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.
1. Animal Care and Housing
<ol>
	<li>Obtain 7 S. murinus (3 females and 4 males weighing 70-90 g) from a closed breeding colony.</li>
	<li>Cage the S. murinus individually after weaning (20 d after birth) in plastic cages equipped with a wooden nest box containing paper strips. Keep them in a conventionally conditioned animal room: 23-27 &#176;C, no humidity control, and a 12:12 h light:dark cycle. Supply commercial trout pellets and water ad libitum.</li>
</ol>
2. Tissue Preparation
<ol>
	<li>To prepare 4% (w/v) paraformaldehyde (PFA), dissolve 40 g of PFA in 1,000 mL of 0.01 M Phosphate-Buffered Saline (PBS, pH 7.4). In a fume cupboard, mix using a vortex until the solution is clear. Store the 4% PFA O/N at 4 &#176;C. 
	Caution: Wear appropriate Personal Protective Equipment (PPE) when handling PFA.</li>
	<li>To perfuse and fix the animal, anesthetize it with ether and then give it an intraperitoneal injection of somnopentyl (pentobarbital sodium, 0.6 mL/kg bodyweight).</li>
	<li>After the animal is completely narcotized, open the abdominal cavity with a scalpel, creating a midline abdominal incision 3 cm long. While incising the inferior vena cava for exsanguinating, insert a catheter retrogradely into the abdominal aorta at the level immediately above the bifurcation of this artery into the common iliac arteries.</li>
	<li>Inject somnopentyl (pentobarbital sodium, 1.0 mL/kg bodyweight). After complete euthanasia, perfuse with 0.01 M PBS (pH 7.4) and then with 4% PFA in a fume cupboard.</li>
	<li>After perfusion, inject 2-3 mL of white neoprene latex (dilute white neoprene latex 3:2 with Distilled Water (DW)) to label the blood vessels.</li>
	<li>Extract the abdominal organs, including the liver, gallbladder, common bile duct, duodenum, and pancreas en bloc, with the related nerves and vessels.</li>
	<li>Inject approximately 0.1 mL of blue neoprene latex (add a drop of blue ink to 10 mL of the diluted white neoprene latex) to the gallbladder to label the biliary system.</li>
	<li>Post-fix overnight with 4% PFA at 4 &#176;C for whole-mount immunostaining. 
	Caution: PFA is toxic. Avoid handling PFA directly. PFA should be used in a fume cupboard.</li>
</ol>
3. Whole-mount Immunohistochemistry
Note: Throughout the protocol, including during washing, antibody incubation, and coloration, the specimen must remain on the nutator, gently rocking at RT&#160;or at 4&#176;C.
<ol>
	<li>Day 1: Enzymatic treatment and inactivation of potential endogenous peroxidase
	<ol>
		<li>Wash the prepared specimen with DW 4x for 1 h each at RT in an appropriately sized glass vial. Rock the specimen gently on the nutator to remove the PFA. Avoid damaging the specimen when exchanging solutions.</li>
		<li>To prepare 1% (w/v) orthoperiodic acid, dissolve 1 g of orthoperiodic acid in 100 mL of DW.</li>
		<li>Incubate the specimen in 1% orthoperiodic acid for 20 min at RT to prevent any intrinsic peroxidase reaction.</li>
		<li>Prepare 0.004% (w/v) papain by dissolving 0.004 g of papain in 100 mL of 0.025 mol/L Tris-HCl buffer (pH 7.6).</li>
		<li>Wash the specimen with DW for 10 min at RT.</li>
		<li>Incubate the specimen in freshly prepared 0.004% papain for 2 h at 37 &#176;C in a constant-temperature bath with gentle rocking.</li>
		<li>Wash the specimen with DW for 50-60 min at RT.</li>
		<li>Store the specimen in 4% PFA at 4 &#176;C O/N.</li>
	</ol>
	</li>
	<li>Day 2: Enzymatic treatment
	<ol>
		<li>Wash the stored specimen with DW 4 times for 1 h each at RT.</li>
		<li>Incubate the specimen in freshly prepared 0.004% papain for 2 h at 37 &#176;C in a constant-temperature bath with gentle rocking.</li>
		<li>Wash the specimen with DW for 50-60 min at RT. Store the specimen in 4% PFA at 4 &#176;C O/N.</li>
	</ol>
	</li>
	<li>Day 3: Freezing treatment
	<ol>
		<li>Wash the stored specimen with DW 4x for 1 h each at RT, as above.</li>
		<li>Prepare 2.5% (w/v), 5% (w/v), and 10% (w/v) sucrose by dissolving 2.5 g, 5 g, and 10 g of sucrose, respectively, in 100 mL of 0.01 M PBS (pH 7.4).</li>
