Name: preparation of mouse brain tissue for immunoelectron microscopy
Uploaded: 2010-07-20T17:45:00
Description: Watch this Scientific Journal Video about Preparation of Mouse Brain Tissue for Immunoelectron Microscopy at JoVE.com

<p class="jove_content">We thank Shao-Ming Lu and Harris A. Gelbard for the use of a peristaltic pump and vibratome, as well as Karen L. Bentley and Gayle Schneider at the Electron Microscope Research Core Facility of the University of Rochester Medical Center for technical assistance. This work was funded by grants from the NIH (EY019277), Whitehall Foundation, Burroughs Wellcome Fund, and the Alfred P. Sloan Foundation to A.K.M.. M.-&Egrave;.T. is funded by a Fonds de la recherche en sant&eacute; du Qu&eacute;bec (FRSQ) postdoctoral training award.</p>

<p class="jove_title">1. Animal perfusion</p>
<ol>
     <li>On the day before perfusion, prepare:<br />
          - 2 L of phosphate buffer (PB; 100 mM, pH 7.4) and 1 L of sodium phosphate-buffered saline (PBS; 0.9% NaCl in 50 mM PB, pH 7.4) in double distilled water. Store the PBS and 1L of PB at 4&deg;C. These will be used for perfusion and pre-embedding immunocytochemistry.<br />
          - 1L of 4.0% paraformaldehyde (PFA, pH 7.4) fixative solution, which will enable the perfusion of 6 mice. For that purpose, heat up 1 L of PB under a ...

<ol>
	<li>Venable, J. H., Coggeshall, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+lead+citrate+stain+for+use+in+electron+microscopy.">A simplified lead citrate stain for use in electron microscopy.</a> <em style='font-style: italic'>J Cell Biol</em>. <b>25</b>, 407-407 (1965).</li><li>Riad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatodendritic+localization+of+5-HT1A+and+preterminal+axonal+localization+of+5-HT1B+serotonin+receptors+in+adult+rat+brain.">Somatodendritic localization of 5-HT1A and preterminal axonal localization of 5-HT1B serotonin receptors in adult rat brain.</a> <em style='font-style: italic'>J Comp Neurol</em>. <b>417</b> (2), 181-181 (2000).</li><li>Tremblay, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+EphA4+in+axon+terminals+and+dendritic+spines+of+adult+rat+hippocampus.">Localization of EphA4 in axon terminals and dendritic spines of adult rat hippocampus.</a> <em style='font-style: italic'>J Comp Neurol</em>. <b>501</b> (5), 691-691 (2007).</li><li>Tremblay, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+course+of+EphA4+cellular+and+subcellular+localization+in+the+postnatal+rat+hippocampus.">Developmental course of EphA4 cellular and subcellular localization in the postnatal rat hippocampus.</a> <em style='font-style: italic'>J Comp Neurol</em>. <b>512</b> (6), 798-798 (2009).</li><li>Bouvier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-synaptic+and+post-synaptic+localization+of+EphA4+and+EphB2+in+adult+mouse+forebrain.">Pre-synaptic and post-synaptic localization of EphA4 and EphB2 in adult mouse forebrain.</a> <em style='font-style: italic'>J Neurochem</em>. <b>106</b> (2), 682-682 (2008).</li><li>Bouvier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EphA4+is+localized+in+clathrin-coated+and+synaptic+vesicles+in+adult+mouse+brain.">EphA4 is localized in clathrin-coated and synaptic vesicles in adult mouse brain.</a> <em style='font-style: italic'>J Neurochem</em>. , (2010).</li></ol>

<p class="jove_content">Here we have described a protocol for preparation of mouse brain tissue for immunoelectron microscopy that provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection, when the procedures are rigorously performed.</p>
<p class="jove_content">Combined with TEM, this method enables to distinguish cellular elements with few distinctive features and identification criteria. In particular, the immunoperoxidase-DAB staining of Iba1 enables to identify microglial perikarya and...

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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>Sodium pentobarbital (Nembutal)</td>
<td>&nbsp;</td>
<td>Ovation Pharmaceuticals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Filter paper 315  24 cm</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>28331-081</td>
<td>&nbsp;</td>
<tr>
<td>*Acrolein purum (&ge;95%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich (Fluka)</td>
<td>01680</td>
<td>&nbsp;</td>
<tr>
<td>Sodium borohydride (&ge;98%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich (Aldrich)</td>
<td>452173</td>
<td>&nbsp;</td>
<tr>
<td>*Prilled paraformaldehyde (PFA) (95%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>441244</td>
<td>&nbsp;</td>
<tr>
<td>Normal goat serum</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
<td>005-000-121</td>
<td>&nbsp;</td>
<tr>
<td>Rabbit anti-Iba1 primary antibody</td>
<td>&nbsp;</td>
<td>Wako</td>
<td>019-19741</td>
<td>Stored at -20&deg;C, 1:1 in glycerol</td>
</tr>
<tr>
<td>Biotin-SP-conjugated AffiniPure F(ab&#x2019;)2 fragment goat anti-rabbit IgG, Fc fragment specific</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
<td>111-066-046</td>
<td>Stored at -20&deg;C, 1:1 in glycerol</td>
</tr>
<tr>
<td>Peroxidase-conjugated streptavidin</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
<td>016-030-084</td>
<td>Stored at -20&deg;C, 1:1 in glycerol</td>
</tr>
<tr>
<td>*Osmium tetroxide 4% solution</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>19150</td>
<td>Light sensitive</td>
</tr>
<tr>
<td>Propylene oxide (&ge;99%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich (Fluka)</td>
<td>82325</td>
<td>&nbsp;</td>
<tr>
<td>Polypropylene disposable beakers (50 mL)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>01-291-10</td>
<td>&nbsp;</td>
<tr>
<td>Aluminium weigh dishes (70 mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>NC9261784</td>
<td>&nbsp;</td>
<tr>
<td>Durcupan epoxy resin</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>14040</td>
<td>&nbsp;</td>
<tr>
<td>Embedding mold</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>70907</td>
<td>&nbsp;</td>
<tr>
<td>DPX mountant for histology</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich (BioChemika)</td>
<td>44581</td>
<td>&nbsp;</td>
<tr>
<td>Gelatin subbed slides</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>100241-864</td>
<td>&nbsp;</td>
<tr>
<td>ACLAR embedding films (7.8 mm)</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>50425</td>
<td>&nbsp;</td>
<tr>
<td>Capsule mold</td>
<td>&nbsp;</td>
<td>Electron microscopy Sciences</td>
<td>70150</td>
<td>&nbsp;</td>
<tr>
<td>High-performance super glue</td>
<td>&nbsp;</td>
<td>Corporate Express</td>
<td>LOC30379</td>
<td>&nbsp;</td>
<tr>
<td>Perfect loop for ultra thin sections</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>70944</td>
<td>&nbsp;</td>
<tr>
<td>Superfrost slides</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-178-277</td>
<td>&nbsp;</td>
<tr>
<td>Diamond knife ultra 45&deg; (2.5-3.5 mm)</td>
<td>&nbsp;</td>
<td>Diatome</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gelatin from cold water fish skin (~45%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich (Sigma)</td>
<td>G7765</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
			<p class="jove_content">*One should always work under a fume hood and wear nitrile gloves when handling acrolein, paraformaldehyde, and osmium and should also dispose of their waste properly.</p>		</div>
		

preparation of mouse brain tissue for immunoelectron microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

Watch this Scientific Journal Video about Preparation of Mouse Brain Tissue for Immunoelectron Microscopy at JoVE.com

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

<p class="jove_content">We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.</p>

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

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