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

<p class="jove_step">Fixation</p>
<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, <strong>only </strong>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.<br /><br /></li>
<li>Aspirate most of FA and add Dent's fixative up to the top of the ...

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		<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

<p class="jove_content">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.</p>

Research

JoVE Journal

Biology

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

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, ...

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 ...

Microinjection of Xenopus Laevis Oocytes

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 <em>Xenopus laevis</em>

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 ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in <em>Xenopus laevis</em>

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 ...

Production of Transgenic <em>Xenopus laevis</em> by Restriction Enzyme Mediated Integration and Nuclear Transplantation

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 ...

Facial Transplants in <em>Xenopus laevis</em> Embryos

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. ...

Dissection of <em>Xenopus laevis</em> Neural Crest for <em>in vitro</em> Explant Culture or <em>in vivo</em> Transplantation

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 ...

Using plusTipTracker Software to Measure Microtubule Dynamics in <em>Xenopus laevis</em> Growth Cones

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 ...