We thank Dr. Roger Tsien for providing us with the miniSOG constructs. Dr. Xuejun Sun for help with the TEM. Lisa Lem, and Peter Shipple from the Cross Cancer Institute for supplying oxygen. Hilmar Strickfaden holds a postdoctoral fellowship by the Alberta Cancer foundation and was supported by the Bayrische Forschungsallianz. This work was supported by grants from the Canadian Institutes of Health Research and Alberta Cancer Foundation.

1.代miniSOG细胞系<ol><li>生长的U2OS（人骨肉瘤）细胞在含2ml即补充有10％胎牛血清（FBS），在37℃下在湿润的培养箱中5％的CO 2低葡萄糖Dulbecco改良的Eagle培养基（DMEM）的35mm培养皿气氛。 </li><li>转染的细胞时，它们是80％汇合通过脂质转染法，用纯化的转染质量（A269 / 280比率1.8-2.0）包含miniSOG和mCherry序列质粒构建根据转染试剂19的制造商的协议标签修复蛋白。该miniSOG表达质粒可以从钱学森实验室订购<a href="http://www.tsienlab.ucsd.edu/Samples.htm">:http://www.tsienlab.ucsd.edu/Samples.htm</a> </li><li>排序的细胞通过荧光活化细胞分选后两天转染表达重组蛋白与miniSOG和mCherry标记。收集细胞用，以避免被过度表达20个细胞中间荧光强度。 </li><li>培养上在10ml的DMEM培养基是补充有10％FBS和选择剂的G418（遗传霉素）的0.6微克/毫升10cm皿的阳性细胞。 </li><li>后可见菌落都出现在平板上，画面mCherry阳性菌落使用倒置荧光显微镜和绘制圆周围（上板的底部）具有永久性标记。使用低倍率的空气物镜（ 如 10倍），并选择使用无菌技术克隆通过仔细刮殖民地断，慢慢地吸吮他们进入无菌1000微升的枪头。 </li><li>扩展所选择的菌落，并利用在步骤1.1补充有10％二甲亚砜中描述的生长培养基冻结其中某些部分向下。通过他们成长在无菌18×18 平方毫米盖玻片，并将其暴露于测试细胞DRF形成辐射2戈瑞。经照射的细胞稳定表达miniSOG mCherry标记的修复蛋白必须显示双链断裂的典型焦图案特性时使用的过滤器设置用于成像mCherry用荧光显微镜显现。 </li></ol> 2.细胞培养<ol><li>培养U2OS细胞稳定表达miniSOG和mCherry在步骤1.1中概述的条件下标记修复蛋白。细胞生长至80％汇合在含2ml DMEM的35毫米直径的玻璃底菜。确保，重视盖玻片到塑料和所使用的塑料的胶水是耐水性和醇溶液和耐使用的树脂！ </li><li>之前进行的实验中，概述了大约1mm 2，其中包含的细胞通过使用荧光显微镜来识别感兴趣的区域，用无菌金刚石笔刮擦表达异位蛋白质的小区域。或者使用玻璃底菜牛逼帽子包含与内置于盖玻片预蚀刻的网格系统盖玻片。 注意:如果使用的是高品质的相关显微镜相结合ESI和荧光显微图片21，确保盖玻片厚度与油浸目标一致。它应具有的厚度号1.5（0.16-0.18毫米）。选择一个面积接近盘的中心区域进行审查的TEM，以避免与附近的边缘可能发生的树脂的聚合（见点4.10-4.13）。 </li></ol> 3. DNA损伤诱导<ol><li>照射细胞用γ辐射，可使用radiomimetic药物，或诱导由激光微照射的伤害上的共焦显微镜（例如，如在18,22或23中所述 ）。 </li><li>在这个例子中，使用405nm的溶胶照射细胞用γ照射的2和6戈瑞铯-137辐射源或激光器microirradiate共聚焦显微镜的致敏细胞20分钟，用0.​​5微克/毫升赫斯特之后的ID激光器。 </li></ol> 4.样品制备<ol><li>固定细胞用1ml的4％多聚甲醛小心在0.1M二甲胂酸钠缓冲液或者在0.1M磷酸盐缓冲液pH 7.4，在室温30分钟在黑暗中。 注意:多聚甲醛和砷酸钠是有毒的！戴上手套和处置有毒物质充分。 </li><li>用4ml的0.1M二甲胂酸钠缓冲液pH 7.4或磷酸盐缓冲液pH 7.4洗涤细胞两次。 </li><li>对待固定的细胞用2ml的50mM甘氨酸，5mM的氨基三唑和在0.1M二甲胂酸钠缓冲液或0.1M磷酸缓冲液（pH7.4）中的10mM氰化钾小心（检查pH值！）处理30分钟，以封闭未反应醛基和以抑制由血红素基团所产生的活性氧，从而可以提高的背景。 注意:氰化钾是剧毒！ W¯¯耳手套，工作引擎盖下和处置有毒物质充分。该反应对pH非常敏感，因此，重要的是将pH核实和准确。 </li><li>记录被在上一个倒置荧光显微镜步骤2.2在二维或三维中选择，如果相关显微术所需的区域。 </li><li>通过将10毫克的二氨基联苯胺盐酸盐片剂到2ml微量离心管中制备一1mg / ml的二氨基联苯胺盐酸小心溶液。添加980微升蒸馏水H 2 O的和20μl浓盐酸（11.65米）的并混合直至溶液为浅棕色并且是半透明的。稀释此溶液在0.1M磷酸盐或0.1M的二甲胂酸钠缓冲液中至1毫克/毫升的最终浓度，同时调整用氢氧化钠将pH值至pH 7.0和7.6之间（pH为7.4，推荐）。 注意:二氨基联苯胺是有毒的！戴上手套和处置有毒物质充分。 </li><li>对于光氧化河E放置用2ml的1毫克/毫升二氨基联苯胺盐酸盐在0.1M二甲胂酸钠缓冲液或磷酸盐缓冲液的缓冲液中。避光该溶液冷却下来在冰上4℃下 ，以提高氧的溶解度。通过该溶液鼓泡氧气饱和与氧的溶液（使用插入到溶液中并连接到一个氧气瓶的软管）。再次检查pH值，并在必要时进行调整。 注:它是用于光氧化反应的pH为7.0和pH 7.6之间至关重要。 </li><li>支座与固定的细胞的培养皿小心地配备了40倍的油浸物镜透镜倒置荧光显微镜，并将其移动到感兴趣的区域。通过使用滤镜立方体的GFP或CFP激发与蓝光miniSOG标签。 MiniSOG有一个最大激发在448 nm的肩膀，在473±10。 </li><li>即使在绿色荧光miniSOG公顷继续与照明š完全消失，直到聚合二氨基联苯胺的棕色光氧化产物出现在所传送的光信道。当氧化产物是可见的，在大多数的细胞，关闭荧光照明，停止光氧化。 注:光氧化最多可能需要取决于物镜，滤波器的传输波长和效率，以及照明源数分钟。 </li><li>后缀的细胞用1ml的2％戊二醛的0.1M二甲胂酸钠pH 7.4的缓冲液或磷酸盐缓冲液pH 7.4 30分钟。 </li><li>固定细胞与0.1％-0.5％的四氧化锇将膜20分钟在0.1M二甲胂酸钠缓冲液pH 7.4或0.1M磷酸盐缓冲液pH 7.4。 注:建议使用来稳定该膜所需的四氧化锇的可能的最低浓度。由于聚合的DAB析出物具有高密度的氮气，它可以直接用ESI，强烈的被检测锇染色是没有必要的，以确定所述miniSOG标记蛋白，可能是有害的ESI。四氧化锇很毒！在通风橱中操作和处置有毒废物充分。 </li><li>通过乙醇系列使用30％，50％，70％，90％，98％的100％乙醇的步骤（每次5分钟）脱水，所述细胞。接着，孵育细胞以1:1混合100％乙醇和丙烯酸树脂（LR白），并把该盘到振荡器上4小时孵育细胞至少在100另外4小时前，以促进渗透到细胞％丙烯酸树脂（LR白）。 </li><li>为了使树脂聚合，切断标记的2 ml离心管的盖用​​刀片和涂层与丙烯酸树脂加速器边缘。确保该加速器仅覆盖轮缘和不流入管内。随后，小心地充满管大约三分之二与使用巴氏吸管的LR白树脂。