sections onto slides.</li>
	<li>Bake the slides in a 60 &#176;C oven for 3-5 h. Avoid baking for too long, which may lead to the loss of the antigens.</li>
	<li>Place the deparaffinized slides into xylene (2 changes, 15 min each).</li>
	<li>Hydrate the slides in absolute ethanol, 95%, 80%, and 75% ethanol for 5 min each. Then, rinse them in reverse osmosis (RO)-purified water for another 5 min.</li>
</ol>
3. Antigen Retrieval
<ol>
	<li>Make working dilutions for calretinin retrieval by mixing 4 mL of EDTA antigen retrieval buffer with 200 mL of RO-purified water. Make working dilutions for S100 and PGP9.5 retrieval by mixing 1 mL of citric acid sodium citrate buffer (100x) with 99 mL of RO-purified water. 
	NOTE: Calretinin can be retrieved preferentially by EDTA retrieval solution. However, citric acid sodium citrate buffer is more suitable for the retrieval of S100 and PGP9.5.</li>
	<li>Place the slides in a coplin staining jar. Fill the jar with retrieval solution and place it into a preheated boiling water bath. After boiling for 20 min, remove the jar from the water bath and let it cool to room temperature. The cooling process takes approximately 10 min. Remove the slides and gently rinse them once with phosphate-buffered saline (PBS).</li>
	<li>Draw a circle with a marker pen around the tissue and prepare a wet box for the following procedures.</li>
</ol>
4. Blocking
<ol>
	<li>Make working dilutions by mixing 3 mL of 30% H2O2 with 27 mL of RO-purified water.</li>
	<li>Add two or three drops of 3% H2O2 solution dropwise onto the tissue to block peroxidates. Add the H2O2 solution promptly to ensure that the solution will not overflow from the circle. Perform blocking of peroxidates in a 37 &#176;C incubator for 20 min.</li>
	<li>Rinse the slides gently with PBS (three washes, 5 min each).</li>
	<li>Block the slides for 20 min at 37 &#176;C with 5% bovine serum albumin (BSA, prepared by mixing 1.5 g of BSA powder with 30 mL of RO-purified water). 
	NOTE: Blocking with 3% H2O2 can prevent 95% of nonspecific staining and blocking with 5% BSA can prevent the other 5% of nonspecific staining. Combine the two methods to obtain a reliable result.</li>
</ol>
5. Antigen-antibody Reaction
<ol>
	<li>Remove the 5% BSA and add 50 &#956;L of primary antibody (calretinin, S100, or PGP9.5) to the slide. Incubate overnight at 4 &#176;C.</li>
	<li>Wash the slide with PBS (three times, 3 min each).</li>
	<li>Add 50 &#956;L of polymer enhancer (reagent A; see Table of Materials) to the section and incubate it for 20 min in a 37 &#176;C incubator. Then, wash the slide with PBS (three times, 3 min each).</li>
	<li>Add 50 &#956;L of secondary antibody B (goat anti-rabbit/mouse IgG secondary antibody) to the section and incubate for 30 min in a 37 &#176;C incubator. Wash the section with PBS (three times, 5 min each).</li>
</ol>
6. 3,3-Diaminobenzidine and Hematoxylin Staining
<ol>
	<li>Add 850 &#956;L of RO-purified water to a 1.5 mL tube and add the staining reagents (see Table of Materials) in the order A, B, and C. Add 50 &#956;L of each reagent to obtain a total of 1 mL of 3,3-diaminobenzidine (DAB) working solution. If there are precipitates, use the solution after filtration.</li>
	<li>Add a drop of DAB working solution to the tissue section and stain for 3-10 min. Incubate the slides in DAB at room temperature until brown staining is detected. Monitor carefully using a brightfield microscope. Keep the DAB working solution away from light. Then, wash the slide with PBS for 5 min.</li>
	<li>Add a drop of hematoxylin to counterstain the nuclei for approximately 1 min. Then, hold the coplin jar and wash it under a trickle of water for approximately 30-40 s until the excess dye is removed. Be careful not to damage the tissue sections.</li>
	<li>Immerse the RSB slides in a coplin jar containing PBS for 25-30 s. Then, differentiate in 1% hydrochloric acid-ethanol for 10 s. Wash the slides with PBS for 1 min.</li>
	<li>Dehydrate the slides in the reverse order of hydration: 75%, 80%, 95%, and absolute ethanol (5 min each).</li>
	<li>Expose the slides to xylene (2 changes, 5 min each).</li>
	<li>Mount the coverslip using ultraclean mounting. Allow slides to dry before visualization using a microscope.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoperoxidase+stains+of+ganglion+cells+and+abnormal+mucosal+nerve+proliferations+in+Hirschsprung's+disease.">Immunoperoxidase stains of ganglion cells and abnormal mucosal nerve proliferations in Hirschsprung's disease.</a> Human Pathology. 19 (4), 432-437 (1988).</li><li>Monforte-Mu&#241;oz, H., Gonzalez-Gomez, I., Rowland, J. M., Landing, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+submucosal+nerve+trunk+caliber+in+aganglionosis:+a+&amp;#34;positive&amp;#34;+and+objective+finding+in+suction+biopsies+and+segmental+resections+in+Hirschsprung's+disease.">Increased submucosal nerve trunk caliber in aganglionosis: a &amp;#34;positive&amp;#34; and objective finding in suction biopsies and segmental resections in Hirschsprung's disease.</a> Archives of Pathology &amp; Laboratory Medicine. 122 (8), 721-725 (1998).</li><li>Bachmann, L., 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=Immunohistochemical+panel+for+the+diagnosis+of+Hirschsprung&#39;s+disease+using+antibodies+to+MAP2,+calretinin,+GLUT1+and+S100.">Immunohistochemical panel for the diagnosis of Hirschsprung&#39;s disease using antibodies to MAP2, calretinin, GLUT1 and S100.</a> Histopathology. 66 (6), 824-835 (2015).</li><li>Sams, V. R., Bobrow, L. G., Happerfield, L., Keeling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+PGP9.5+in+the+diagnosis+of+Hirschsprung's+disease.">Evaluation of PGP9.5 in the diagnosis of Hirschsprung's disease.</a> Journal of Pathology. 168 (1), 55 (1992).</li><li>Huang, Y., Anupama, B., Zheng, S., Xiao, X., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+of+enteric+nerve+markers+and+nerve+innervation+in+total+colonic+aganglionosis.">The expression of enteric nerve markers and nerve innervation in total colonic aganglionosis.</a> International Journal of Surgical Pathology. 19 (3), 303 (2011).</li></ol>

