Name: immunocytochemistry: human neural stem cells
Uploaded: 2007-08-24T00:00:00
Description: Watch this Scientific Journal Video about Immunocytochemistry: Human Neural Stem Cells at JoVE.com

<p>作者要感谢在儿童医院，奥兰治县研究院提供封口膜染色技术的初步指导hNSPC文化和加里Bassell博士在埃默里大学的菲利普H ·施瓦茨博士国家人类神经干细胞资源。 </p>

<p class="jove_title">第1天：准备德国盖玻片和种子细胞</p><p class="jove_content"> <em>注</em> ：德国盖玻片往往是最好培养初级的神经干细胞和神经元。我们使用下面的清洗方案，提高细胞的粘附和铺展。在一个很小的机架位置盖玻片盖玻片两侧，将允许液体访问。首先，在10分钟的1％Liquinox孵化盖玻片，其次是3用去离子水洗涤。确保没有肥皂泡保持。接下来，在1M盐酸，30分钟的孵育单用去离子水洗涤3。干盖玻片在65 ° C?...

<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>这一协议描述为hNSPCs的免疫程序，最大限度地减少了所需的抗体量，并提供可靠的的细胞染色。所描述的程序是最好的细胞内的抗原，但可以修改染色细胞表面的分子或增强细胞骨架的染色。 </p>

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		<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>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>
		</table>
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immunocytochemistry: human neural stem cells

<p class="jove_content">免疫细胞化学是一个非常强大的，相当简单的方法，确定存在的，亚细胞定位，利益，最常见的在培养细胞中的蛋白质，的抗原的相对丰度。此协议提供了一个易于遵循一系列的措施，将使研究人员能够节省小学和中学的抗体，而其染色质量高，重现性好定性和定量数据。这个协议有两个方面，以帮助保护抗体染色所需的量。其一，细胞是生长在小的，圆形盖玻片，放置在一个组织的文化板块井。盖玻片上的细胞固定后，可以删除从该板块的水井。抗体染色，与细胞的盖玻片上的封口膜的小滴的抗体溶液倒到第二件的封口膜覆盖，以防止干燥。使用这种方法，只〜25μl抗体溶液的需要为每个盖玻片（或样品）进行染色。本协议描述了人类神经干/前体细胞（hNSPCs）免疫，但可用于许多其他类型的细胞。 </p>

Watch this Scientific Journal Video about Immunocytochemistry: Human Neural Stem Cells at JoVE.com

Immunocytochemistry: Human Neural Stem Cells (Video) | JoVE

<p class="jove_content">免疫细胞化学是一个功能强大的方法来确定是否存在，亚细胞定位和兴趣在培养的细胞抗原的相对丰度。该协议提出了一个易于遵循的一系列步骤，使一个保护抗体染色。</p>

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