This work was supported bythe American Pediatric Surgical Association Foundation Award (AG) and the National Institutes of Health K08DK098271 (AG).

The University of Wisconsin Animal Care and Use Committee approved all procedures.
1. Preparation of Solutions
<ol>
	<li>Use 1x phosphate buffered saline (PBS) as dissection buffer and rinsing solution.</li>
	<li>Prepare 30% sucrose by weighing 30 g of sucrose and place into a bottle. Add 99 ml of 1x PBS and add 1 ml 10% sodium azide. Mix thoroughly until all of the sucrose is dissolved. Store at 4 &#176;C until required.</li>
	<li>Prepare Blocking solution by mixing 1x PBS, 3% bovine serum albumin (BSA) and 0.1% Triton-X-100. Mix thoroughly and store in the fridge until needed.</li>
	<li>Prepare 8% paraformaldehyde (PFA) solution in 1x PBS by weighing the appropriate amount of PFA into 1x PBS and then incubate at 65 &#176;C until it is completely dissolved. Store in 25 ml aliquots in the -20 &#176;C freezer until needed. Dilute to 4% PFA in 1x PBS on the day of use.</li>
</ol>
2. Embryo and Gut Dissection
<ol>
	<li>In accordance with Institutional Animal Care and Use Committee approved protocols, euthanize timed pregnant mouse and transfer the uterus into a 60 mm Petri dish containing ice cold 1x PBS.</li>
	<li>Under a dissection microscope using sharp scissors, cut the uterine wall open to expose the embryos. Remove the embryos from the uterus and place into a clean 60 mm Petri dish containing ice cold 1x PBS.</li>
	<li>Euthanize each embryo by decapitation in ice-cold 1x PBS. If you are using transgenic mice containing fluorescent proteins, under fluorescence illumination, identify the positive transgenic embryos.</li>
	<li>Dissect the GI tract from each embryo using a dissection microscope. Using fine forceps, orient the embryo such that the left side is facing upwards and the right side is against the bottom of the Petri dish. Remove the upper body wall from the embryo to expose the internal organs. Insert fine forceps between the dorsal body wall and the internal organs. Cross the forceps against each other in a scissor-like cutting action to remove the internal organs from the embryo.</li>
	<li>Further sub-dissect the GI tract away from the surrounding organs and then place each GI tract into a 1.5 ml microcentrifuge tube containing ice-cold 1x PBS.</li>
</ol>
3. Fixation of GI Tracts
<ol>
	<li>Rinse each GI tract 3 times with ice-cold 1x PBS and then replace with 4% PFA. Fix the GI tracts in the 1.5 ml microcentrifuge tubes on a rocking platform at RT&#160;for 1.5 hr. Rinse the GI tracts 3 times for 5 min at RT and then for 1 hr on the rocking platform. At this stage, store the GI tracts at 4 &#176;C in 30% sucrose in 1x PBS containing 0.1% sodium azide until needed. 