The authors have nothing to disclose.

Here we described a procedure using three different immunohistochemical antibodies to stain RBS sections for the diagnosis of HD. The sensitivity of our diagnostic protocol was 96.49% (95% CI, 0.88-0.99), and the specificity was 100% (95% CI, 0.97-1.00).
The most critical steps of the protocol are RSB and antigen-antibody reaction. The size of the biopsy determines the accuracy of the staining. A small biopsy will not provide enough tissue to make a precise diagnosis, while a large biopsy leads to a higher risk of bleeding. As a matter of experience, biopsies with a diameter of 2 mm are the optimal size. For the antigen-antibody reaction, proper preservation of the antibody cannot be ignored. Following the protocol step by step and gently working are needed. Reverse osmosis-purified water is recommended in all steps, yielding a clearer field for observation and making it easier for pathologists to make an accurate diagnosis.
Contrast enema, anorectal manometry, and biopsy with histology are three preoperative diagnostic methods that have been used in recent years. According to Takawira et al.&#39;s research, biopsy with histology has the highest sensitivity and specificity8. Meier-Ruge et al. first reported the use of acetylcholinesterase (AChE) staining on frozen rectal suction tissue to help diagnose HD in 19729. This staining method detects hypertrophic nerve trunks in the submucosa. After that report, many institutions around the world started to use this method as an adjunct for the diagnosis of HD. Due to its diagnostic limitations, researchers started to concentrate on other markers that can also stain the ganglion cells and nerve fibers in formalin-fixed and paraffin-embedded specimens. In 2004, Barshack et al. found that the loss of calretinin expression may help diagnose aganglionic segments in HD10. Kapur et al. and Guinard-Samuel et al. repeated the protocol in 2009 and concluded that immunohistochemical staining for calretinin was superior to staining for acetylcholinesterase11,12. In 1988, S100 immunostaining for the diagnosis of HD was first introduced by Robey et al.13. Monforte-Mu&#241;oz et al. repeated the method in 1998 and found that 90% of their 20 Hirschsprung cases had nerve fibers wider than 40 &#956;m14. Therefore, S100 immunostaining may serve as an ancillary diagnostic method in cases without ganglion cells. Furthermore, Bachmann et al. noticed that immunohistochemical staining for MAP2, calretinin, GLUT1, and S100 could obtain a reliable result as accurate as frozen section techniques15. However, in 1992, Sams et al. evaluated the value of PGP9.5 in the diagnosis of HD16. Although S100 can detect more lamina propria nerve fibers, it has a higher false-negative rate. Therefore, we chose to use PGP9.5 and S100 together for the immunohistochemical diagnosis of HD, as previous studies recommended. Many cases of staining for neuron-specific enolase in transitional zones also showed positive ganglion cells in the myenteric and submucosal plexus, but these cells were sparse and small12. Similarly, calretinin-positive ganglion cells are small and have a clustered formation. A previous study indicated that some slightly thickened nerve trunks could also be detected in the transitional segments of HD, but with total colonic aganglionosis, the nerve trunks may be within normal limits (&#60; 40 &#956;m)17. We should analyze the results case by case and combine these results with clinical manifestations to avoid misdiagnosis.
This protocol could not represent the final pathological diagnosis, so we carried out a minimum of one year of thorough clinical follow-up for each child. Additionally, this protocol requires more samples to prove its reliability.
In conclusion, the diagnosis of HD by RSB is a convenient method that provides an ideal histological diagnosis with acceptable minimal injury. The sensitivity is closely related to whether the site of sampling is correct. Immunohistochemical staining of RSB for calretinin, S100, and PGP9.5 is sensitive and specific in the detection of ganglion cells and hypertrophic nerve trunks. The combination of these three markers offers a more reliable diagnostic method for HD.

