<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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<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

<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>

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

Research

JoVE Journal

Biology

13.5K visualizações.  University of California, Irvine (UCI). Perfis plásticos manter morfologia do tecido verdadeiro em seções de tecido fino que pode ser imunocoloração com fluorescentes anticorpos secundários, tornando este método mais útil do que seções parafina ou congelados para muitos tipos de tecido. O método para a coloração, a incorporação de plástico, e secção é demonstrada neste vídeo.