Name: using a whole-mount immunohistochemical method to study the innervation of the biliary tract in suncus murinus
Uploaded: 2017-06-15T12:30:00
Description: Watch this Scientific Journal Video about Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus at JoVE.com

The authors have no acknowledgements.

All experimental procedures were approved by the Tokyo Metropolitan University Institutional Animal Care and Use Committee (IACUC). The animals were housed and handled in accordance with the Guide for the Care and Use of Laboratory Animals and the Guide for the Care and Use of Experimental Animals of the Canadian Council on Animal Care. We used animals bred and maintained at the Functional Morphology Laboratory, Department of Frontier Health Sciences, Tokyo Metropolitan University, Japan.
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This work describes the experimental procedure for the visualization of the innervation of the extrahepatic biliary tract using an antibody against neurofilament protein. This protocol was adapted from the protocol described by Kuratani and Tanaka1,2,3.
For this experiment, plan the timeline for each of the experiments, as the whole-mount staining approach continues for 2-3 weeks. The vessel containin...

The house musk shrew, Suncus murinus, belongs to the order Insectivora and the family Soricidae. This tiny mammal is distributed throughout Southeast Asia and inhabits houses and grassy areas that are situated near human habitations or cattle pens4. This species exhibits general morphological characteristics more similar to humans than other laboratory animals, such as the mouse, rat, and rabbit5. Previously, whole-mount immunostaining with a peripheral neuron marker for S. murinus neurofilament protein (NFP) was used to label peripheral nerves in the pancreas6, major duodenal papilla7, pylorus8, gallbladder5, and extrahepatic biliary tract9.
NFP is a macromolecular complex that is part of the mature neuronal cytoskeleton. Neurofilament-related proteins mediate interactions between NFP and the zygosome. Both enzyme function and the structure of linker proteins are regulated by NFP10. The NFP complex is made of three polypeptides: NF-L (70 kDa), NF-M (150-160 kDa), and NF-H (200 kDa). NFP can be found in neuronal axons in both the central and peripheral nervous system. Anti-human NFP antibody has been shown to label axons of the central and peripheral nervous system. Anatomical and electrophysiological investigations have demonstrated that the sphincter, gallbladder, and proximal gastrointestinal tract are connected11,12,13,14. However, the morphological features of their neuronal connections have not yet been defined.
This work demonstrates the use of a whole-mount immunohistochemical staining method to label the innervation of the S. murinus biliary tract with an NFP antibody.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">orthoperiodic acid</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">162-00732</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">papain</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">164-00172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bovine serum albumin (BSA)</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">010-15131</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sodium azide</td>
			<td style="width:71px;">Nacalai Tesque</td>
			<td style="width:119px;">312-33</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">neurofilament protein (NFP) antibody</td>
			<td style="width:71px;">Dako</td>
			<td style="width:119px;">M0762, lot 089, clone: 2F11</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">peroxidase-conjugated affinity-purified sheep anti-mouse IgG-HRP</td>
			<td style="width:71px;">MBL</td>
			<td style="width:119px;">Code 330</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3,3&#39;-diaminobenzidine tetrahydrochloride (DAB)</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">349-00903</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">imidazole</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">I-0125</td>
			<td></td>
		</tr>
	</tbody>
</table>

using a whole-mount immunohistochemical method to study the innervation of the biliary tract in suncus murinus

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the biliary tract in Suncus murinus (S. murinus&#160;). First, the specimen was dissected from S. murinus and fixed in 4% paraformaldehyde (PFA). Second, an enzymatic treatment and potential endogenous peroxidase inactivation were performed. The specimen was then exposed to the primary antibody, anti-neurofilament protein antibody, for 3-6 days. It was then incubated with the secondary antibody conjugated with horseradish peroxidase. The color reaction was revealed by reacting the specimen with a 3,3&#39;-diaminobenzidine (DAB) substrate. This method can be applied to analyze the innervation of all visceral organs. Furthermore, this protocol can also be adapted to test other neuronal antibodies, but optimization of the antibodies should be done first. This method was originally introduced by Kuratani and Tanaka1,2,3.

Figures 1-4 show typical results for NFP-positive nerve fibers in the extrahepatic biliary tract in S. murinus. The antibody against NFP reproducibly labeled the innervation in the entire image of the extrahepatic biliary tract (Figure 1), gallbladder (Figure 2), upper bile duct (Figure 3), and duodenal papilla area (Figure 4), ...

Watch this Scientific Journal Video about Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus at JoVE.com

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

A whole-mount immunohistochemical approach, to visualize neurofilament protein expression in the extrahepatic biliary tract in Suncus murinus. is presented here. This protocol can be used to analyze the innervation of all visceral organs in S. murinus or other species.

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the ...

The overall goal of this procedure is to employ whole-mount immunohistochemistry staining for the visualization of the nerve distribution within the biliary tract of Suncus murinus. This method can be used to analyze the nerve distribution of the visceral organs of many species, particularly irregular or small organs that are difficult to assess by standard methods. The main advantages of this technique are that the operation is simple.It does not require complicated procedures and it has a high success rate. Demonstrating the procedure with me will be Yidan Dai, a graduate student, and Shuang-Qin Yi, a research fellow, both from my laboratory. Begin by flushing the animal with individual PBS and 4%paraformaldehyde perfusions in a fume cupboard.Then inject two to three milliliters of white neoprene latex through the perfusion catheter into the abdominal aorta to label the blood vessels. And when all of the tissues of interest have been labeled, extract the abdominal organs and their related nerves and vessels. Then inject approximately 0.1 milliliters of blue neoprene latex into the gallbladder to label the biliary system and post-fix the tissues in 4%paraformaldehyde overnight at four degrees Celsius for whole-mount immunostaining.The next morning, transfer the sample into a glass vial for four one-hour room temperature washes in distilled water on a nutator. After the last wash, incubate the tissues in freshly-prepared 1%orthoperiodic acid for 20 minutes at room temperature to prevent any intrinsic peroxidase reactions. Next, wash the sample in distilled water for 10 minutes at room temperature, followed by incubation in freshly-prepared 0.004%papillon for two hours in 37 degrees Celsius constant temperature water bath with gentle rocking.At the end of the incubation, wash the specimen with distilled water for 50 to 60 minutes. Then store the tissues in 4%paraformaldehyde at four degrees Celsius overnight. The next morning, wash the sample in distilled water as just demonstrated followed by another two-hour incubation in freshly-prepared papillon.Wash the sample for another 50 to 60 minutes in distilled water, followed by overnight fixation in 4%paraformaldehyde. On the third morning, wash the tissues four times in distilled water, followed by immersion in three sequential ascending 30-minute sucrose incubations. Then freeze the sample at minus 20 degrees Celsius for 30 to 60 minutes, followed by complete thawing at room temperature.After the third freeze-thaw cycle, transfer the sample into 2%Triton X-100 for a four degree Celsius overnight incubation. The next morning, transfer the sample into a container of the primary antibody of interest for gentle rocking on the nutator at four degrees Celsius for three to six days according to the size of the specimen. After the appropriate labeling period, remove the unbound primary antibody with four one-hour washes in PBS at room temperature and label the specimen with the appropriate secondary antibody for three days with gentle rocking at four degrees Celsius.At the end of the secondary antibody labeling period, wash the specimen four times in PBS. Then incubate the tissues in 10 microliters of 30%hydrogen peroxide in 100 milliliters of freshly-prepared 0.002%DAB coloration solution at four degrees Celsius for 16 to 72 hours with gentle rocking. When the optimal staining intensity has been reached, transfer the specimen into 0.04%sodium azide in PBS to stop the coloration reaction.Then carefully immerse the sample in a 10 centimeter Petri dish containing fresh PBS and capture whole-mount images of the tissues using a camera mounted on a stereo microscope. In addition to visualization of the innervation of the extrahepatic biliary tract, labeling with antibody against neurofilament positive nerve fibers also allows the unambiguous identification of the running and distribution density of the vagus nerve along the esophagus and into the stomach. Labeling the blood vessels of the gallbladder with white neoprene latex reveals thin nervous fibrosis in high contrast to the surrounding tissues with a higher density of innervation observed in the neck of the gallbladder than in the fundus.In the upper bile duct, two types of nerve bundles are labeled, the fine nerve bundles which form an irregular and dense network of nerves and run adhesively residing on and in the biliary tract and the thicker neural bundles that are distributed parallel to the surface of the biliary tract. Labeling of the common bile duct with blue neoprene latex and the vessels with white neoprene latex allows visualization of the thin nervous fibers at the end of the common bile duct and the duodenal papilla area. Before attempting this procedure, take care to play out the timeline for each of the experiments as the entire process requires two to three weeks to be completed.To avoid damaging the specimen when changing the solutions, always pour out the solutions instead of removing the specimen with forceps. After watching this video, you should be able to apply this technique to the labeling of a range of nerves within various visceral organs.

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