Hirschsprung&#39;s disease (HD) is a common congenital gut intestinal disorder characterized by a lack of ganglion cells in different segments of the distal intestinal tract1. The human enteric nervous system is formed when the invasion of the embryonic neural cells is completed. If there is a disturbance of the process and the invasion fails to complete, the distal intestine of the newborn becomes aganglionic2. This potentially fatal condition is called Hirschsprung&#39;s disease. Proliferation, motility, and gut growth are the three main components of successful colonization.
Traditional hematoxylin and eosin (H&#38;E) staining of a limited submucosal biopsy cannot attain as satisfactory of a result as H&#38;E staining of a full-thickness tissue obtained from surgery. Additionally, acetylcholinesterase (AChE) staining of rectal suction tissue is theoretically challenging due to its inadequate sensitivity, which is 91%, and complex processing of frozen sections3,4. Several other immunohistochemical markers of ganglion cells and nerve fibers that can be stained in formalin-fixed and paraffin-embedded specimens are gradually becoming the mainstream HD diagnostics. Calretinin is a vitamin D-dependent calcium-binding protein that is not expressed in the myenteric and submucosal plexus of HD-affected segments5. S100 protein is expressed in cells derived from the neural crest, such as nerve fibers and glial cells, which often present neural hypertrophy in the submucosal tissue of HD-affected segments6. Protein gene product 9.5 (PGP9.5) reliably stains nerve fibers and ganglion cells; PGP9.5 staining acts as a supplement to calretinin staining, especially in cases of isolated hypoganglionosis. Double staining with S100 and PGP9.5 can decrease the false-negative rate and increase the sensitivity. As a prerequisite, the current study aims to ensure adequate specificity and high sensitivity of this novel diagnostic method. Our novel protocol used all three markers for the discrimination of aganglionic intestine and hypertrophic nerve fibers. A prospective study of 318 children was performed by our lab and previously published without a detailed protocol7. The detailed protocol and precautions are discussed in this article. Any neonates who suffered from a severe defecation problem since birth or children with chronic constipation excluding other common diseases are potential candidates for rectal suction biopsy (RSB). Our novel protocol is suitable for staining not only RSBs but also full-thickness biopsies or surgical specimens to make a final diagnosis.