请注意，该树脂不会进入接触机智H于轮辋的加速器！ </li><li>删除渗入细胞在培养皿的树脂和颠倒放置皿到垂直竖立的离心管，使得该管的宽度几乎完全填满了盘的玻璃覆盖的观察窗。 </li><li>等待1至2分钟的加速器以密封管和盖玻片，然后反转管，使得在管中的树脂现已覆盖细胞。 </li><li>放置与管盘放入烘箱，在60℃，并固化它12小时。 </li><li>当块被治愈，删除与从烘箱所附树脂填充的离心管盘和分离的离心管从盘。促进在液氮和热水通过冷冻 - 解冻循环的分离。 </li><li>丢弃玻璃底培养皿，并仔细剖开的离心管，用刀片以除去该块。 </li><li>标签永久性标记块。 </li><li>使用刀片为三M中的嵌段，使得只是包含先前标记与金刚石或钨笔的区域（或含有与感兴趣的细胞中的区域）中的1毫米2区保持。 </li><li>安装块在超微，具有修边刀修剪块切成约50nm的超薄切片，使用金刚石刀。 </li><li>拿起高传输300目网格的部分。这些网格有非常小的尘棒，从而覆盖更少细胞中所关注的区域。 </li><li>涂层，使用约0.2-0.4纳米碳的碳涂布机，以帮助TEM的电子束下稳定它们的网格的部分。 </li></ol> 5.电子显微镜<ol><li>加载网格成配备有能量过滤器的TEM。调整显微镜并根据制造商的说明，将能量过滤后，检查对切片中的细胞低倍率模式（通常传输），并比较它们与荧光数据在适当的时候（在步骤4.4获得）。 </li><li>一旦与有趣的功能（DNA损伤曲目或DNA修复灶）原子核被发现，切换到能过滤模式并记录厚度​​图。 注:此过程包括零丢失和未经过滤的图像的记录。厚度达0.3平均自由程（在入射光束的电子的30％得到由样品散射）是足够薄，以产生良好的元素映射。 </li><li>元素磷和氮的使用控制所使用的显微镜​​的能量过滤器的软件的记录比率的地图。记录在175电子伏特能量损失的磷地图后边缘图像以20电子伏特的预边缘图像，在120电子伏特能量损失的狭缝宽度，也以20电子伏特的狭缝宽度。在氮地图，记录后的边缘图像在447伏特与35伏特和预边缘图像的狭缝宽度在358电子伏特，也为35电子伏特的狭缝宽度。 注:由于成像元件的含量（phosphOrús酒店位和氮）是相对低的（约1％），选择"比率图像"（定性元素地图）比"3窗口方法"（定量元素地图），以产生具有更好的信噪比的图像。 </li></ol> 6.图像处理<ol><li>在图像处理软件可以处理所获取的图像的文件格式 （例如，数字显微）打开元素比地图。氮地图复制到红色通道和磷地图成RGB图像的绿色通道，然后叠加并对准地图。 </li><li>合成图像导出为标记的图像文件格式（TIFF）文件。在图像处理的软件，可以使用层（如，Photoshop中）打开图像。 </li><li>通过重新缩放的最大值和最小值在图像中的8位数据组（0到255）调整各信道的动态范围。 </li><li>减去从氮地图中的磷含量（使用"层"的窗口），以产生一个定性图，显示蛋白质的分布。 </li><li>转换在上一步分为"索引彩色"创建的核酸（磷）和蛋白质（氮）的地图和在该地图表示核酸分布和青色查找表CMYK颜色空间创建一个黄色的查找表以显示非核蛋白地图。颜色被选择为产生在两个元素地图之间的最大对比度。 </li><li>创建一个新的形象，呈现出进口磷和蛋白质的不同层的分布地图。使用以便在"屏幕"透明模式，看看这两个层相互叠加。 </li><li>打开在步骤5.2获得的零损失图像。此图像表示记录区，看起来像一个常规的TEM图像的图像，但只包含通过试样已通过而不与样品碰撞的电子。出口此图片为TIFF图像。 </li><li>在照片编辑器中打开导出的零损耗的图像，并将其复制到图像显示的地图元素的最上层。对齐使用"屏幕"透明模式下的图像。 </li><li>任选阈零损失使图像段源自所述miniSOG信号。添加得到的图像作为一个单独的层，如上所述。 </li></ol>