	NOTE: Alternatively, store the embryos in 30% sucrose for up to one year without any effect on the integrity of the tissues. Storage of the samples in 30% sucrose allows later processing of the samples either for immunostaining or into OCT for cryo-sectioning. Alternatively, proceed with the immunostaining protocol detailed below.</li>
</ol>
4. Immunostaining Protocol
<ol>
	<li>If samples have been stored in 30% sucrose, rinse them 3 times for 20 min in 1x PBS on a rocking platform.</li>
	<li>Place the GI tracts into blocking solution on a rocking platform for 1h at RT.</li>
	<li>Remove the blocking solution and incubate the GI tracts with the appropriate amount of primary antibodies diluted in blocking solution for either 4 hr at RT or O/N&#160;at 4 &#176;C on a rocking platform. Use 1:1,000 dilution of human anti-Hu antibody (serum obtained from patient), 1:1,000 dilution of chicken anti-green fluorescent protein (GFP) antibody and 1:100 dilution of goat anti-ChAT antibody. 
	NOTE: We utilize a human anti-Hu antibody that was obtained locally from a patient, however, anti-Hu antibodies are commercially available, for example, mouse anti-Hu, (use at 1:500 dilution).</li>
	<li>Rinse the GI tracts in 1x PBS 3 times for 5 min and then for 1 hr at RT on a rocking platform.</li>
	<li>Replace the 1x PBS with secondary antibodies diluted 1:500 in blocking solution on a rocking platform for either 4 hr at RT or O/N&#160;at 4 &#176;C. Use donkey anti-human amine-reactive dye such as Dylight, donkey anti-chicken Cy2 and donkey anti-goat Cy5. If using the mouse anti-Hu antibody then use a 1:500 dilution of donkey anti-mouse amine-reactive dye 405. 
	NOTE: The intensity of the endogenous GFP and tdTomato expression and the clarity of immunostaining increase with developmental age. We typically immunostain embryonic guts individually in 0.2 ml tubes with 150 &#956;l of staining solution in order to reduce the volume of antibody used and also to ensure efficient staining of the tissue.</li>
	<li>Rinse the GI tracts in 1x PBS 3 times for 5 min and then for 1 hr at RT on a rocking platform.</li>
	<li>Place a few drops of fluorescence mounting medium with DAPI onto a glass slide. Immerse the GI tract into the fluorescence mounting medium and add a cover glass directly on top of the tissue. The fluorescence mounting medium -G is a water-soluble compound that provides a semi-permanent seal. 
	NOTE: Use fluorescence mounting medium such as Vectashield which is a glycerol based mounting medium that prevents fading and photobleaching of antibodies. Both of these products enable tissues to be stored for long periods of time at 4 &#176;C.</li>
	<li>Capture images of each of fluorophore using a confocal microscope. Use excitation wavelengths for fluorophores and filters employed presented in Table 3.</li>
	<li>Perform computer-aided image analysis using appropriate software depending on the type of confocal used to capture images.</li>
</ol>