		<li>Immerse the specimen in 2.5% (w/v), 5% (w/v), and 10% (w/v) sucrose for 30 min each.</li>
		<li>Freeze the specimen at -20 &#176;C for 30 - 60 min and then completely thaw it at RT; repeat the cycle 3x.</li>
		<li>To prepare 2% Triton X-100 (v/v), add 2 mL of Triton X-100 to 100 mL of 0.01 M PBS (pH 7.4).</li>
		<li>Store the specimen in 2% Triton X-100 at 4 &#176;C O/N.</li>
	</ol>
	</li>
	<li>Day 4: Primary antibody incubation
	<ol>
		<li>Prepare the dilution solution of the primary antibody by dissolving 0.2 g of Bovine Serum Albumin (BSA), 0.3 mL of Triton X-100, and 0.1 g of sodium azide in 100 mL of 0.01 M PBS (pH 7.4). 
		Caution: Sodium azide is acutely toxic. Inhalation and touching must be avoided. Wear appropriate PPE.</li>
		<li>Dilute the primary antibody (NFP) 1:600 in the above dilution buffer (step 3.4.1). Incubate the specimen with NFP antibody at 4 &#176;C for 3-6 days, keeping the specimen gently rocking on the nutator. Larger specimens will require increased incubation times.</li>
	</ol>
	</li>
	<li>Secondary antibody incubation
	<ol>
		<li>Wash the specimen in PBS 4x for 1 h each at RT to remove unbound primary antibody.</li>
		<li>Prepare the dilution solution for the secondary antibody by dissolving 0.2 g of BSA and 0.3 mL of Triton X-100 in 100 mL of 0.01 M PBS (pH 7.4).</li>
		<li>Dilute the secondary antibody (peroxidase-conjugated, affinity-purified sheep anti-mouse IgG-HRP) 1:600 in the dilution buffer (step 3.5.2). Incubate the specimen with the secondary antibody at 4 &#176;C for 3 d, and keep the specimen rocking gently on the nutator.</li>
	</ol>
	</li>
	<li>Coloration
	<ol>
		<li>Prepare DAB coloration solution by adding 0.002 g of 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) to 100 mL of 0.05 mol/L Tris-HCl buffer (pH 7.6) under a fume cupboard, keeping the solution in the dark.</li>
		<li>Wash the specimens at RT in PBS 4x for 1 h each.</li>
		<li>Add 10 &#181;L of 30% H2O2 in freshly prepared DAB coloration solution and incubate with the solution at 4 &#176;C O/N or for 3 d while rocking gently on the nutator.</li>
		<li>Stop the reaction by placing the specimen in PBS with 0.04% sodium azide when the optimal staining intensity is reached. 
		NOTE: Avoid the use of sodium azide until this step, as it inhibits horseradish peroxidase.</li>
	</ol>
	</li>
	<li>Imaging the stained tissue
	<ol>
		<li>Carefully transfer the specimen to a petri glass dish (5 cm high, 10 cm in diameter) containing PBS. Capture whole-mount images using a camera mounted on a stereomicroscope. 
		NOTE: Whole-mount stained tissue should be imaged while immersed completely in PBS on a transparent glass dish.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Kuratani, S., Tanaka, S., Ishikawa, Y., Zukeran, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+development+of+the+hypoglossal+nerve+in+the+chick+embryo+as+observed+by+the+whole-mount+nerve+staining+method.">Early development of the hypoglossal nerve in the chick embryo as observed by the whole-mount nerve staining method.</a> Am. J. Anat. 182 (2), 155-168 (1988).</li><li>Kuratani, S., Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+development+of+the+avian+vagus+nerve+with+special+reference+to+the+morphological+innervation+of+heart+and+lung.">Peripheral development of the avian vagus nerve with special reference to the morphological innervation of heart and lung.</a> Anat. Embryol. (Berl). 182 (5), 435-445 (1990).</li><li>Kuratani, S., Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+development+of+avian+trigeminal+nerves.">Peripheral development of avian trigeminal nerves.</a> Am. J. Anat. 187 (1), 65-80 (1990).</li><li>Yi, S. Q., 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=House+musk+shrew,+Suncus+murinus:+a+novel+and+natural+obesity-resistant+animal+model.">House musk shrew, Suncus murinus: a novel and natural obesity-resistant animal model.</a> Obes. Metab. 6, 22-28 (2010).</li><li>Yi, S. Q., 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=Surgical+anatomy+of+innervation+of+the+gallbladder+in+humans+and+Suncus+murinus+with+special+reference+to+morphological+understanding+of+gallstone+formation+after+gastrectomy.">Surgical anatomy of innervation of the gallbladder in humans and Suncus murinus with special reference to morphological understanding of gallstone formation after gastrectomy.