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	<li>Schermelleh, L., Heintzmann, R., Leonhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+super-resolution+fluorescence+microscopy.">A guide to super-resolution fluorescence microscopy.</a> The Journal of Cell Biology. 190, 165-175 (2010).</li><li>Bazett-Jones, D. P., Hendzel, M. J., Kruhlak, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometric+analysis+of+protein-+and+nucleic+acid-based+structures+in+the+cell+nucleus.">Stoichiometric analysis of protein- and nucleic acid-based structures in the cell nucleus.</a> Micron (Oxford, England: 1993). 30, 151-157 (1999).</li><li>Michael J Hendzel, F. M. B., Bazett-Jones, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Visualization+of+a+Protein+Nuclear+Architecture.">Direct Visualization of a Protein Nuclear Architecture.</a> Molecular biology of the cell. 10, 2051 (1999).</li><li>Ottensmeyer, F. P., Andrew, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+microanalysis+of+biological+specimens+by+electron+energy+loss+spectroscopy+and+by+electron+spectroscopic+imaging.">High-resolution microanalysis of biological specimens by electron energy loss spectroscopy and by electron spectroscopic imaging.</a> Journal of ultrastructure research. 72, 336-348 (1980).</li><li>Leapman, R. D., Ornberg, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+electron+energy+loss+spectroscopy+in+biology.">Quantitative electron energy loss spectroscopy in biology.</a> Ultramicroscopy. 24, 251-268 (1988).</li><li>Simon, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+spectroscopic+imaging.">Electron spectroscopic imaging.</a> Ultrastructural pathology. 11, 705-710 (1987).</li><li>Egerton, 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> Electron Energy-Loss Spectroscopy in the Electron Microscope. , (2011).</li><li>Aronova, M. A., Kim, Y. C., Zhang, G., Leapman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+and+thickness+correction+of+EFTEM+phosphorus+maps.">Quantification and thickness correction of EFTEM phosphorus maps.</a> Ultramicroscopy. 107, 232-244 (2007).</li><li>Hopkins, C., Gibson, A., Stinchcombe, J., Futter, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+molecules+employing+horseradish+peroxidase+as+reporter+enzyme+for+protein+localization+in+the+electron+microscope.">Chimeric molecules employing horseradish peroxidase as reporter enzyme for protein localization in the electron microscope.</a> Methods in enzymology. 327, 35-45 (2000).</li><li>Shu, X., 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=A+Genetically+Encoded+Tag+for+Correlated+Light+and+Electron+Microscopy+of+Intact+Cells,+Tissues,+and+Organisms.">A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms.</a> PLoS biology. 9, e1001041 (2011).</li><li>Gaietta, G., 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=Multicolor+and+electron+microscopic+imaging+of+connexin+trafficking.">Multicolor and electron microscopic imaging of connexin trafficking.</a> Science (New York, NY). 296, 503-507 (2002).</li><li>Fernandez-Capetillo, O., Celeste, A., Nussenzweig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focusing+on+foci:+H2AX+and+the+recruitment+of+DNA-damage+response+factors.">Focusing on foci: H2AX and the recruitment of DNA-damage response factors.</a> Cell cycle (Georgetown, Tex). 2, 426-427 (2003).</li><li>Haaf, T., Golub, E. I., Reddy, G., Radding, C. M., Ward, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+foci+of+mammalian+Rad51+recombination+protein+in+somatic+cells+after+DNA+damage+and+its+localization+in+synaptonemal+complexes.">Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes.</a> Proceedings of the National Academy of Sciences of the United States of America. 92, 2298-2302 (1995).</li><li>Maser, R. S., Monsen, K. J., Nelms, B. E., Petrini, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hMre11+and+hRad50+nuclear+foci+are+induced+during+the+normal+cellular+response+to+DNA+double-strand+breaks.">hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks.</a> Molecular and Cellular Biology. 17, 6087-6096 (1997).</li><li>Bekker-Jensen, S., Mailand, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+function+of+DNA+double-strand+break+repair+foci+in+mammalian+cells.">Assembly and function of DNA double-strand break repair foci in mammalian cells.</a> DNA repair. 9, 1219-1228 (2010).</li><li>Chapman, J. R., Sossick, A. J., Boulton, S. J., Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA1-associated+exclusion+of+53BP1+from+DNA+damage+sites+underlies+temporal+control+of+DNA+repair.">BRCA1-associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair.</a> Journal of cell science. 125, 3529-3534 (2012).</li><li>Dellaire, G., Kepkay, R., Bazett-Jones, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+imaging+of+changes+in+the+structure+and+spatial+organization+of+chromatin,+gamma-H2A.X+and+the+MRN+complex+within+etoposide-induced+DNA+repair+foci.">High resolution imaging of changes in the structure and spatial organization of chromatin, gamma-H2A.X and the MRN complex within etoposide-induced DNA repair foci.</a> Cell cycle (Georgetown, Tex). 8, 3750-3769 (2009).</li><li>Kruhlak, M. J., 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=Changes+in+chromatin+structure+and+mobility+in+living+cells+at+sites+of+DNA+double-strand+breaks.">Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks.</a> The Journal of Cell Biology. 172, 823-834 (2006).</li><li>Goodson, H. V., Dzurisin, J. S., Wadsworth, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+stable+cell+lines+expressing+GFP-tubulin+and+photoactivatable-GFP-tubulin+and+characterization+of+clones.">Generation of stable cell lines expressing GFP-tubulin and photoactivatable-GFP-tubulin and characterization of clones.</a> Cold Spring Harbor protocols. 2010, (2010).</li><li>Zeyda, M., Borth, N., Kunert, R., Katinger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+sorting+conditions+for+the+selection+of+stable,+high-producing+mammalian+cell+lines.">Optimization of sorting conditions for the selection of stable, high-producing mammalian cell lines.</a> Biotechnology progress. 15, 953-957 (1999).</li><li>Dellaire, G., Nisman, R., Bazett-Jones, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+light+and+electron+spectroscopic+imaging+of+chromatin+in+situ.">Correlative light and electron spectroscopic imaging of chromatin in situ.</a> Methods in enzymology. , 375-456 (2004).</li><li>Campbell, S., Ismail, I. H., Young, L. C., Poirier, G. G., Hendzel, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycomb+repressive+complex+2+contributes+to+DNA+double-strand+break+repair.">Polycomb repressive complex 2 contributes to DNA double-strand break repair.</a> Cell cycle. 12, 2675-2683 (2013).</li><li>Mortusewicz, O., 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=Recruitment+of+RNA+polymerase+II+cofactor+PC4+to+DNA+damage+sites.">Recruitment of RNA polymerase II cofactor PC4 to DNA damage sites.</a> J Cell Biol. 183, 769-776 (2008).</li><li>Bekker-Jensen, S., 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=Spatial+organization+of+the+mammalian+genome+surveillance+machinery+in+response+to+DNA+strand+breaks.">Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks.</a> The Journal of Cell Biology. 173, 195-206 (2006).</li><li>Ferrando-May, E., 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=Highlighting+the+DNA+damage+response+with+ultrashort+laser+pulses+in+the+near+infrared+and+kinetic+modeling.">Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling.</a> Frontiers in genetics. 4, 135 (2013).</li><li>Lukas, 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=53BP1+nuclear+bodies+form+around+DNA+lesions+generated+by+mitotic+transmission+of+chromosomes+under+replication+stress.">53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress.</a> Nature. 13, 243-253 (2011).</li><li>Harrigan, J. A., 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=Replication+stress+induces+53BP1-containing+OPT+domains+in+G1+cells.">Replication stress induces 53BP1-containing OPT domains in G1 cells.</a> The Journal of Cell Biology. 193, 97-108 (2011).</li><li>Aronova, M. A., 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=Reprint+of+'Three-dimensional+elemental+mapping+of+phosphorus+by+quantitative+electron+spectroscopic+tomography+(QuEST).">Reprint of 'Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST).</a> J. Struct. Biol. 161, 322-335 (2007).</li><li>Fussner, E., 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=Constitutive+heterochromatin+reorganization+during+somatic+cell+reprogramming.">Constitutive heterochromatin reorganization during somatic cell reprogramming.</a> The EMBO journal. 30, 1778-1789 (2011).</li><li>Kepkay, R., Attwood, K. M., Ziv, Y., Shiloh, Y., Dellaire, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KAP1+depletion+increases+PML+nuclear+body+number+in+concert+with+ultrastructural+changes+in+chromatin.">KAP1 depletion increases PML nuclear body number in concert with ultrastructural changes in chromatin.</a> Cell cycle. 10, 308-322 (2011).</li></ol>

