Preparing Adhesion Chambers (the day before the injection/dissection)
Adhesion Assay Chambers
If you have a conventional compound microscope (optical locates above the stage), it is ncecessary to make a low-height adhesion chamber as follows. If using the inverted-optics microscope, then "NUNC Lab-Tek Chambered Coverglass" can just be used,&nbsp;and skip the steps #1 - 2.
<ol>
<li>Attach one of "press-to-seal silicon insulator" on a 40 x 24mm cover slip. Firmly press the insulator to the cover slip on clean benchtop and look from the other side to make sure that the insulator is completely attached without any air-bubbles. </li>
<li>Optional: Put the adhesion chambers in a glass Petri dish with Whatman filter paper and autoclave in dry-cycle to sterilize. Cool them down to RT.</li>
</ol>
Coating the Chamber
<ol>
<li>Put a 200ml droplet of working solution of cadherin (10mg/ml) or fibronectin (20mg/ml) onto the center of an autoclaved adhesion chamber, using sigmacoated yellow tips. Incubate at RT for 4 hours. </li>
<li>Take out the cadherin or fibronectin solution and transfer to a 1.5ml tube. This solution can be reused for next two weeks. </li>
<li>Dispense 500ml of 4% BSA in 1x MBS-H to block the chamber. Incubate at RT for 1 hour. </li>
<li>Aspirate the BSA solution. Wash the chamber with 500ml of 1x MBS-H twice. After the second wash, aspirate most of MBS-H (not completely), and store in a sealed container at 4&ordm;C. This adhesion chamber is good for the next 2 - 3 days.</li>
</ol>
Dissection and Dissociation of Animal Cap Explants
<ol>
<li>Incubate the injected embryos in 0.1x MBS-H, diluted from the autoclaved 1x MBS-H, as non-autoclaved medium sometime carries little germs that can disturb the dissociated animal cap cells. </li>
<li>Make 1x Barth's agarose plates and CMF-MBS agarose plates with 60mm dishes, at least as many as the number of your samples. Pour autoclaved 1x MBS-H to 1x Barth's plates. For CMF-MBS plates, pour 10ml of CMF-MBS. </li>
<li>When the embryos become stage 8.5, start dissecting the animal caps in a 1x Barth's plate. 10 - 15 explants per samples are usually enough for an experiment. </li>
<li>Transfer the animal cap explants to a CMF-MBS plate. Incubate the explants for 45 - 60 minutes until the explants are completely dissociated. Gentle agitations in every 15 minutes will help the dissociation.</li>
</ol>
Cell Adhesion Assay
<ol>
<li>Seeding the cells: Dispense 500ml of 1x MBS-H to the adhesion chamber prepared in the steps in the sections above,&nbsp;Adhesion Assay Chambers and Coating the Chamber. Transfer the dissociated animal cap cells from the CMF-MBS plate to the adhesion chamber using P200 with a sigmacoated and chopped-off yellow tips. </li>
<li>Optional: If possible, carefully suck up the medium up to approximately 2/3 to halfway, without exposing cells to the air. Add approximately the same amount of fresh MBS-H, to make sure that the medium contains sufficient Ca2+ and Mg2+. </li>
<li>Incubate at RT for 45 - 90 minutes. Check the cells occasionally to see if they are attaching to the bottom surface. </li>
<li>Now you can take the picture of the cells under a microscope. </li>
<li>Put the adhesion chamber on a grid slide glass. Align one of the corners of the adhesion chamber to a corner of the grid slide. Count and record the number of the attached cells in several different grids. </li>
<li>Pour 1L of MBS-H in a large plastic bucket (e.g., Tupperware, etc.). Gently put the adhesion slide chamber to the bottom of the bucket. Rotate it on the orbital shaker for five minutes at low speed (50 - 60rpm). </li>
<li>Put the adhesion chamber back on the grid slide, aligning the same corner of the chamber to the same corner of the grid slide, as was done&nbsp;in step #5 of this section, Cell Adhesion Assay. Count the number of cells which remain attached on the same grid that you have counted before the washing. Calculate the percentage of cells that remain attached after the wash.</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>1x MBS-H</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Modified Barth&#x2019;s medium: 1L recipe
5.14g		NaCl
75mg		KCl
0.2g		NaHCO3
0.2g		MgSO4&#x2022;7H2O
60mg		CaCl2&#x2022;2H2O
80mg		Ca(NO3)2&#x2022;4H2O
2.38g		HEPES, free-acid
Adjust the pH to 7.4 with NaOH. Autoclave.
</td>
</tr>
<tr>
<td>CMF-MBS	</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>This is Ca2+/Mg2+-free version of MBS-H. 500ml recipe :
2.57g		NaCl
37.5mg		KCl
0.1g		NaHCO3
1.19g		HEPES, free-acid
Adjust the pH to 7.4 with NaOH. Autoclave.
</td>
</tr>
<tr>
<td>1% agarose/1x Barth&#x2019;s plates</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>as many as the number of samples</td>
</tr>
<tr>
<td>1% agarose/CMF-MBS plates</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>as many as the number of samples</td>
</tr>
<tr>
<td>E-Cadherin/Fc chimera </td>
<td>Reagent</td>
<td>Sigma</td>
<td>E-2278</td>
<td>Dissolve 0.1mg of solid E-cadherin in 100ul of 1x MBS-H containing 40% glycerol to make 100x master stock (1.0mg/ml). Store it in &#x2013;20C. Make 1/100 dilution with 1x MBS-H to make 10ug/ml working solution just before you use. You need 200ul per samples of this working solution.</td>
</tr>
<tr>
<td>Fibronectin, 0.1% solution </td>
<td>Reagent</td>
<td>Sigma</td>
<td>F-1141</td>
<td>This pre-made solution can be used as 50x stock. Make 1/50 dilution with 1x MBS-H make 20ug/ml working solution just before you use. You need 200ul per samples of this working solution.
I do NOT recommend using solid Fibronectin, because it is difficult to dissolve at 0.1% concentration to make the stock solution. Although it is more expensive, this pre-made 0.1% solution is better to obtain consistent results.
</td>
</tr>
<tr>
<td>4% BSA /MBS-H</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>BSA: molecular biology grade fraction V</td>
</tr>
<tr>
<td>Sigmacoted yellow tips </td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>No.1 cover slips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>40x24mm or 60x24mm</td>
</tr>
<tr>
<td>Press-to-seal silicon insulator</td>
<td>Tool</td>
<td>Glace Bio-Labs</td>
<td>JTR20-2.5</td>
<td>2.5mm depth x 20mm diameter </td>
</tr>
<tr>
<td>NUNC Lab-Tek Chambered Coverglass</td>
<td>Tool</td>
<td>Fisher</td>
<td>12-565-471</td>
<td>2wells/slides </td>
</tr>
<tr>
<td>Grid slide glass</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>or Finder graticule for cell counting</td>
</tr>		</tbody>
		</table>
		</div>

dissection of organizer and animal pole explants from xenopus laevis embryos and assembly of a cell adhesion assay

Watch this Scientific Journal Video about Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay at JoVE.com

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.

dissection-organizer-animal-pole-explants-from-xenopus-laevis-embryos

Research

JoVE Journal

Biology

10.6K 次观看.  University of California, Irvine (UCI). 本视频演示所使用的技术准备从非洲爪蟾胚胎，包括使用的眉刀的组织者和动物极植 - 一个专门的清扫工具的眉。也给出了组装粘附实验的协议，目前表面上的组织者或动物极细胞，适当发展的关键，关键粘附分子的存在探头。

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

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

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.

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 ...

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.

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

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 ...