</a> World J. Gastroenterol. 13 (14), 2066-2071 (2007).</li><li>Yi, S. Q., 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=Anatomical+study+of+the+pancreas+in+the+house+musk+shrew,+Suncus+murinus,+with+special+reference+to+the+blood+supply+and+innervation.">Anatomical study of the pancreas in the house musk shrew, Suncus murinus, with special reference to the blood supply and innervation.</a> Anat. Rec. A Discov. Mol. Cell Evol. Biol. 273 (1), 630-635 (2003).</li><li>Yi, S. Q., 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=Surgical+anatomy+of+the+innervation+of+the+major+duodenal+papilla+in+humans+and+Suncus+murinus,+from+the+perspective+of+preserving+innervation+in+organ-saving+procedures.">Surgical anatomy of the innervation of the major duodenal papilla in humans and Suncus murinus, from the perspective of preserving innervation in organ-saving procedures.</a> Pancreas. 30 (3), 211-217 (2005).</li><li>Yi, S. Q., 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=Surgical+anatomy+of+the+innervation+of+the+pylorus+in+humans+and+Suncus+murinus,+in+relation+to+the+surgical+technique+for+pylorus-preserving+pancreatoduodenectomy.">Surgical anatomy of the innervation of the pylorus in humans and Suncus murinus, in relation to the surgical technique for pylorus-preserving pancreatoduodenectomy.</a> World J. Gastroenterol. 12 (14), 2209-2216 (2006).</li><li>Yi, S. Q., Ren, K., Kinoshita, M., Takano, N., Itoh, M., Ozaki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innervation+of+Extrahepatic+Biliary+Tract,+With+Special+Reference+to+the+Direct+Bidirectional+Neural+Connections+of+the+Gall+Bladder,+Sphincter+of+Oddi+and+Duodenum+in+Suncus+murinus,+in+Whole-Mount+Immunohistochemical+Study.">Innervation of Extrahepatic Biliary Tract, With Special Reference to the Direct Bidirectional Neural Connections of the Gall Bladder, Sphincter of Oddi and Duodenum in Suncus murinus, in Whole-Mount Immunohistochemical Study.</a> Anat. Histol. Embryol. 45 (3), 184-188 (2016).</li><li>Wang, H., 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=Neurofilament+proteins+in+axonal+regeneration+and+neurodegenerative+diseases.">Neurofilament proteins in axonal regeneration and neurodegenerative diseases.</a> Neural Regen. Res. 7 (8), 620-626 (2012).</li><li>Wyatt, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+of+the+sphincter+of+Oddi+to+the+stomach,+duodenum+and+gall-bladder.">The relationship of the sphincter of Oddi to the stomach, duodenum and gall-bladder.</a> J. Physiol. 193 (2), 225-243 (1967).</li><li>Mawe, G. M., Kennedy, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duodenal+neurons+provide+nicotinic+fast+synaptic+input+to+sphincter+of+Oddi+neurons+in+guinea+pig.">Duodenal neurons provide nicotinic fast synaptic input to sphincter of Oddi neurons in guinea pig.</a> Am. J. Physiol. 277, G226-G234 (1999).</li><li>Seo, J. H., Cho, S. S., Lee, I. S., Lee, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+and+neuropeptidergic+properties+of+the+duodenal+neurons+projecting+to+the+gallbladder+in+the+golden+hamster.">Anatomical and neuropeptidergic properties of the duodenal neurons projecting to the gallbladder in the golden hamster.</a> Arch. Histol. Cytol. 65 (4), 317-321 (2002).</li><li>Padbury, R. T., Furness, J. B., Baker, R. A., Toouli, J., Messenger, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+of+nerve+cells+from+the+duodenum+to+the+sphincter+of+Oddi+and+gallbladder+of+the+Australian+possum.">Projections of nerve cells from the duodenum to the sphincter of Oddi and gallbladder of the Australian possum.</a> Gastroenterology. 104 (1), 130-136 (1993).</li><li>White, J. J., Reeber, S. L., Hawkes, R., Sillitoe, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wholemount+immunohistochemistry+for+revealing+complex+brain+topography.">Wholemount immunohistochemistry for revealing complex brain topography.</a> J. Vis. Exp. (62), e4042 (2012).</li></ol>

