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 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 ...

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6.7K Views.  Tokyo Metropolitan University. 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.

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

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...