<ol>
	<li>Gershon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+determinants+of+the+independence+and+complexity+of+the+enteric+nervous+system.">Developmental determinants of the independence and complexity of the enteric nervous system.</a> Trends Neurosci. 33 (10), 446-456 (2010).</li><li>Amiel, J., Sproat-Emison, E., 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=Hirschsprung+disease,+associated+syndromes+and+genetics:+a+review.">Hirschsprung disease, associated syndromes and genetics: a review.</a> J Med Genet. 45 (1), 1-14 (2008).</li><li>Erickson, C. S., Barlow, A. J., 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=Colonic+enteric+nervous+system+analysis+during+parenteral+nutrition.">Colonic enteric nervous system analysis during parenteral nutrition.</a> J Surg Res. 184 (1), 132-137 (2013).</li><li>Erickson, C. S., Zaitoun, I., Haberman, K. M., Gosain, A., Druckenbrod, N. R., Epstein, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sacral+neural+crest-derived+cells+enter+the+aganglionic+colon+of+Ednrb(-/-)+mice+along+extrinsic+nerve+fibers.">Sacral neural crest-derived cells enter the aganglionic colon of Ednrb(-/-) mice along extrinsic nerve fibers.</a> J Comp Neurol. 20 (3), 620-632 (2011).</li><li>Zaitoun, I., Erickson, C. S., 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=Altered+neuronal+density+and+neurotransmitter+expression+in+the+ganglionated+region+of+Ednrb+null+mice:+implications+for+Hirschsprung's+disease.">Altered neuronal density and neurotransmitter expression in the ganglionated region of Ednrb null mice: implications for Hirschsprung's disease.</a> Neurogastroenterol Motil. , (2013).</li><li>Margolis, K. G., Stevanovic, K., 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=Enteric+neuronal+density+contributes+to+the+severity+of+intestinal+inflammation.">Enteric neuronal density contributes to the severity of intestinal inflammation.</a> Gastroenterology. 141 (2), 588-598 (2011).</li><li>Qu, Z. -. D., Thacker, M., Castelucci, P., Bagy&#225;nszki, M., Epstein, M. L., Furness, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+analysis+of+neuron+types+in+the+mouse+small+intestine.">Immunohistochemical analysis of neuron types in the mouse small intestine.</a> Cell Tissue Res. 334 (2), 147-161 (2008).</li><li>Bian, X. -. C., Bornstein, J. C., Bertrand, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+transmission+at+functionally+distinct+synapses+in+descending+reflex+pathways+of+the+rat+colon.">Nicotinic transmission at functionally distinct synapses in descending reflex pathways of the rat colon.</a> Neurogastroenterol Motil. 15 (2), 161-171 (2003).</li><li>Johnson, C. D., Epstein, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+and+polyvalent+antiserum+to+chicken+choline+acetyltransferase.">Monoclonal antibodies and polyvalent antiserum to chicken choline acetyltransferase.</a> J Neurochem. 46 (3), 968-976 (1986).</li><li>Tooyama, I., Kimura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protein+encoded+by+an+alternative+splice+variant+of+choline+acetyltransferase+mRNA+is+localized+preferentially+in+peripheral+nerve+cells+and+fibers.">A protein encoded by an alternative splice variant of choline acetyltransferase mRNA is localized preferentially in peripheral nerve cells and fibers.</a> J Chem Neuroanat. 17 (4), 217-226 (2000).</li><li>Koga, T., Bellier, J. -. P., Kimura, H., Tooyama, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoreactivity+for+Choline+Acetyltransferase+of+Peripheral-Type+(pChAT)+in+the+Trigeminal+Ganglion+Neurons+of+the+Non-Human+Primate+Macaca+fascicularis.">Immunoreactivity for Choline Acetyltransferase of Peripheral-Type (pChAT) in the Trigeminal Ganglion Neurons of the Non-Human Primate Macaca fascicularis.</a> Acta histochemica et cytochemica. 46 (2), 59-64 (2013).</li><li>Sang, Q., Young, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+identification+and+chemical+coding+of+cholinergic+neurons+in+the+small+and+large+intestine+of+the+mouse.">The identification and chemical coding of cholinergic neurons in the small and large intestine of the mouse.</a> Anat Rec. 251 (2), 185-199 (1998).</li><li>Lei, J., Howard, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+deletion+of+Hand2+in+enteric+neural+precursor+cells+affects+its+functions+in+neurogenesis,+neurotransmitter+specification+and+gangliogenesis,+causing+functional+aganglionosis.">Targeted deletion of Hand2 in enteric neural precursor cells affects its functions in neurogenesis, neurotransmitter specification and gangliogenesis, causing functional aganglionosis.</a> Development (Cambridge, England). 138 (21), 4789-4800 (2011).</li><li>Erickson, C. S., Lee, S. J., Barlow-Anacker, A. J., Druckenbrod, N. R., Epstein, M. 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D., Corfield, 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=Children+with+Hirschsprung's+disease+have+an+abnormal+colonic+mucus+defensive+barrier+independent+of+the+bowel+innervation+status.">Children with Hirschsprung's disease have an abnormal colonic mucus defensive barrier independent of the bowel innervation status.</a> J Pediatr Surg. 32 (8), 1206-1210 (1997).</li><li>Puig, I., Champeval, D., De Santa Barbara, P., Jaubert, F., Lyonnet, S., Larue, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+Pten+in+the+mouse+enteric+nervous+system+induces+ganglioneuromatosis+and+mimics+intestinal+pseudoobstruction.">Deletion of Pten in the mouse enteric nervous system induces ganglioneuromatosis and mimics intestinal pseudoobstruction.</a> J Clin Invest. 119 (12), 3586-3596 (2009).</li></ol>