The authors declare no conflicts of interest.

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 containing the tissue must be rotated on a nutator at all times, except during the freeze/thaw process. Particularly during incubation with the primary and secondary antibodies, the specimen was set in a cold storage chamber while rotating on a nutator to ensure the uniform exposure of the specimen to the reaction solutions. Several solutions &#8211; 1% (w/v) orthoperiodic acid, 0.004% (w/v) papain, and the dilution buffer for the primary/secondary antibodies &#8211; must be made fresh for each experiment. Throughout the protocol, to avoid touching and damaging the specimen when changing solutions, the solutions should be poured out instead of being removed with forceps15.
After fixing with PFA, the samples should be washed sufficiently with DW or PBS. In addition, the enzymatic treatment with the papain incubation is important. The heat and the enzyme used for antigen retrieval help to prepare the tissue for antibody penetration. To enhance the penetration of the solutions, the outside membrane of the tissue should be disrupted by a freezing treatment at -20 &#176;C for 30 min or -80 &#176;C O/N15.
It is important to completely submerge the experimental specimen in adequate volumes of buffer solution to ensure that the solution can incubate the whole specimen. The incubation time of the primary antibody must be empirically determined for different tissues and sample sizes. Usually, it must be 3 days for a 2-3 cm sample. The optimal dilution must be empirically determined for each antibody. A dilution at 1:600 is recommended for both the primary and secondary antibodies when using the appropriate antibody buffer.
This work describes, in detail, the protocol of a versatile whole-mount immunohistochemical approach to reveal neurofilament protein expression in the biliary tract of S. murinus. This method can be used to analyze the innervation of all visceral organs in many species. Furthermore, this protocol can also be adapted to test other neuronal antibodies, but optimization of the antibodies should be performed.
However, the technique was limited in labeling shallow nervous tissue and in constructing a three-dimensional model of innervation. Also, it could not identify the characteristics of the nervous fibers (i.e.&#160;sympathetic or parasympathetic, motor or sensory, etc.).

