Name: assessing phagocytic clearance of cell death in experimental stroke by ligatable fluorescent probes
Uploaded: 2014-05-27T13:00:00
Description: Watch this Scientific Journal Video about Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes at JoVE.com

这项研究是由赠款R01NS062842从神经混乱国家学院和健康（VVD）的行程，国家机构和补助R21 NS064403从神经混乱国家学院和中风，国家通过ARRA卫生研究院（VVD）和R21支持生物医学成像和生物工程的EB006301研究所，美国国立卫生研究院（VVD）。

该方法适用于甲醛固定，石蜡包埋的组织切片。该技术是在这里的应用程序演示实验中风大鼠。 1，对酪胺信号放大（TSA）系统组件的准备虽然该协议为基础的强化中使用的标签的最后阶段，准备用于该反应可能需要超过1个小时。因此，这是方便的开始标签之前必须进行的全港性系统评估的试剂。 <ol><li>使荧光酪胺原液。使用与TSA标记试剂盒将各小瓶?...

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	<li>Jin, R., Yang, G., Li, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+mechanisms+in+ischemic+stroke:+role+of+inflammatory+cells.">Inflammatory mechanisms in ischemic stroke: role of inflammatory cells.</a> J Leukoc Biol. 87, 779-789 (2010).</li><li>Schilling, M., Besselmann, M., M&#252;ller, M., Strecker, J. K., Ringelstein, E. B., Kiefer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predominant+phagocytic+activity+of+resident+microglia+over+hematogenous+macrophages+following+transient+focal+cerebral+ischemia:+an+investigation+using+green+fluorescent+protein+transgenic+bone+marrow+chimeric+mice.">Predominant phagocytic activity of resident microglia over hematogenous macrophages following transient focal cerebral ischemia: an investigation using green fluorescent protein transgenic bone marrow chimeric mice.</a> Exp Neurol. 196 (2), 290-297 (2005).</li><li>Faustino, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cells+contribute+to+endogenous+brain+defenses+after+acute+neonatal+focal+stroke.">Microglial cells contribute to endogenous brain defenses after acute neonatal focal stroke.</a> J Neurosci. 31 (36), 12992-13001 (2011).</li><li>Thored, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+term+accumulation+of+microglia+with+proneurogenic+phenotype+concomitant+with+persistent+neurogenesis+in+adult+subventricular+zone+after+stroke.">Long term accumulation of microglia with proneurogenic phenotype concomitant with persistent neurogenesis in adult subventricular zone after stroke.</a> Glia. 57 (8), 835-849 (2009).</li><li>Sierra, A., Abiega, O., Shahraz, A., Neumann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Janus-faced+microglia:+beneficial+and+detrimental+consequences+of+microglial+phagocytosis.">Janus-faced microglia: beneficial and detrimental consequences of microglial phagocytosis.</a> Front Cell Neurosci. 7 (6), (2013).</li><li>Samejima, K., Earnshaw, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trashing+the+genome:+Role+of+nucleases+during+apoptosis.">Trashing the genome: Role of nucleases during apoptosis.</a> Nat.Rev. Mol. Cell Biol. 6 (9), 677-688 (2005).</li><li>Minchew, C. L., Didenko, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+probes+detecting+the+phagocytic+phase+of+apoptosis:+enzyme-substrate+complexes+of+topoisomerase+and+DNA.">Fluorescent probes detecting the phagocytic phase of apoptosis: enzyme-substrate complexes of topoisomerase and DNA.</a> Molecules. 16 (6), 4599-4614 (2011).</li><li>Shuman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+classes+of+DNA+end-joining+reactions+catalyzed+by+vaccinia+topoisomerase.">Two classes of DNA end-joining reactions catalyzed by vaccinia topoisomerase.</a> Sci. Rep. 267, 16755-16758 (1992).</li><li>Barcia, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROCK/Cdc42-mediated+microglial+motility+and+gliapse+formation+lead+to+phagocytosis+of+degenerating+dopaminergic+neurons+in+vivo.">ROCK/Cdc42-mediated microglial motility and gliapse formation lead to phagocytosis of degenerating dopaminergic neurons in vivo.</a> Sci. Rep. . 2, 809 (2012).</li><li>Didenko, V. V., Ngo, H., Minchew, C. L., Boudreaux, D. J., Widmayer, M. A., Baskin, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+irreparable+ischemic+damage+in+brain+by+selective+labeling+of+double-strand+blunt-ended+DNA+breaks.">Visualization of irreparable ischemic damage in brain by selective labeling of double-strand blunt-ended DNA breaks.</a> Mol. Med. 8, 818-823 (2002).</li></ol>

在这段视频中，我们证明，在组织切片格式，如何标记吞噬细胞活跃其中透明细胞死亡局灶性脑缺血。所提出的技术是专门标签只有那些废物管理细胞已吞噬并积极消化细胞尸体的第一种方法。这使得它相对于对吞噬细胞，其不具有这种能力的其它标记的现有方法是有利的。 该方法使用吞噬活性的一般和选择性DNA为基础的标记 - 双链，钝核吞噬消化过程中废物管理细胞吞噬?...