The authors have nothing to disclose.

ESI可作为极好的工具用于检查染色质和核糖核蛋白的不同状态在细胞核中，因为它是能够专门映射是富含磷的区域。它深刻增强细节，可以用电子显微镜来获得并且不依赖于非特异性对比方法的量。 ESI的一个缺点是，它需要比在相同的加速电压常规的TEM较薄部分。这可以通过在与连续切片或使用层析方法28来克服。 虽然大量的信息可以通过查看磷和氮的分布来获得?...

尽管近年来在1光学显微镜显著的进步，细胞生物学家仍在遭受分辨率的差距。这限制涉及大分子复合物之间的协调相互作用中基本的细胞过程中的结构-功能关系的了解（例如，在染色质重塑，DNA修复，转录的RNA和DNA复制）。虽然透射电子显微镜（TEM）■提供所要求的分辨率，已经挑战来定义这些过程无法的结构因为标注的特定蛋白质，同时还能够以确定的可视化结构的生化成分。在没有内部的膜，以帮助区分核结构中，核一直特别具有挑战性。电子光谱成像（ESI）通过允许DNA，RNA和PROT的同时检测和分化解决某些限制EIN基核结构2-5。 电子光谱成像: 为了映射以高灵敏度和分辨率在电子显微镜下元素分布，可以用一个图像光谱仪，其选择已通过与元素的在样本 6内壳层电子相互作用非弹性散射电子。因为能量的单元特定的量被丢失原子在试样的电离的结果，这些电子可以被分离并使用被附接到电子显微镜的分光计可视化。因此，已经相互作用与试样的电子的频谱分析揭示关于样品7的元素组成的定性和定量的信息。电子通过检体传递时不损失能量被发现在电子能量损失的"零损耗峰"谱。这些电子的丰度是相关的试样的质量，密度和厚度，并包括通过检体通过，而不碰撞与样品或通过试样通道中失去能量的电子。这个信息可以是为特定的元素本的原子数，在试样8的绝对定量是有用的。 因为生物样品包括主要光元件很差偏转电子在TEM的入射光束，染色方法使用重金属盐具有以生成所述样品中的对比度得以应用。特异性大多数这些造影剂和无法的可视化多个污点其中特异性是可能的缺乏在细胞核的研究已限制常规电子显微镜的值。 ESI有超过常规的TEM显著的优点，特别是对细胞核的结构的研究。有可能利用DNA和含有RNA的大分子复合的富磷性质区分蛋白质复合核蛋白复合物和解决基于核酸它们的密度不同核蛋白复合物。剩余的生物材料可以根据它的丰度氮气进行成像。不同的解剖结构中的映射只是这两种元素的分布和相对丰度的分析为我们提供了许多关于核信息。例如，它是容易识别染色质，并在地图上的核糖体代表磷丰度。所述interchromatin空间，核孔复合物，和核机构，另一方面，可以容易地在该氮地图图像中检测到。 迷你单态氧发生器系统（miniSOG） 尽管ESI代表一种强大的技术原位染色质结构来研究，因为它需要的优势在磷和氮之间的元素组成的特征比率，元素组成通常不能使用的蛋白质复合物的不同群体之间进行区分。抗体标记的小的金颗粒在纳米范围内已被广泛地用于绘制单个分子的位置。由于金颗粒通常连接到一个二级抗体，它将出现的周围由第一抗体检测的表位大约20nm的圆周内。在后包埋样品，抗体只能检测是在部分的表面露出的表位。虽然这是可能的，以证明抗原的存在，并将其涉及到细胞的特定解剖结构，所获得的信息是不完整的，因为大部分的表位是由树脂遮蔽。预嵌入技术，其使用类似的协议与用于荧光显微镜，在整个允许访问抗原样品的整个深度，但所需要的使抗体穿透细胞通常需要去除脂膜，并删除无法固定醛组分通透步骤。此外，优选的醛固定液超微结构保存，戊二醛，通常破坏的表位，因此，多聚甲醛，通常需要。这是在蛋白质 - 蛋白质交联效果较差。抗体的被标记有金纳米颗粒的另一缺点是，金是创建一个强烈的对比，可以掩盖样品，它具有弱对比度性的结构细节非常电子致密材料。 绿色荧光蛋白（GFP）作为表达的蛋白质标签的出现改变了使用荧光显微镜的解答在细胞生物学的问题。标记的蛋白质与小荧光域允许其在体内的分布制图</e米&gt;而不需要透过程，可以改变结构。为了翻译用蛋白质标记映射蛋白质分布在细胞中，以电子显微镜的优雅原则，需要一个系统，该系统能够产生一个信号接近感兴趣的结构，并产生以TEM对比度。聚合的二氨基联苯胺]是经常用于组织学检测抗体结合的污点。此染色是使用辣根过氧化物酶（HRP）缀合的第二抗体通常沉积。尽管反应产生一个可靠的结果，HRP是不是在细胞9的胞质溶胶催化活性。 HRP的反应产物也可以扩散远离一代的网站，这样它们的分辨率比纳米金法10恶化。为了绕开这些问题，FLASH / ReAsH系统的开发11。它由重组融合蛋白，其拥有四半胱氨酸基序。这个主题可以结合一个biarsenic荧光。当激发时，结合的闪光或ReAsH是能够产生反应性高的单重态氧，因此photoconvert二氨基联苯胺（DAB）插入在标签的蛋白质的位点紧接该沉淀的聚合物。民建联聚合物可染色用四氧化锇，这是电子致密，因而可以用于映射重组融合蛋白在TEM的分布。 在2011年，舒等人 10提交的miniSOG系统，它由来自拟南芥的黄素蛋白是荧光，并且能够在与448纳米的蓝色光激发而产生很多单线态氧自由基衍生的小106个氨基酸融合标记的。这些单线态氧自由基可用于光氧化二氨基联苯胺，以形成上和标签蛋白，这是比免疫标记的抗体11相当接近的表面附近的聚合物。虽然闪存/ ReAsH系统需要使氟铬和二氨基联苯胺成执行光转化前的细胞中，miniSOG系统只需要二氨基联苯胺和轻，此外是大约两倍于聚合二氨基联苯胺作为有效。在这里，miniSOG采用与ESI的组合，以图的DNA修复灶的超微结构。 DNA修复灶（DRF） 未修复DNA双链断裂构成严重威胁到细胞，因为它们可以导致易位和遗传信息的丢失。反过来，这可导致衰老，癌症和细胞死亡。参与DNA双链断裂修复，许多蛋白积聚在病灶周围的DNA双链组装打破12-14。虽然它们的功能是未知的，它们所代表的部位在包含DNA双链断裂，并且是DNA双链断裂修复的部位的核心。 DNA修复FOCI（DRF）的特点是荧光显微镜和它们作为生物标志物用于DNA损伤12,15。它们大量相对于双链断裂的大小和被认为是相对均匀的，直到最近的超高分辨率显微镜研究揭示在每个聚焦16个子条块分子的一些证据。为了了解这些位点的组织，有必要以可视化的所有潜在的生物结构的相对于彼此。这不能用荧光显微镜下进行，但有可能通过电子显微镜17,18。这里，描述了一种方法，结合了电子光谱成像与miniSOG方法，来说明这种组合方法的潜力，以探索的DNA双链断裂修复的超微结构。