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><tbody><tr><td>orthoperiodic acid</td><td>Wako</td><td>162-00732</td><td></td></tr><tr><td>papain</td><td>Wako</td><td>164-00172</td><td></td></tr><tr><td>bovine serum albumin (BSA)</td><td>Wako</td><td>010-15131</td><td></td></tr><tr><td>sodium azide</td><td>Nacalai Tesque</td><td>312-33</td><td></td></tr><tr><td>neurofilament protein (NFP) antibody</td><td>Dako</td><td>M0762, lot 089, clone: 2F11</td><td></td></tr><tr><td>peroxidase-conjugated affinity-purified sheep anti-mouse IgG-HRP</td><td>MBL</td><td>Code 330</td><td></td></tr><tr><td>3,3&#039;-diaminobenzidine tetrahydrochloride (DAB)</td><td>Wako</td><td>349-00903</td><td></td></tr><tr><td>imidazole</td><td>Sigma</td><td>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), with high specificity and minimal background.
For all tissues of the specimen, regardless of shape and size, the tegmental nerves can be dyed at the same time. In addition to the innervation of the extrahepatic biliary tract, the running and distribution density of the vagus of the esophagus and the stomach were exhibited unambiguously (Figure 1).
As the blood vessels of the gallbladder were labeled with white neoprene latex, thin nervous fibroses were clearly demonstrated with high contrast. The density of innervation was higher in the neck than in the fundus of the gallbladder (Figure 2).
In the upper bile duct, two types of nerve bundles were labeled. The fine nerve bundles formed an irregular and dense network of nerves, ran adhesively, and resided on/in the biliary tract; the thicker neural bundles were distributed parallel to the surface of the biliary tract (Figure 3).
The common bile duct was labeled with blue neoprene latex and the vessels with white neoprene latex. The thin nervous fibers at the end of the common bile duct and the duodenal papilla area were clearly demonstrated with high contrast (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55483/55483fig1.jpg" /> 
Figure 1: NFP-positive Nerve Fibers in the Biliary Tract, the Esophagus, and the Stomach. Arrows show the vagus running along the esophagus to the stomach. CBD, Common Bile Duct; Es, esophagus; St, stomach. Scale bar = 1,300 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55483/55483fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55483/55483fig2.jpg" /> 
Figure 2: NFP-positive Nerve Fibers in the Gallbladder. The blood vessels of the gallbladder were labeled with white latex. Abundant innervation occurred in the neck of the gallbladder (arrows). Scale bar = 1,000 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55483/55483fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55483/55483fig3.jpg" /> 
Figure 3: NFP-positive Nerve Fibers in the Upper Bile Duct. Two types of nerve bundles were observed. One type was the fine nerve bundles that ran adhesively and resided on/in the extrahepatic biliary tract (arrows); the other type was thicker neural bundles that were distributed parallel to the surface of the extrahepatic biliary tract and ran between the gallbladder and duodenum (triangles). PV, Portal Vein. Scale bar = 650 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55483/55483fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55483/55483fig4.jpg" /> 
Figure 4: NFP-positive Nerve Fibers in the Duodenal Papilla Area. The common bile duct was labeled with blue latex and the vessels with white latex (*). Arrows show the innervation of the end of the common bile duct, and triangles show the innervation of the duodenal papilla area (P), which comes from the common bile duct and vessels. Scale bar = 1,000 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55483/55483fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