缺血性中风引起的影响脑组织的深刻变化。它发起大量的细胞死亡，从而导致小胶质细胞来源的居民吞噬细胞迅速激活。它也由各种类型的血源性专业吞噬细胞包括中性粒细胞，巨噬细胞，树突状细胞和肥大细胞1,2衬托缺血性脑浸润。 目前仍有争论，这是否反应缺血性损伤起到积极的或消极的作用。虽然吞噬中风后，因为它清除死细胞和抑制炎症可以是有益的，它也产生有毒的活性氧自由基影响神经元的存活和加剧组织损伤1-5。 虽然几种类型的吞噬细胞浸润缺血性脑，不是所有的人都被吞噬细胞的尸体和再生过程1-3结算方式参与废物管理。此CReates选择性识别废物管理的细胞，进行细胞死亡在中风的吞噬细胞清除的要求。当成像这样的活性废物管理信元，它也是重要的回答他们是如何高效率地降低了吞噬细胞尸体的问题。在吞噬死亡细胞的DNA的有效和完全降解非常重要，因为它可以防止自身免疫和病理核材料6的释放。 在这里，我们提出了使用特定的DNA断裂为主动吞噬细胞的新标志物探针。这些签名里面休息职能废物管理细胞吞噬细胞核破裂过程中独家生产。因此，探针选择性标记，只有那些吞噬细胞吞噬，并积极消化细胞尸体。他们还允许观察吞噬后的强度和DNA分解完成。该探针是有用的在强度和effici的评价ENCY吞噬细胞清除的。 新探针依赖于非蛋白质为基础的标记，因此可以特别有利的行程，其中广泛的缺血性损伤可破坏细胞形态或耗尽基于蛋白质标志物，特别是内芯缺血区的研究。 该技术的原理是在图1，图中示出了由酶牛痘拓扑异构酶（VACC TOPO）连接发夹形的寡核苷酸探针 到5&#39;OH DNA末端的DNA消化7时溶酶体的DNase II产生。

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			<td height="34" style="height:34px;width:312px;">Name of the reagent or material</td>
			<td style="width:99px;">Company</td>
			<td style="width:157px;">Catalogue number</td>
			<td style="width:129px;">Comments (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td>JT Baker</td>
			<td>9490-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td>Roche</td>
			<td>3115879001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS Buffer</td>
			<td>Invitrogen</td>
			<td>AM9624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vaccinia Topoisomerase I</td>
			<td colspan="2">Vivid Technologies</td>
			<td>5 pmol/&#956;L stock&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">12-Base Overhang Suicide Oligonucleotide</td>
			<td>IDT DNA</td>
			<td></td>
			<td>5&#8217;-AAG GGA CCT GCB GCA GGT CCC TTG ATA CGA TTC TA -3&#8217; ; B &#8211; Biotin-dT; 100 pmol/&#956;L stock in ddH2O&#160;&#160;</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Adapter Oligonucleotide</td>
			<td>IDT DNA</td>
			<td></td>
			<td>5&#8217;-TAG AAT CGT ATC-3&#8217;; 100 pmol/&#956;L stock in ddH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 M Tris-HCl, PH 7.4</td>
			<td>Lonza</td>
			<td>51237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TSA, Fluorescein System&#160;</td>
			<td>PerkinElmer&#160;</td>
			<td>NEL701A001KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 M NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO&#160;</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20&#160;</td>
			<td>Sigma</td>
			<td>P9416-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Coverslips</td>
			<td></td>
			<td></td>
			<td style="width:129px;">22x22mm or 22x40mm glass or plastic coverslips. Plastic coverslips are preferable during the reaction, as they are easier to remove from the section.</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;">Rat Brain Sections</td>
			<td></td>
			<td></td>
			<td style="width:129px;">Rat brain taken 48 hours after reproduction of ischemic stroke as described in 11. 5-6&#956;m-thick sections cut from paraformaldehyde-fixed, paraffin-embedded tissue blocks. Use charged and precleaned slides.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital Video camera and software</td>
			<td>Micromax</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Software</td>
			<td>Metamorph</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing phagocytic clearance of cell death in experimental stroke by ligatable fluorescent probes

我们描述局灶性脑缺血的吞噬细胞清除的可视化一个新的组织化学方法。该方法允许消除死细胞的行程中任何细胞谱系的废物管理吞噬细胞的研究。虽然不同的起源，有能力吞噬了无数的细胞存在于脑缺血，其中只有一部分积极吞噬和消化细胞的尸体。选择性可视化，量化和这种积极的吞噬废物管理的分析是评估大脑反应缺血帮助。高效的细胞死亡的间隙是由缺血性脑损伤的恢复很重要，因为它开辟了道路，为后续的再生过程。未能清理尸体会导致因使用非降解的DNA和蛋白质毒性反应。所描述的过程使用的荧光探针由病毒拓扑异构酶选择性地连接到吞噬过程中所有的吞噬细胞产生的特征DNA断裂和细胞消化缺血不可逆的损坏。该方法用于脑反应缺血性损伤的调查的新工具。

该技术用于标记活性废物管理吞噬细胞的原理示于图1的示意图显示出，在三个步骤中检测所得。第一步骤包括探测激活（ 图1A）;在第二步骤中被激活的探测器被连接到在组织切片上（ 图1C）的特定DNA断裂;第三步骤包括荧光信号放大（ 图1D）。在一个更详细的解释，检测去如下： 1，探头激活 。之前，将连接反?...

Watch this Scientific Journal Video about Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes at JoVE.com

Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

我们提出了选择性的一个新的荧光技术在主动吞噬细胞原位标记，它明确了在中风细胞的尸体。该方法对于评估脑反应缺血重要的，因为目前在缺血性脑吞噬细胞的只有一小部分参与细胞死亡的间隙。

评估细胞死亡的吞噬细胞清除由Ligatable荧光探针试验行程

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination ...

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