<table><tbody><tr><td>35 mm glass bottom dishes</td><td>MatTek</td><td>P35G-1.5-14-C</td><td></td></tr><tr><td>Gridded 35 mm glass bottom dishes</td><td>MatTek</td><td>P35G-2-14-CGRD&amp;nbsp;</td><td>not made for immersion objectives</td></tr><tr><td>LR White: acryl resin</td><td>emsdiasum</td><td>14381</td><td></td></tr><tr><td>LR White accelerator</td><td>emsdiasum</td><td>14385</td><td></td></tr><tr><td>3,3&amp;prime;-Diaminobenzidine tetrahydrochloride 10 mg tablets</td><td>Sigma</td><td>D5905</td><td></td></tr><tr><td>Slim Bar Grids 300 Mesh</td><td>SPI</td><td>1161123</td><td></td></tr><tr><td>Tungsten-Point Lab Pen</td><td>emsdiasum</td><td>41148</td><td></td></tr><tr><td>Osmium Tetroxide</td><td>emsdiasum</td><td>19100</td><td></td></tr><tr><td>Carbon Coater 208 carbon</td><td>Cressington</td><td></td><td></td></tr><tr><td>Ultra microtome Leica EM UC6</td><td>Leica</td><td></td><td></td></tr><tr><td>Photoshop CS5</td><td>Adobe</td><td></td><td>might even work with older versions</td></tr><tr><td>Digital Micrograph V.2.30</td><td>Gatan</td><td></td><td>might even work with older versions</td></tr><tr><td>Hoechst 33342</td><td>Sigma</td><td>H-1399</td><td></td></tr><tr><td>Effectene</td><td>Quiagen</td><td></td><td></td></tr><tr><td>MiniSOG-Constructs</td><td>Tsien-Lab</td><td></td><td>&lt;a href=&quot;mailto:tsienlab@yahoo.com&quot;&gt;tsienlab@yahoo.com&lt;/a&gt;</td></tr><tr><td>MDC1 miniSOG mCherry</td><td></td><td></td><td></td></tr><tr><td>53BP1 miniSOG mCherry</td><td></td><td></td><td></td></tr><tr><td>Rad52 miniSOG mCherry</td><td></td><td></td><td></td></tr><tr><td>cesium 137 radiation source &quot;MARK 1&quot;</td><td>&amp;nbsp;(J.L. Shepherd &amp;amp; Associated)</td><td></td><td></td></tr><tr><td>ImageJ/FiJi</td><td>open source</td><td></td><td>http://fiji.sc/Fiji</td></tr><tr><td>2 ml Eppendorf tubes</td><td>Fisherbrand</td><td>05-408-146</td><td></td></tr><tr><td>Diamond Knife ultra 35&amp;deg;</td><td>Diatome</td><td></td><td></td></tr><tr><td>Trimming Knife ultratrim</td><td>Diatome</td><td></td><td></td></tr><tr><td>Sodium cacodylate trihydrate</td><td>emsdiasum</td><td>12300</td><td>Caution Toxic!</td></tr><tr><td>Glutaraldehyde EM Grade 8%</td><td>emsdiasum</td><td>16020</td><td>Caution Toxic!</td></tr><tr><td>Sodium phosphate dibasic</td><td>emsdiasum</td><td>21180</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>emsdiasum</td><td>21190</td><td></td></tr><tr><td>Paraformaldehyde</td><td>emsdiasum</td><td>19202</td><td></td></tr><tr><td>Osmium tetroxide 4% solution</td><td>emsdiasum</td><td>19150</td><td></td></tr><tr><td>Inverted Fluorescence Micoscope Axiovert 200M</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisherbrand</td><td>A142-212</td><td></td></tr><tr><td>Sodium Hydroxide Solution 10M</td><td>Fluka</td><td>72068</td><td></td></tr><tr><td>Oxygen</td><td>Medigas</td><td></td><td></td></tr><tr><td>3-Amino-1,2,4-triazole</td><td>Sigma</td><td>A8056</td><td></td></tr><tr><td>Potassium cyanide</td><td>Sigma</td><td>207810</td><td>Caution Toxic!</td></tr><tr><td>Ethanol</td><td>emsdiasum</td><td>15058</td><td>Caution Toxic!</td></tr><tr><td>Razor blade Single Edge Carbon Steel</td><td>emsdiasum</td><td>71960</td><td>Caution Sharp!</td></tr><tr><td>DMEM</td><td>Sigma</td><td>D 5546</td><td></td></tr><tr><td>FBS</td><td>Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>G418</td><td>Life Technologies</td><td>11811-023</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Transmission Electron Microscope 200 kV</td><td>JEOL</td><td>2100</td><td></td></tr><tr><td>GIF Tridiem 863 Energy filter</td><td>Gatan</td><td></td><td></td></tr></tbody></table>

visualization of minisog tagged dna repair proteins in combination with electron spectroscopic imaging (esi)

极限到光学分辨率和鉴定特定蛋白质种群透射电镜的挑战已经在细胞生物学障碍。许多现象无法通过体外分析在简化的系统进行说明，并且需要额外的结构信息在原位 ，特别是在1毫微米和0.1微米之间的范围内，以便被完全理解。这里，电子光谱成像，透射电子显微技术，允许蛋白质和核酸的分布同时映射，和表达标签，miniSOG，被组合以研究DNA双链断裂修复病灶的结构和组织。

ESI 与常规的TEM图像比较所述核（ 图1）的ESI 图像 （例如， 图7-1）中揭示了解剖结构，可以很容易分辨的显着增加。染色质脊显示为黄色，这是很容易识别在小鼠细胞中的chromocenters。核仁可以容易地从染色质区分通过圆形结构和不同的颜色，由于预组装的核糖体已经含有高量的蛋白质，其是富含氮，除了核糖核酸，其中富?...

Watch this Scientific Journal Video about Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI at JoVE.com

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

在联合电子能谱成像miniSOG标记的DNA修复蛋白可视化（ESI）

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

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Biology

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

10.0K 次观看.  University of Alberta. Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

视频: Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

科研

生物化学

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

405 Nm 激光微辐照对 DNA 损伤的蛋白质招募研究

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

遗传学

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

基于免疫荧光显微镜的瞬时和持久双链 dna 损伤 dna 修复过程的表征

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

癌症研究

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

可视化的活细菌细胞蛋白-DNA相互作用使用光活化的单分子跟踪

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

免疫与感染

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate identification and localization of proteins, glycans, and small molecules. While this technique is well-established in two-dimensional (2D) cell culture, less is known about its use in three-dimensional (3D) cell models. Ovarian cancer organoids are 3D tumor models that recapitulate tumor cell clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Thus, they are superior to cell lines for the evaluation of drug sensitivity and functional biomarkers. Therefore, the ability to utilize immunofluorescence on primary ovarian cancer organoids is extremely beneficial in understanding the biology of this cancer. The current study describes the technique of immunofluorescence to detect DNA damage repair proteins in high-grade serous patient-derived ovarian cancer organoids (PDOs). After exposing the PDOs to ionizing radiation, immunofluorescence is performed on intact organoids to evaluate nuclear proteins as foci. Images are collected using z-stack imaging on confocal microscopy and analyzed using automated foci counting software. The described methods allow for the analysis of temporal and special recruitment of DNA damage repair proteins and colocalization of these proteins with cell-cycle markers.