using-whole-mount-immunohistochemical-method-to-study-innervation

Nanyang Technological University

Research

JoVE Journal

Biology

6.8K Views.  Tokyo Metropolitan University.  Een gehele-mount immunohistochemische aanpak, om de neurofilament eiwit expressie in het extrahepatische galweg in Suncus murinus te visualiseren. Wordt hier gepresenteerd. Dit protocol kan gebruikt worden om de innervering van alle viscerale organen in S. murinus of andere soorten te analyseren. 

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

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

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

Cancer Research

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Neuroscience

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Immunology and Infection

Immunohistochemistry and Immunocytochemistry: Tissue Imaging via Light Microscopy

Source: Michael S. Lee1 and Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201
Immunohistochemistry (IHC) and immunocytochemistry (ICC) are techniques used to visualize the expression and localization of specific antigens using antibodies. The first published use of IHC was in 1941 when Albert Coons used the technique to visualize the presence of pneumococcal antigen in tissue sections from mice infected with Pneumococcus (1). The name, immunohistochemistry, is derived from the roots &#34;immuno-,&#34; in reference to antibodies, and &#34;histo-,&#34; in reference to the tissue sections used in IHC. The root &#34;cyto-&#34; in immunocytochemistry highlights the key difference between ICC and IHC. Whereas IHC uses sections of whole tissue, ICC uses cells that have been isolated from tissue or grown in culture. The difference in samples used means sample preparation technically differs between IHC and ICC, but otherwise the protocols for ICC and IHC are identical and one will find the terms are frequently used interchangeably.
In both IHC and ICC, antibodies with either chemical or fluorescent tags, such as peroxidase or rhodamine, respectively, are used to visualize the distribution of any antigen of interest through specific binding of the tagged antibody to the antigen. In the case of IHC, thin slices of tissue are immobilized on a slide to maintain the structure of the tissue before being stained, allowing the visualization of antigens in the context of whole tissues (Figure 1). In the case of ICC, cells are distributed evenly on a slide before being stained, allowing the visualization of antigen distribution within individual cells but not within the structure of any specific tissue. Due to the similarities between the two protocols, this protocol will focus on IHC to address the additional complexities of sample preparation involved in IHC.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10498/10498fig1.jpg" /> 
Figure 1: Outline of IHC Protocol. Visual outline of an IHC protocol for paraffin-embedded tissue dissected from a mouse. This protocol uses a biotinylated secondary antibody and strepavidin-HRP to visualize the location of antibody binding. Other options, such as fluorescently tagged antibodies, are also possible. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/10498/10498fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The first major decision when performing IHC is how to prepare the tissue sections in order to maintain the structure of the tissue throughout the staining process. The two main choices are formalin-fixed sections of paraffin-embedded tissue or fresh sections of frozen tissue. There is no simple answer as to which method to use as it depends on what downstream analysis will be conducted. Formalin-fixation of paraffin embedded tissues is generally thought to better preserve tissue morphology for optimal imaging while freezing fresh tissue can preserve protein function for subsequent assays outside of IHC. In addition, fresh frozen tissue sections have been shown to be more suitable for gene expression analysis (2). A third consideration is whether or not the antibodies for your antigen of interest are suitable for either fixed or frozen tissue sections, as some antibodies have only been optimized for a specific type of section and may not work for others. Finally, one also needs to determine how long they need to store the tissue sections, as fresh frozen samples must be kept at -80&#176;C and may not last beyond one year while fixed sections can be stored for much longer at room temperature. These are a few of the major considerations for determining whether to use formalin-fixed sections of paraffin-embedded tissue or fresh sections of frozen tissue. Ultimately, if one has enough tissue, it may be best just to have some of both.
In this experiment, we set out to determine if cyclin D1 expression was increased in enlarged spleens from a spontaneous mouse model of lymphoma development. Splenic tissue samples were first isolated from either wild-type mice, transgenic mice that do not have lymphoma, or transgenic mice that have spontaneously developed lymphoma. The spleen tissue samples were fixed in paraformaldehyde, embedded in paraffin, sectioned, stained using a mouse anti-cyclin D1 primary antibody followed by a horse anti-mouse secondary antibody, and developed using 3,3-diaminobenzidine (DAB). The sections were then counterstained in Harris Hematoxylin Solution and then the sections were imaged at 20X magnification.
Reagents
Paraffin-Embedded Sections
<ol type="a">
	<li>4% Paraformaldehyde (PFA)</li>
	<li>Ethanol (anhydrous denatured, histological grade 100%, 95%, 80%, 75%, and 50%). Can be diluted from 100% stock using double distilled water (ddH2O)</li>
	<li>Xylene</li>
	<li>IHC compatible glass slide to ensure tissue section remains attached throughout the entire procedure. IHC compatible glass slides have a specialized coating and are readily available from multiple retailers. If performing ICC, use a chambered slide. Chambered slides allow for cells to be seeded in the chambers and placed in the incubator until cells attach to the slide and reach proper confluency, at which point the chambers can be removed and staining can proceed in the same manner as IHC.</li>
	<li>Paraffin</li>
	<li>0.3% Hydrogen peroxide (H2O2)/methanol: To prepare, add 1 mL 30% H2O2 to 99 mL methanol. Store at -20&#176;C</li>
	<li>Antigen retrieval buffer: IHC citrate buffer pH 6.0</li>
</ol>
Fresh Frozen Sections
<ol type="a">
	<li>Optimal cutting temperature (OCT) embedding compound</li>
	<li>Optimal fixative: 4% PFA or acetone that has been cooled to -20&#176;C</li>
</ol>
Staining
<ol type="a">
	<li>Blocking buffer: Should be determined by user. One example is horse serum diluted in 1X PBS</li>
	<li>Diluted primary antibody: see manufacturer specifications</li>
	<li>Diluted biotinylated secondary antibody: see manufacturer specifications</li>
	<li>Diluted avidin-Horseradish Peroxidase (HRP): Only for peroxidase visualization. See manufacturer specifications.</li>
	<li>DAB or another compatible substrate</li>
	<li>Counterstain (optional)</li>
	<li>Ethanol (anhydrous denatured, histological grade 100% and 95%)</li>
	<li>Xylene</li>
	<li>Organo/Limonene Mount</li>
</ol>

Immunohistochemistry &amp; Immunocytochemistry: Tissue Imaging

Source: Michael S. Lee1 and Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and ...