<table><tbody><tr><td>calretinin antibody</td><td>MXB Biotechnologies</td><td>MAB-0716 170416405c</td><td>antibody: primary antibody</td></tr><tr><td>S-100 antibody</td><td>MXB Biotechnologies</td><td>Kit-0007</td><td>antibody: primary antibody</td></tr><tr><td>PGP9.5 antibody</td><td>Shanghai long island antibody Co. Ltd</td><td>R-0457-03</td><td>antibody: primary antibody</td></tr><tr><td>enhancer reagent</td><td>MXB Biotechnologies</td><td>KIT-9902-A 170416405a</td><td>antibody: secondary antibody A</td></tr><tr><td>Goat anti-Rabbit/Mouse IgG Secondary Antibody</td><td>MXB Biotechnologies</td><td>KIT-9902-B</td><td>antibody: secondary antibody B</td></tr><tr><td>DAB staining kit (containing reagent A B and C)</td><td>MXB Biotechnologies</td><td>DAB-0031</td><td>staining kit</td></tr><tr><td>Heat incubator</td><td>Shanghai yiheng instrument Co. Ltd</td><td>DHP-9082</td><td>instrument</td></tr><tr><td>Rbi2 suction rectal biopsy system</td><td>Aus Systems Pty Ltd, South Australia, Australia</td><td>CP1200 HP1000 SS1000</td><td>instrument</td></tr><tr><td>microtome</td><td>Leica</td><td>leica RM2016</td><td>instrument</td></tr><tr><td>citric acid sodium citrate buffer(100X)</td><td>MXB Biotechnologies</td><td>MVS-0101</td><td>antigen retrieval buffer</td></tr><tr><td>pathological tissue dehydrator</td><td>wuhan junjie electronic Co. Ltd</td><td>JT-12F</td><td>instrument</td></tr></tbody></table>

diagnosis of hirschsprung's disease by immunostaining rectal suction biopsies for calretinin, s100 protein and protein gene product 9.5

Hirschsprung&#39;s disease (HD) is a congenital intestinal disease that is clinically manifested as an inability to pass meconium in infants or as long-term constipation in children. Rectal suction biopsy (RSB) to determine the absence of ganglion cells and neural hypertrophy is the most accurate test for the diagnosis of HD at present. Traditional hematoxylin-eosin staining lacks sensitivity and specificity. Acetylcholinesterase staining cannot be widely used due to its complex process. Our novel protocol of immunostaining for calretinin, S100 protein, and protein gene product 9.5 (PGP9.5), which we conducted on RSBs, exhibits high sensitivity and specificity rates of 96.49% (95% confidence interval, 0.88-0.99) and 100% (95% confidence interval, 0.97-1.00), respectively. The HD-affected segments often present as the absence of the expression of calretinin, S100 protein, and PGP9.5, which are markers of neural hypertrophy in the submucosal tissue. This protocol describes the detailed operating process of this new diagnostic method.

In total, 318 patients were enrolled in our study. All of the patients underwent RSB, and the tissues were stained for calretinin, S100, and PGP9.5. The diagnosis based on our novel protocol was HD in 97 cases, non-HD in 213 cases, and suspected HD in 8 cases. Among the 132 surgical patients, 99 patients were diagnosed with HD by immunostaining of full-thickness specimens after surgery. S100 and PGP9.5 staining showed that 92% and 93% of the 99 patients, respectively, had hypertrophied nerve trunks. The other 83 of the 186 patients underwent full-thickness biopsies and showed ganglion cells. A total of 103 patients were clinically cured by conservative therapies, and HD was excluded. Of the 8 patients with suspected HD, 3 patients were categorized as short-segment HD, and the other 5 patients were categorized as non-HD.
According to our results, 97 patients were diagnosed by RSB initially. Detailed information is shown in Table 1. The protocol of immunohistochemical staining for calretinin, S100, and PGP9.5, which we conducted on RSBs, has high sensitivity and specificity rates of 96.49% (95% confidence interval [CI], 0.88-0.99) and 100% (95% CI, 0.97-1.00), respectively. The positive predictive value is 94.3% (95% CI, 0.87-0.99), and the negative predictive value is 99.1% (95% CI, 0.95-1.00).
Figure 1A and 1B represent typical results after immunostaining for calretinin. Many ganglion cells, which express calretinin, can be found in the normal tissues. However, no ganglion cells were detected in any area of the tissue from the HD segment. As shown in Figure 1C-F, S100 and PGP9.5 immunostaining shows hypertrophied nerve trunks in the submucosa, while only granular staining of some small nerve twigs was observed in the normally innervated intestine. In our study, hypertrophic nerve trunks are nerve fibers with a diameter of &#8805; 40 &#956;m.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58799/58799fig1.jpg" /> 
Figure 1: Immunostaining of rectal suction biopsies for calretinin, PGP9.5, and S100. Immunostaining for calretinin in HD-affected tissue (A) and in ganglion cells of myenteric plexuses in normally innervated tissue (B). No calretinin staining was detected in the myenteric plexuses of the HD-affected segment. PGP9.5 staining in HD-affected tissue showed dense hypertrophic nerve fibers in the submucosa (C) in contrast to nerve fibers in the normal tissues (D).(E) Dense and prominent S100 immunostaining showed hypertrophic nerve trunks in the submucosa of HD-affected tissue. (F) Normal nerve fibers in the submucosa. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58799/58799fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>protein markers</td>
			<td colspan="2">HD(99)</td>
			<td colspan="2">Non-HD(219)</td>
		</tr>
		<tr>
			<td></td>
			<td>ganglion cells (+)</td>
			<td>Hypertrophic nerve trunks (+)</td>
			<td>ganglion cells (+)</td>
			<td>Hypertrophic nerve trunks (+)</td>
		</tr>
		<tr>
			<td>calretinin</td>
			<td>2</td>
			<td>&#8212;</td>
			<td>214</td>
			<td>&#8212;</td>
		</tr>
		<tr>
			<td>S100</td>
			<td>&#8212;</td>
			<td>91</td>
			<td>&#8212;</td>
			<td>3</td>
		</tr>
		<tr>
			<td>PGP9.5</td>
			<td>&#8212;</td>
			<td>92</td>
			<td>&#8212;</td>
			<td>4</td>
		</tr>
	</tbody>
</table>
Table 1: Immunohistochemical staining scores for the three protein markers in rectal suction biopsies.