The authors have nothing to disclose.

Our laboratory and others have shown that intestinal defects in HSCR are not restricted to the aganglionic colon but extended proximally, even into the ganglionated small intestine5,15,16. These alterations include changes in ENS neuronal density and neurotransmitter phenotype and may account for dysmotility that has been observed in patients with HSCR. We have utilized the above techniques in our efforts to understand the determinants of ENS formation. Specifically, these techniques have been employed to visu...

A functioning Enteric Nervous System (ENS), which controls motility, nutrient absorption, and local blood flow, is essential to life1. The ENS is formed by neural crest cells (NCC) that proliferate, migrate and colonize the gut, where they differentiate into ganglia containing neurons and glial cells. Hirschsprung&#8217;s Disease (HSCR, Online Mendelian Inheritance in Man), a multigeneic congenital disorder with an incidence of 1 in 4,000 live births, can be considered the prototypic disease for studying disrupted ENS formation. In HSCR, NCC fail to migrate to and colonize variable lengths of the distal hindgut2. Additionally, other common gastrointestinal (GI) developmental defects in the pediatric population, such as anorectal malformations, intestinal atresias, and motility disorders are associated with disturbances in basic ENS functions, and are likely associated with subtle, underappreciated, anatomic changes and functional changes in the ENS3-6. Therefore, techniques that allow us to understand the developmental determinants of ENS formation may shed light on the pathogenesis and potential treatment of pediatric GI tract disorders.
Following migration and colonization, NCC differentiates into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons comprise approximately 60% of enteric neurons7, and can be detected by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for the excitatory neurotransmitter acetylcholine. Historically, attempts to visualize cholinergic neurons were confounded by differing antigen specificity of antibodies directed against central nervous system (CNS) ChAT versus peripheral nervous system (PNS) ChAT8-10. However, antibodies directed against placental ChAT recognize both central and peripheral ChAT11-13, and we have recently described techniques that allow for visualization of ENS cholinergic neurons with high sensitivity earlier in development than has been achieved with ChAT reporter lines14.
Here, we present a technique for dissecting, fixing and immunostaining of the murine embryonic GI tract to visualize ENS neurotransmitter expression in neurons. For these studies, we have utilized ChAT-Cre mice mated with R26R:floxSTOP:tdTomato animals to produce ChAT-Cre;R26R:floxSTOP:tdTomato mice (defined as ChAT-Cre tdTomato throughout the manuscript). These animals were then mated with homozygous ChAT-GFP reporter mice, to obtain mice expressing both fluorescent reporters that detect ChAT expression14. These two reporter animals are on a C57BL/6J background and are commercially available (Jackson Laboratories, Bar Harbor, ME).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Oxoid</td>
			<td>BR0014G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>S2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Fisher</td>
			<td>BP9221</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher</td>
			<td>BP1605</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dishes</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence scope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine science tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tubes</td>
			<td>VWR</td>
			<td>20170-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>SouthernBiotech, Birmingham, AL</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>16004-330</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>Nikon A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Elements</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

We have previously described the generation of mice expressing both GFP and tdTomato fluorescent reporters that detect ChAT expression14. Briefly, ChAT-Cre mice were mated with R26R:floxSTOP:tdTomato animals to produce ChAT-Cre;R26R:floxSTOP:tdTomato mice (called ChAT-Cre tdTomato). These animals were then mated with homozygous ChAT-GFP reporter mice. Embryos were isolated and tissues were dissected prior to being fixed and immunostained as above, with the antibodie...

Watch this Scientific Journal Video about Immunostaining to Visualize Murine Enteric Nervous System Development at JoVE.com

Immunostaining to Visualize Murine Enteric Nervous System Development | Developmental Biology | Video

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. 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 ...

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11.3K Views.  University of Wisconsin School of Medicine and Public Health. 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.

Video: Immunostaining to Visualize Murine Enteric Nervous System Development

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila larvae, and fish, which all engage in undulatory movements (like crawling or swimming) as their primary mode of locomotion. However, a more sophisticated understanding of the individual motor neuron (MN) specification&#8212;at least in terms of informing disease therapies&#8212;demands an equally tractable system that better models the complex appendage-based locomotion schemes of vertebrates. The adult Drosophila locomotor system in charge of walking meets all of these criteria with ease, since in this model it is possible to study the specification of a small number of easily distinguished leg MNs (approximately 50 MNs per leg) both using a vast array of powerful genetic tools, and in the physiological context of an appendage-based locomotion scheme. Here we describe a protocol to visualize the leg muscle innervation in an adult fly.

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Here we describe a protocol to visualize the axonal targeting with a florescent protein in adult legs of Drosophila by fixation, mounting, imaging, and post-imaging steps.

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Immunostaining to Visualize Murine Enteric Nervous System Development

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Chapters in this video

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Technique to Target Microinjection to the Developing Xenopus Kidney

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells