Fixation
<ol>
<li>Transfer whole embryos or dissected explants into MEMFA or 3.7% FA+PBS in a small glass scintillation vial (Fisher cat# 03-339-25B). Incubate vial on the nutator at RT, only for 2 hours, and not more than that. Do not&nbsp;leave the embryo in FA for more than 2 hours. Longer incubation in FA will make the embryonic tissue harder and result in a poor penetration of antibodies in the detection process. </li>
<li>Aspirate most of FA and add Dent's fixative up to the top of the vial (~ 4.5ml). Gently invert the vial for a few times to wash the embryos in Dent's, exchange with another 4.5ml of fresh Dent's, and incubate it in -20&ordm;C for at least 48 hours. Overnight is NOT enough. This step makes the embryos more permeable for the antibody. Embryos can be kept in this condition for several weeks.</li>
</ol>
Immunofluorescence
<ol>
<li>Rehydrate the specimen. Incubate it in a graded series of methanol, as follows: 
<table style="width: 171px; height: 119px;" border="0" cellpadding="3">
<tbody>
<tr>
<td>75% MeOH/H2O</td>
<td>10min</td>
</tr>
<tr>
<td>50% MeOH/PBS</td>
<td>10min</td>
</tr>
<tr>
<td>25% MeOH/PBS</td>
<td>10min</td>
</tr>
<tr>
<td>PBS</td>
<td>10min</td>
</tr>
<tr>
<td>PBS</td>
<td>10min</td>
</tr>
</tbody>
</table>
 </li>
<li>Make 1.5% agarose/PBS plates with 60mm plastic dishes. Pour PBS on top of the agarose after it has become solid. </li>
<li>Hemi-sectioning of the embryos: Put the embryos in a 1.5% agarose/PBS plate. Grab an embryo with a pair of forceps and cut it in half through the center of the embryo using a thin razor blade. Choose the ones that you have successfully cut in half with a straight cutting plane. <img src="/files/ftp_upload/old/188_2008_7_10_3/188_figure-01.jpg" alt="Figure 1" width="591" height="193" /> </li>
<li>Transfer the bisected embryos into a glass scintillation vial with PBT. Exchange with 4.5ml of 20% goat serum+PBT. Incubate at RT for 2 hours on a nutator (Blocking). </li>
<li>Prepare an appropriate dilution of the primary antibody in PBT. 0.5ml of this per samples is needed. For instance, I used 1/200 dilution of rabbit anti-C-cadherin pAb to study the subcellular localization of C-cadherin in 5mm plastic sections. </li>
<li>Aspirate the blocking solution and add 0.5ml of the diluted primary antibody. Put the vials in stand-up position on a Styrofoam tube holder. Incubate overnight at 4&ordm;C on a nutator. </li>
<li>Remove the diluted primary antibody (this diluted primary antibody can be saved and reused in the next 1-2 weeks). Wash with 4.5ml of PBT for 4 times (40-60 minutes each) at RT. </li>
<li>Exchange with 0.5ml of 1/100 dilution of Biotin-conjugated secondary antibody in PBT. Incubate overnight at 4&ordm;C on a nutator. Cover the vials with aluminum foil to protect biotin from light. </li>
<li>From this point, care should be taken to protect the sample from light, i.e., keep the vials under some cover or aluminum foil. </li>
<li>Remove the Biotin-secondary antibody (this can be also reused in the next 1-2 weeks). Wash with or 4.5ml of PBT for 4 times (40-60 minutes each) at RT. </li>
<li>Exchange with 0.5ml of 1/200 dilution of fluorescently-labeled (FITC, Texas-red, etc.) Streptavidin in PBT. Incubate overnight at 4&ordm;C on a nutator. </li>
<li>Remove the streptavidin (this can be reused, too). Wash with 4.5ml of PBT for 3 times (15 - 30 minutes each) at RT. </li>
<li>Fix the embryos in 4.5ml of 3.7% FA+PBS for 30 minutes at RT. </li>
<li>Wash with 4.5ml of PBS twice. </li>
<li>Dehydrate the specimen. Incubate it in a graded series of ethanol, as follows: 
<table style="width: 172px; height: 80px;" border="0">
<tbody>
<tr>
<td>30% EtOH/H2O&nbsp;</td>
<td>10 min</td>
</tr>
<tr>
<td>50% EtOH/H2O</td>
<td>10 min</td>
</tr>
<tr>
<td>75% EtOH/H2O</td>
<td>10 min</td>
</tr>
<tr>
<td>100% EtOH</td>
<td>10 min</td>
</tr>
</tbody>
</table>
 </li>
<li>The specimen can be stored in 100% EtOH at 4&ordm;C with a cover to protect it from light.</li>
