Name: immunocytochemistry: human neural stem cells
Uploaded: 2007-08-24T00:00:00
Description: Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

<p class="jove_content">The authors would like to acknowledge Dr. Philip H. Schwartz of the National Human Neural Stem Cell Resource at the Children's Hospital, Orange County Research Institute for providing hNSPC cultures and Dr. Gary Bassell at Emory University for initial instruction in the parafilm staining technique.</p>

<p class="jove_title">Day 1: Preparing German Glass Coverslips and Seeding Cells</p>
<p class="jove_content"><em>Note</em>: German glass coverslips are often preferable for culturing primary neural stem cells and neurons. We use the following cleaning protocol to improve cell adhesion and spreading. Position coverslips in a small rack that will allow liquid access to both sides of the coverslip. First, incubate coverslips in 1% Liquinox for 10 minutes, followed by 3 washes with deionized water. Make sure that no soap bubbles remain. Next, incub...

<ol>
	<li>Flanagan, L. A., Chou, J., Falet, H., Neujahr, R., Hartwig, J. H., Stossel, T. P., Filamin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamin+A,+the+Arp2/3+complex,+and+the+morphology+and+function+of+cortical+actin+filaments+in+human+melanoma+cells.">Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells.</a> <em style='font-style: italic'>J. Cell Biol</em>. <b>155</b>, 511-518 (2001).</li><li>Flanagan, L. A., Rebaza, L. M., Derzic, S., Schwartz, P. H., Monuki, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+neural+precursor+cells+by+laminin+and+integrins.">Regulation of human neural precursor cells by laminin and integrins.</a> <em style='font-style: italic'>J. Neurosci. Res</em>. <b>83</b>, 845-856 (2006).</li><li>Flanagan, L. A., Ziaeian, B., Palmer, T., Schwartz, P. H., Loring, J. F., Wesselschmidt, R. W., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+analysis+of+stem+cells.">Immunocytochemical analysis of stem cells.</a> <em style='font-style: italic'>In Human Stem Cell Manual: A Laboratory Guide</em>. , (2007).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passaging+Human+Neural+Stem+Cells.">Passaging Human Neural Stem Cells.</a> <em style='font-style: italic'>Journal of Visualized Experiments</em>. <b>7</b>, (2007).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+Human+Neural+Stem+Cells.">Counting Human Neural Stem Cells.</a> <em style='font-style: italic'>Journal of Visualized Experiments</em>. <b>7</b>, (2007).</li></ol>

<p class="jove_content">This protocol describes an immunostaining procedure for hNSPCs that minimizes the volume of antibody necessary and gives reliable cell staining.  The procedure as described is best for intracellular antigens, but can be modified to stain cell surface molecules or to enhance staining of the cytoskeleton.</p>

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>German glass (Deutsche Spiegelglas) coverslips</td>
<td>Tool</td>
<td>Carolina Biological Supply Company</td>
<td>WW-63-3029</td>
<td>12,15,18, 22, 25 mm coverslips available</td>
</tr>
<tr>
<td>Liquinox phosphate-free detergent</td>
<td>Reagent</td>
<td>Sigma</td>
<td>Z273279</td>
<td>very viscous</td>
</tr>
<tr>
<td>Poly-D-lysine hydrobromide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
<tr>
<td>Laminin, natural mouse</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>23017-015</td>
<td>&nbsp;</td>
<tr>
<td>EMEM (1X)</td>
<td>Reagent</td>
<td>Mediatech</td>
<td>MT-10-010-CV</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>24 well plate</td>
<td>Tool</td>
<td>Fisher</td>
<td>08-772-1</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Fisher</td>
<td>BP151-500</td>
<td>very viscous</td>
</tr>
<tr>
<td>Paraformaldehyde</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P6148</td>
<td>potential carcinogen, use caution when using it in powder and solution form</td>
</tr>
<tr>
<td>Bovine Serum Albumin, IgG-free</td>
<td>Reagent</td>
<td>Jackson Immunoresearch</td>
<td>001-000-161</td>
<td>&nbsp;</td>
<tr>
<td>Hoechst 33342</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>H-1399</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield</td>
<td>Reagent</td>
<td>Vector Laboratories</td>
<td>H-1000</td>
<td>viscous</td>
</tr>		</tbody>
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immunocytochemistry: human neural stem cells

<p class="jove_content">Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of an antigen of interest, most commonly a protein, in cultured cells.  This protocol presents an easy-to-follow series of steps that will enable researchers to conserve primary and secondary antibodies while getting high quality, reproducible qualitative and quantitative data out of their staining.  There are two aspects of this protocol that help to conserve the volume of antibody necessary for staining.  For one, the cells are grown on small, circular coverslips that are placed in wells of a tissue culture plate.  After fixation, the cells on coverslips can be removed from the wells of the plate. For antibody staining, the coverslip with cells is inverted onto a small drop of antibody solution on parafilm and is covered with a second piece of parafilm to prevent drying.  Using this method, only ~25  &mu;l of antibody solution is needed for each coverslip (or sample) to be stained. This protocol describes immunostaining of human neural stem/precursor cells (hNSPCs), but can be used for many other cell types.</p>

<p class="jove_content">Immunocytochemistry is a powerful method to determine the presence, subcellular localization, and relative abundance of an antigen of interest in cultured cells.  This protocol presents an easy-to-follow series of steps that will enable one to conserve antibodies and get the most out of one's staining.</p>

Immunocytochemistry: Human Neural Stem Cells

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

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