Name: plastic embedding and sectioning of xenopus laevis embryos
Uploaded: 2007-04-29T00:00:00
Description: Watch this Scientific Journal Video about Plastic Embedding and Sectioning of Xenopus laevis Embryos at JoVE.com

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 ...

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		<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

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

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 ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

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. ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...