</ol>
Infiltration
<ol>
<li>Make Technovit 7100 infiltration mix. I usually take 15ml of the base liquid in a 50ml conical tube, add 150mg of hardener I and mix it by rocking on a nutator for 15 minutes. This infiltration mix can be kept at 4&deg;C for up to one month. </li>
<li>Infiltrate the specimen with the infiltration mix by exchanging with a graded series of Technovit infiltration mix/EtOH combo, as follows: <ol>
<li>50% Technovit/EtOH -&nbsp;1 hour </li>
<li>100% Technovit -&nbsp;1 hour </li>
<li>100% Technovit - overnight </li>
</ol></li>
<li>The specimen can be stored in the 100% infiltration mix for up to one month at 4&ordm;C. Put a cover on the samples to protect&nbsp;them from light.</li>
</ol>
Embedding
<ol>
<li>Make Technovit 7100 embedding mix: Take 0.75ml of the infiltration mix in a 1.5ml tube and add 50ml of hardener II. Mix it by inverting the tube several times. </li>
<li>Using a plastic transfer pipette, take one specimen infiltrated overnight into 0.5ml thin wall PCR tube. Remove most of the supernatant. </li>
<li>Add 0.25ml of the embedding mix made in step #1 of this section, "Embedding",&nbsp;to the specimen. Orient the bisected embryo to make the dissection plane facing to the bottom of the tube, by gently nudging with a dull-tip dissection needle (wooden-handle dissection needle). </li>
<li>Close the cap and incubate the tube in a dark place for approximately four hours to allow the plastic polymerize. </li>
<li>Make Technovit 3040 mix. Take 1 gram of the powder to a weighing dish, and add 0.5ml of the liquid. Immediately, mix powder and liquid using a bluetip. Pour on top of the hardened spacimen. This yellow Technovit 3040 will prevent the entire plastic block from coming out from the tube.</li>
</ol>
Sectioning
<ol>
<li>Wipe Histoknife H with xylene (EtOH is often not good enough) and set it on your microtome. Angle the blade at approximately 45&deg;. Wipe the stage area in front of the blade with xylene, too, because the cleanness of this area is critical to the quality of sectioned slices. </li>
<li>Cut the side of the tube and peel the tip off using a cardboard knife. <img src="/files/ftp_upload/old/188_2008_7_10_5/188_figure-02.jpg" alt="Figure 2" width="818" height="243" /> </li>
<li>Set the specimen into the chuck holder of the microtome. </li>
<li>Attach a "press-to-seal" silicon insulator on a slide glass to make a mounting chamber for section slices. Firmly press the insulator to the slide on clean benchtop and look from the other side to make sure that the insulator is completely attached without any air-bubbles. Pour clean and warm dH2O (~ 40&deg;C) to the pool on the slide using a Pasteur pipette, up to the level where it can be seen that the water surface becomes flat, not concave or convex in shape. </li>
<li>Start making section slices. For immunofluorescence studies, I make 5 mm section slices. For in situ hybridization samples, the thickness of the slices can be adjusted in the range of ~ 3 - 8 mm, depending on the purpose and the intensities of the signals. </li>
<li>Carry the section slices from the stage in front of the blade to the mounting chamber with water, using a small paintbrush. Hold the paintbrush with a slice on top of the water pool and drop the slice down to the water by knocking it with a dull-tip dissection needle. The slice will expand into a circular shape as soon as it hits the surface of the water. </li>
<li>When most of the water surface area of the mounting chamber is filled with section slices, suck up almost half of the water using a Pasteur pipette. Incubate the slide on a slide warmer overnight to make the sample completely dry. </li>
<li>Counter-stain with 1ml of DAPI (0.1mg/ml in TE) for five minutes. Aspirate DAPI and dry the sample. </li>
<li>Remove the silicone rubber insulator from the slide. Now pictures can be taken of the sample. Store the sample at&nbsp;4&deg;C, protected from light.</li>
</ol>