Watch this Scientific Journal Video about Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5 at JoVE.com

Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5

This protocol describes the process of immunostaining rectal suction biopsies for calretinin, S100 protein, and protein gene product 9.5. This novel adjuvant diagnostic method for Hirschsprung&#39;s disease has preferable sensitivity and specificity rates.

Hirschsprung&#39;s disease (HD) is a congenital intestinal disease that is clinically manifested as an inability to pass meconium in infants or as ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

13.3K Views.  Huazhong University of Science and Technology. This protocol describes the process of immunostaining rectal suction biopsies for calretinin, S100 protein, and protein gene product 9.5. This novel adjuvant diagnostic method for Hirschsprung's disease has preferable sensitivity and specificity rates.

Video: Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5

Immunostaining to Visualize Murine Enteric Nervous System Development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must ...

Developmental Biology

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Immunology and Infection

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

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

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

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

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

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

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

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

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

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

Medicine

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 ...

Biology

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

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

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

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

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

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

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

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

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

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

Hematoxylin and Eosin Staining to Assess Colon Damage in DSS-Induced Colitis

Source: Du, Y., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58668">Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model.</a> J. Vis. Exp. (2018)
This video demonstrates the hematoxylin and eosin (H&#38;E) staining method to evaluate colon damage in a murine model of DSS-induced colitis &#8212; an inflammatory bowel disease. Upon isolating the colon from a DSS-treated mouse and obtaining sections of the tissue, H&#38;E staining of the sections helps identify the inflammation-mediated tissue damage.

Source: Du, Y., et al. Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host-Pathogen Interaction

Characterization of Small Bowel Neuroendocrine Tumor Spheroids Using Immunofluorescence

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/60303/establishment-characterization-small-bowel-neuroendocrine-tumor'>Source: Ear, P. H., et. al. Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids. J. Vis. Exp. (2019)</a>This video demonstrates the immunofluorescence staining of small bowel neuroendocrine tumor spheroids. The spheroids are fixed, permeabilized, and blocked and are then incubated with primary and secondary antibodies. A dye is added to stain nuclei before mounting and visualizing them under a fluorescent microscope.

Source: Ear, P. H., et. al. Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids. J. Vis. Exp. (2019)This video demonstrates ...

Neuropathology

Tumors

Assessment of Chemical-Induced Colitis via Immunohistochemical Analysis of Colon Tissue Sections

Source: Du, Y. W. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58668">Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model.</a>&#160;J. Vis. Exp.&#160;(2018)
This video demonstrates the assessment of chemical-induced colitis via immunohistochemical staining of mouse colon tissue. In a stained tissue section, the presence of a large number of immune-responsive cells in the damaged tissue represents the infiltration of immune cells during colitis.

Source: Du, Y. W. et al., Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model.&#160;J. Vis. ...

Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5

Summary

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Chapters in this video

Immunostaining to Visualize Murine Enteric Nervous System Development

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

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

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

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

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

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

Hematoxylin and Eosin Staining to Assess Colon Damage in DSS-Induced Colitis

Characterization of Small Bowel Neuroendocrine Tumor Spheroids Using Immunofluorescence

Assessment of Chemical-Induced Colitis via Immunohistochemical Analysis of Colon Tissue Sections