Name: three-dimensional confocal analysis of microglia/macrophage markers of polarization in experimental brain injury
Uploaded: 2013-09-04T13:00:00
Description: Watch this Scientific Journal Video about Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury at JoVE.com

Stefano Fumagalli is a fellow of the Monzino Foundation.

1。免疫荧光下面的协议是从心脏灌注小鼠（20毫升的PBS，0.1摩尔/升，pH为7.4，随后加入50毫升PBS中冷却的多聚甲醛中4％）中获得冠状脑冷冻切片进行。灌注后，大脑小心移除，并转移到30％蔗糖的PBS中于4℃过夜冷冻保护。 45℃下进行3分钟被密封到小瓶中并储存在-70℃直至使用前 - 大脑，然后迅速通过浸没在冷冻中的异戊烷。冠状脑冷冻切片（20微米）被连续切割，并进行免疫?...

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我们在座的免疫为基础的协议，其次是三维共聚焦分析作为一个强大的方法，调查国产化的M / M型标志物和共表达到缺血区域（更详细的分析见文献6）。此方法结合了M / M活化与三维共焦成像相关的标记物的特异性染色。抗体，血清和fluorconjugated工作稀释度的微调提供了最佳的信号被调查指标的信噪比。图像处理以获得三维渲染允许在细胞群标志物表达的定义，使得所获取的图像表示的M /...

急性脑损伤，小胶质细胞迅速激活并发生剧烈的形态和表型的变化1-3。这种内在的反应相关联招聘血液出生的巨噬细胞迁移到损伤脑实质4,5。小胶质细胞和巨噬细胞的抗原性是无法区分的作用（以下称为M / M）在脑损伤仍有争议。越来越多的研究表明，与什么所述外围巨噬细胞，小胶质细胞和脑招募巨噬细胞可以假定不同的表型，其极端对应于经典的促炎性毒性（M1）或抗炎保护（M2）的表型。在不同的激活状态，包括亲或抗炎因子分泌，神经营养分子和溶酶体活性释放的特征的表型标志物的特定图案，其表达依赖于时间周围环境的演变。这些M / M表型在脑损伤的特征仍然是一知半解。我们使用PMCAO的一套行之有效的小鼠模型来分析的M / M表达和进化中风后。这里提出了一种基于免疫的协议旨在让洞察特定的M / M型标志，其本地化和细胞共同表达在缺血区的外观。我们研究了相关的不同的激活状态，或表型的分子数，即细胞CD11b，白细胞和M / M激活/招聘6-8的一种广泛使用的标记表示的表面标志物CD68溶酶体6,7和的Ym1分泌的标志蛋白替代性活化（M2）巨噬细胞表达和相关的回收和功能恢复9-10。 当两个标记都表达了相同的细胞，但在不同亚细胞区室，独自一人共定位可能不会有太大的INFormative。在这种情况下，可以通过使用单一的平面视图和三维效果进行共表达的分析。在这里，我们描述了一个协议来获取标记共表达的透彻三维分析。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		 </tr>
		 <tr>
			<td>Rat Anti-mouse CD11b</td>
			<td>Kindly provided by Dr. A. Doni, Istituto Clinico Humanitas, Milan, Italy</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rat Anti-mouse CD68 </td>
			<td>AbD Serotec</td>
			<td>MCA 1957</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rabbit Anti-mouse Ym1 </td>
			<td>Stem Cell Technologies</td>
			<td>1404</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Hoechst 33342</td>
			<td>Life technologies</td>
			<td>H21492</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mouse Anti-Neural Nuclei (NeuN)</td>
			<td>CHEMICON</td>
			<td>MAB377</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Biotinilated Goat Anti-Rat antibody</td>
			<td>Jackson Immuno Research</td>
			<td>112-065-143</td>
			<td></td>
		 </tr>
		 <tr>
			<td>TSA Cyanine 5 System </td>
			<td>Perkin Elmer</td>
			<td>NEL705A001KT</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Prolong Gold </td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti-rat alexa 546</td>
			<td>Invitrogen</td>
			<td>A-11081</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti mouse Alexa 488</td>
			<td>Invitrogen</td>
			<td>A-21121</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti-rabbit Alexa 594</td>
			<td>Invitrogen</td>
			<td>A-11037</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Normal Goat Serum</td>
			<td>Vectors Laboratories</td>
			<td>S-1000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Tritin X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma</td>
			<td>P4417-100</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>Cryostat CM1850</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Olympus IX81 confocal microscope </td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>AnalySIS software</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Imaris software 5.0</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Photoshop cs2</td>
			<td>Adobe Systems</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software packages GraphPad Prism version 4.0</td>
			<td>GraphPad Software Inc.</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

three-dimensional confocal analysis of microglia/macrophage markers of polarization in experimental brain injury

脑卒中小胶质细胞/巨噬细胞后（M / M）进行快速活化，包括新颖的表面抗原和生产的建立和维持炎症反应介质的表达显着形态学和表型的改变。新的证据表明，M / M是高塑性细胞急性脑损伤后，可以假定经典的促炎症（M1）或替代消炎（M2）的激活。但是M / M型标志物表达，他们的共存和时间演化的脑损伤的完整的特征是人仍下落不明。 免疫荧光协议专门染色的M / M激活相关标志物可在缺血性脑进行。在这里，我们提出了免疫为基础的协议，其次是三维共聚焦分析，作为一个强大的方法，调查本地化和共表达M / M型标志，如图案细胞CD11b，CD68，的Ym1，在小鼠局灶性脑缺血引起的大脑中动脉（PMCAO）的永久性闭塞模型。污渍区域的二维分析表明，每个标记是关联到一个定义的M / M的形态，并具有一个给定的定位中的缺血性损伤。 M / M的表型标记物的共表达模式可以通过在局部缺血区域的三维共聚焦成像来评估。图像可以在一个确定的容积被收购（10μm的z轴和一个0.23微米的步长大小，对应于180×135×10微米的体积）与连续扫描模式，以尽量减少渗色的效果，并避免波长重叠。图像，然后处理由软件Imaris里的手段来获得三维渲染。三维效果图实视图允许在集群细胞标志物表达的定义。我们表明，M / M具有分化向多个表型的能力，这取决于在病变部位和Ti的位置我受伤后。

当标记协议和共焦的采集进行入缺血区得到的结果的一个例子示于图1A和1B。获得的图像的二维视图显示，在缺血后24小时（A），溶酶体标记CD68（绿色）表示肥厚ameboid CD11b的细胞（红色）出现在缺血中心。在边境区（B）的CD11b阳性细胞显示圆形胞体和积极的CD68分枝过程。 Z轴投影（A和B的右侧和底部）允许的记录是否在?...

Watch this Scientific Journal Video about Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury at JoVE.com

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

一种方式来获得新的见解的大脑炎症反应的复杂性呈现。我们描述了免疫为基础的协议，其次是三维共聚焦分析，以探讨局灶性脑缺血小鼠模型共表达小胶质细胞/巨噬细胞表型标志物的图案。

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