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>PBS</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1L recipe
8.0g		NaCl
0.2g		KCl
2.68g		Na2HPO4&#183;7H2O
0.2g		KH2PO4
The pH is supposed to be 7.4. Adjust the volume to 1L. Autoclave.
</td>
</tr>
<tr>
<td>PBT</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Add 0.5ml of Triton X-100 and 1.0g of BSA (Molecular Biology grade, fraction V is good [>99%], but don't use crude 97% Albumin) to 500ml of autoclaved PBS. Filtrate through 0.2mm filter cup. Store it in 4&#186;C.</td>
</tr>
<tr>
<td>&#183;	Dent&#x2019;s fixative</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Mix 40ml of methanol and 10ml of DMSO. Keep it in &#x2013;20&#176;C.</td>
</tr>
<tr>
<td>3.7% FA+PBS</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Mix 37% formaldehyde and PBS at 1:9 ratios. Make it fresh at each time and discard the left over.</td>
</tr>
<tr>
<td>Biotinylated Anti-Mouse IgG (H+L), made in goat</td>
<td>Ab</td>
<td>Vector Laboratories</td>
<td>BA-9200</td>
<td>&nbsp;</td>
<tr>
<td>Biotinylated Anti-Rabbit IgG (H+L), made in goat</td>
<td>Ab</td>
<td>Vector Laboratories</td>
<td>BA-1000</td>
<td>&nbsp;</td>
<tr>
<td>IG'S HUADS BIOTIN GTXRBT</td>
<td>Ab</td>
<td>Invitrogen</td>
<td>ALI3409</td>
<td>BioSource&#x2122;</td>
</tr>
<tr>
<td>IgG (g), Goat Anti-Mouse</td>
<td>Ab</td>
<td>Invitrogen</td>
<td>M30115</td>
<td>CALTAG&#x2122;</td>
</tr>
<tr>
<td>Fluorescent streptavidin kit</td>
<td>Reagent</td>
<td>Vector Laboratories</td>
<td>SA-1200</td>
<td>Contains streptavidin labeled with three different fluorophores (FITC, Texas Red and AMCA) suitable for this procedure.</td>
</tr>
<tr>
<td>20% goat serum/PBT</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>This will be used as a blocking solution.</td>
</tr>
<tr>
<td>Small paintbrush</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dull-tip dissection needle</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Kulzer Technovit 7100 GMA , 3040</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>7100 GMA , 3040</td>
<td>glycol methacrylate</td>
</tr>
<tr>
<td>Kulzer Tungsten Histoknife H</td>
<td>Tool</td>
<td>Energy Beam Sciences</td>
<td>H1310</td>
<td>&nbsp;</td>
<tr>
<td>75% MeOH/H2O</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50% MeOH/PBS</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>25% MeOH/PBS</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>30% EtOH/H2O</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50% EtOH/H2O</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>75% EtOH/H2O</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>100% EtOH</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Small glass scintillation vials</td>
<td>Tool</td>
<td>Fisher</td>
<td>03-339-25B</td>
<td>&nbsp;</td>
<tr>
<td>0.5ml Thin-wall PCR tubes</td>
<td>Tool</td>
<td>Molecular BioProducts</td>
<td>3430</td>
<td>&nbsp;</td>
<tr>
<td>Press-to-Seal Silicone Rubber Insulators</td>
<td>Tool</td>
<td>Grace Bio-Labs</td>
<td>JTR1L-2.5</td>
<td>32mm L x 19mm W, 2.5mm D </td>
</tr>
<tr>
<td>Thin razor blade</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5% Agarose in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
			Primary antibody against your antigen or epitope tags: Make an appropriate dilution in PBT.
Biotin-conjugated secondary antibody: various vendors sell biotin-conjugated antibodies.
		</div>

plastic embedding and sectioning of xenopus laevis embryos

Watch this Scientific Journal Video about Plastic Embedding and Sectioning of Xenopus laevis Embryos at JoVE.com

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

plastic-embedding-and-sectioning-of-xenopus-laevis-embryos

Research

JoVE Journal

Biology

13.5K Views.  University of California, Irvine (UCI). أقسام البلاستيك الحفاظ على النسيج الحقيقي التشكل في الفروع رقيقة من الأنسجة التي يمكن immunostained مع الأجسام المضادة الثانوية الفلورية ، مما يجعل هذا الأسلوب أكثر فائدة من الفروع جزءا لا يتجزأ من البارافين أو المجمدة لكثير من أنواع الأنسجة. ويتجلى أسلوب التلوين ، ا...

Video: Plastic Embedding and Sectioning of Xenopus laevis Embryos

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Developmental Biology

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 &#176;C. 
Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

Here, we describe a method to transplant and identify human cell spheroids into chick embryos. This xenograft model uses the embryonic microenvironment as a source of instructive signals to assay cell migration, differentiation, and tropism and is especially suited for the study of primary and/or heterogeneous cell populations.

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Neuroscience

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.