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Advanced Biology

Immunology

Immunohistochemical Staining of Nerve Fibers in the Nucleus Basalis of Meyner of the Mouse Brain

Source: Singh, P., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60405">Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain.</a> J. Vis. Exp. (2020)
This video demonstrates the immunohistochemical staining of cholinergic fibers in the nucleus basalis of the Meynert region of mouse brain sections. Sections are blocked and treated with primary against choline acetyltransferase, followed by biotinylated secondary antibodies. Avidin-biotin-peroxidase complexes are added, which bind to secondary antibodies, followed by the chromogenic substrate. Sections are then dehydrated and mounted before visualizing under the microscope.

Source: Singh, P., et al. Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain. J. Vis. Exp. (2020)
...

JoVE Encyclopedia of Experiments

Neuropathology

Neurodegenerative Diseases

An In Vivo Immunofluorescence Localization Method to Study Antibody Biodistribution

Following anesthetization as described in the text protocol, prepare for the tail vein inoculation of the antibody solutions. Disinfect the tail of the animal by wiping three times with an alcohol wipe. Dilate the tail veins by warming with a heat pad for approximately 30 seconds; avoid heating the entire animal. Using a 27-gauge tail vane catheter insert the butterfly needle into one of the two lateral tail veins.
Visualize blood backflow into the catheter to ensure that the needle is properly placed so that the solutions will fully enter the animal versus being captured in the tail.
Use a piece of surgical tape to carefully fix the tail with the inserted needle to the stage. Flush the catheter with 25 microliters of sterile phosphate-buffered saline. Then, inject the antibody solution into the catheter using insulin syringes. Flush the catheter once again with 25 microliters of sterile PBS. Remove the needle from the tail, and apply pressure to stop any bleeding.
Perform humane euthanasia of the mouse, as described in the text protocol, and lay the mouse in the supine position. Use surgical scissors and forceps to excise tumor tissues. Use forceps to grasp only the outer layer of skin between the set of mammary glands closest to the tail, and to make a small incision with a pair of surgical scissors. Introduce the closed scissors into the cut, and slowly open the tip to carefully separate the skin from the underlying abdominal wall membrane, keeping it intact.
Make a vertical incision up the abdomen, continuing to separate the skin from the inner membrane. Between the third and fourth mammary glands, make a horizontal cut across the abdomen, to allow retraction of the skin and visualization of the mammary glands. Grasping each tumor or normal gland with forceps, carefully trim away the attached skin using surgical scissors.
Place excised tissues into tissue disposable base molds that are pre-labeled and filled with optimal cutting temperature embedding medium. Quickly freeze the molds by placement on dry ice. In order to study off-target delivery, excise other tissues or organs of interest. Using a cryostat, section frozen tissue blocks at 10-micron thickness, and place adjacent sections onto pre-labeled adhesion glass slides.
Rinse frozen tissue slides with room temperature PBS for 5 minutes to remove the embedding medium. Demarcate tissue sections with a hydrophobic barrier pen to reduce the volume of solutions needed during staining. Fix the tissue sections with 4% paraformaldehyde solution for 5 minutes. After rinsing the slides in PBS for 5 minutes, permeabilize tissue sections with 0.5% Triton X-100 in PBS for 15 minutes. Rinse the slides again in PBS for 5 minutes.
Block the tissues with PBS containing 3% weight per volume bovine serum albumin and 5% volume per volume goat serum for 1 hour at room temperature. After rinsing the slides in PBS for 5 minutes, incubate the sections with record-keeping primary antibodies, such as a common nuclear, vascular, or cytoplasmic marker. Here, rat anti-mouse CD31 is used at a 1 to 100 dilution in the blocking solution. The slides with primary antibody are left overnight at 4 degrees Celsius, protected from dehydration on a slide tray.
Following incubation, rinse the slides in PBS for 5 minutes three times. Change the PBS each time. Incubate the slides with secondary antibodies to label the primary antibodies. For this application, visualize anti-B7-H3 antibody using AlexaFluor-546 conjugated goat anti-rabbit antibody, and visualize CD31 with AlexaFluor-488 goat anti-rat secondary antibody in blocking solution. Protect the slides from light and dehydration on a slide tray for 1 hour at room temperature.
After rinsing the slides in PBS, as before, apply one drop of the mounting medium into the center of the tissue slice. Carefully place a coverslip avoiding entrapment of air bubbles. Seal the edges of the coverslip with clear nail polish and allow to dry. Proceed with confocal microscopy imaging and quantitative image analysis, as described in the text protocol.

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

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