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

The present protocol describes methods for evaluating DNA damage repair proteins in patient-derived ovarian cancer organoids. Included here are comprehensive plating and staining methods, as well as detailed, objective quantification procedures.

通过免疫荧光测定 可视化 患者来源的卵巢癌类器官中的 DNA 损伤修复蛋白

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate ...

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

由紫外线引起的复制中间体的可视化 E。大肠杆菌使用二维的琼脂糖凝胶电泳分析

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

生物学

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

免疫荧光DNA修复蛋白相互作用的可视化

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

可视化和量化基于内分泌酶的站点特定DNA损伤

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is ...

通过 RPA2 免疫荧光染色检测 G1 期单链 DNA 病灶

Visualizing Single-Stranded DNA Foci in the G1 Phase of the Cell Cycle

DNA has dedicated cellular repair pathways capable of coping with lesions that could arise from both endogenous and/or exogenous sources. DNA repair necessitates collaboration between numerous proteins, responsible for covering a broad range of tasks from recognizing and signaling the presence of a DNA lesion to physically repairing it. During this process, tracks of single-stranded DNA (ssDNA) are often created, which are eventually filled by DNA polymerases. The nature of these ssDNA tracks (in terms of both length and number), along with the polymerase recruited to fill these gaps, are repair pathway-specific. The visualization of these ssDNA tracks can help us understand the complicated dynamics of DNA repair mechanisms.
This protocol provides a detailed method for the preparation of G1 synchronized cells to measure ssDNA foci formation upon genotoxic stress. Using an easy-to-utilize immunofluorescence approach, we visualize ssDNA by staining for RPA2, a component of the heterotrimeric replication protein A complex (RPA). RPA2 binds to and stabilizes ssDNA intermediates that arise upon genotoxic stress or replication to control DNA repair and DNA damage checkpoint activation. 5-Ethynyl-2'-deoxyuridine (EdU) staining is used to visualize DNA replication to exclude any S phase cells. This protocol provides an alternative approach to the conventional, non-denaturing 5-bromo-2'-deoxyuridine (BrdU)-based assays and is better suited for the detection of ssDNA foci outside the S phase.

作者聚焦:在 DNA 修复过程中可视化单链 DNA 以获得治疗见解 

The following protocol presents the detection of single-stranded DNA foci in the G1 phase of the cell cycle utilizing cell cycle synchronization followed by RPA2 immunofluorescent staining.

在细胞周期的 G1 期可视化单链 DNA 病灶

DNA has dedicated cellular repair pathways capable of coping with lesions that could arise from both endogenous and/or exogenous sources. DNA repair ...

在联合电子能谱成像miniSOG标记的DNA修复蛋白可视化（ESI）

摘要

探索更多视频

此视频中的章节

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

405 Nm 激光微辐照对 DNA 损伤的蛋白质招募研究

基于免疫荧光显微镜的瞬时和持久双链 dna 损伤 dna 修复过程的表征

可视化的活细菌细胞蛋白-DNA相互作用使用光活化的单分子跟踪

通过免疫荧光测定可视化患者来源的卵巢癌类器官中的 DNA 损伤修复蛋白

由紫外线引起的复制中间体的可视化 E。大肠杆菌使用二维的琼脂糖凝胶电泳分析

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

免疫荧光DNA修复蛋白相互作用的可视化

可视化和量化基于内分泌酶的站点特定DNA损伤

在细胞周期的 G1 期可视化单链 DNA 病灶

在联合电子能谱成像miniSOG标记的DNA修复蛋白可视化（ESI）

摘要

探索更多视频

此视频中的章节

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

405 Nm 激光微辐照对 DNA 损伤的蛋白质招募研究

基于免疫荧光显微镜的瞬时和持久双链 dna 损伤 dna 修复过程的表征

可视化的活细菌细胞蛋白-DNA相互作用使用光活化的单分子跟踪

通过免疫荧光测定 可视化 患者来源的卵巢癌类器官中的 DNA 损伤修复蛋白

由紫外线引起的复制中间体的可视化 E。大肠杆菌使用二维的琼脂糖凝胶电泳分析

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

免疫荧光DNA修复蛋白相互作用的可视化

可视化和量化基于内分泌酶的站点特定DNA损伤

在细胞周期的 G1 期可视化单链 DNA 病灶

通过免疫荧光测定可视化患者来源的卵巢癌类器官中的 DNA 损伤修复蛋白