This work was supported by a National Institutes of Health grant (GM77311) and a seed grant from The Ohio Cancer Research Associates (to H.A.F.). SM was partially supported by an Up on the Roof fellowship from the Human Cancer Genetics Program of The Ohio State University Comprehensive Cancer Center.

1.细胞培养<ol><li>使用端粒酶逆转录酶（hTERT）永生视网膜色素上皮细胞（hTERT的-RPE1;这里称为RPE1）。 注:RPE1细胞是那些通常用于研究中心粒装配和ciliogenesis近二倍体，未转化的人细胞。这些细胞遵循正常的中心粒复制周期协调与调控细胞周期。 </li><li>通道RPE1细胞的近汇合百毫米细胞培养皿以1:5稀释的原始培养物进含有10ml的新鲜100mm皿的DME / F-12（1:1）培养基补充有10％胎牛血清（FBS），100U / ml青霉素G和100微克/ ml链霉素（完全培养基被称作DME / F-12培养基）。在5％CO 2存在下生长的细胞在37℃。 <ol><li>孵育12毫米圆形盖玻片在50毫升的1N HCl的带盖的玻璃烧杯中，O / N不摇动，在60℃下在水浴或培养箱。 </li><li>自动对焦之三废弃的酸溶液，孵育15分钟，偶尔晃动洗涤盖玻片在100ml蒸馏水洗涤三次。在70％的乙醇和95％的乙醇同样地重复洗涤。 </li><li>通过单独地散布出来以实验室印迹纸在生物安全柜，随后灭菌用UV辐射60分钟，干燥该盖玻片。 </li></ol></li><li>将2-4干盖玻片35毫米的细胞培养皿。稀释纤连蛋白的溶液或纤连蛋白样工程聚合物（原料浓度为1毫克/毫升），以25微克/毫升细胞培养PBS中的浓度。 <ol><li>放置80-100微升的稀释液到每个盖玻片并孵育60分钟以涂覆的盖玻片的顶侧。洗盖玻片三次用PBS加入完全培养基之前。 </li></ol></li></ol> 2.生长的细胞和细胞治疗与蛋白酶体抑制剂<ol><li>通过2×10 5异步成长包含ING RPE1细胞中的每个35mm培养皿盖玻片和成长的细胞在2毫升完全培养基。更换培养液，每24小时用新鲜预热完全培养基。 <ol><li>来分析测试蛋白质的水平中心体蛋白酶体抑制的效果（这里VDAC3或γ微管蛋白），生长细胞两张35毫米的菜肴44小时。替换完全培养基的培养基中，在5微米或0.05％的最终浓度分别添加任MG115或DMSO作为对照的溶剂。与此同时，4小时添加BrdU的给细胞以40μM的终浓度，并培育细胞。 </li><li>转移在24孔板每个盖玻片。加入500微升冷却的甲醇到每个孔中并且孵育所述板在-20℃下10分钟以固定在盖玻片的细胞。立即清洗盖玻片三次，用500μL洗涤缓冲液（1×PBS中含0.5mM的MgCl 2和0.05％的Triton-X 100）。 </li></ol></li><li>收获残留细胞从35毫米培养皿，并离心将细胞在1000×g离心5分钟。使用细胞沉淀来分析由蛋白质印迹（步骤6）的总蛋白。 </li></ol> 3. BrdU的脉冲和大通分析来分析不同细胞周期蛋白水平<ol><li>为了分析蛋白质（此处VDAC3，SAS6或Cep135）的电平的变化，在中心体在细胞周期的不同阶段，在含有盖玻片为44小时2张35毫米培养皿生长的细胞。替换用含有40μM的BrdU完全培养基培养基孵育的细胞在细胞培养孵化4小时。 </li><li>来自一个培养皿，盖玻片转移到一个24孔板中步骤2.1.2描述以固定用冷却的甲醇的细胞。 </li><li>在4小时的BrdU脉冲之后，除去含有的BrdU的培养基，用PBS洗一次细胞，并一次用完全培养基，在不存在的BrdU不同的时间（通常是另一个4小时加入新鲜培养基，再生长的细胞对于如在步骤2.1.2中所述的固定的细胞前RPE1细胞）。 注:对于RPE1细胞，大多数BrdU阳性细胞都在后期S或G2期为4小时追。这必须独立地对每个细胞类型来确定。 </li></ol> 4.免疫染色<ol><li>孵育在200μl封闭缓冲液（2％BSA和0.1％的TritonX-100在1×PBS中），30分钟的盖玻片固定细胞。 <ol><li>孵育细胞与初级抗体的混合物（典型地一个凸起在兔和小鼠另一个凸起）稀释在封闭缓冲液O / N在4℃下在湿润的腔室中。 </li><li>为了使加湿室，把湿纸巾在底部空1000微升枪头盒一半。躺在机架表面封口膜的条带，并发现该抗体溶液的液滴（15-20微升）到封口膜为每个盖玻片培养。倒置盖玻片上的抗体溶液的液滴（使得细胞浸泡）和附近的尖端盒的盖子。 </li><li>再次反转盖玻片，并将其回回到24孔的菜。洗盖玻片，然后孵育它们在150微升二次抗体混合物（此处绿色荧光染料缀合的抗兔和红色荧光染料缀合的抗小鼠稀释于封闭缓冲液）处理1小时，在室温。洗盖玻片三次。 注:荧光团共轭次级抗体是光敏感的。保护过程中的步骤，从光样品4.1.3-5.1.2。 </li></ol></li><li>通过在-20℃下用500微升冷却的甲醇再次固定染色RPE1细胞10分钟，如在步骤2.1.2中所述制备用于抗BrdU染色。三次洗盖玻片。 注意:此固定期间在以下步骤中的酸水解过程固定感兴趣的蛋白质（多个）的初级和次级抗体标记。 <ol><li>孵育细胞在200μl的2N盐酸进行30分钟，在室温。中和用300微升的1M Tris-的Cl，pH值8的d。洗涤细胞用洗涤缓冲液三次。 </li><li>方框用200μl的封闭缓冲液30分钟，在RT下再细胞。 </li><li>孵育细胞与大鼠抗BrdU抗体（稀释于封闭缓冲液），45分钟，在37℃在湿润的腔室中。返回盖玻片回24孔皿中，把它们洗干净，然后孵育蓝色荧光染料缀合的抗大鼠第二抗体（稀释于150微升封闭缓冲液在24孔培养皿1小时，在室温。 </li></ol></li><li>洗盖玻片三次。发现含在玻璃显微镜载玻片抗淬灭剂的安装溶液的液滴（约3-6微升）。倒置盖玻片，与细胞侧朝下，到安装的解决方案。轻轻按压使用清洁的软组织幻灯片盖玻片擦去多余的液体。应用透明指甲油沿盖玻片的边缘，以密封其上的显微镜载玻片。 </li></ol> 5.免疫荧光图像Acquisition与分析<ol><li>使用100X计划的Apo油浸物镜（具有1.4的数值孔径），以获得BrdU阳性RPE1细胞的图像在环境温度下。 <ol><li>把幻灯片上的显微镜（见设备）附能够进行数字成像的相机。对于每一个荧光团，通过手动检查的实验的所有样品确定适当的曝光时间（一般为300-1,000毫秒）。取得的细胞的图像之前，手动确定适当的顶部和底部的焦平面沿Z轴（通常为0.2μm的步长）。获得不同的样品是在使用相同的曝光时间为沿着Z轴的不同的荧光团使用数字显微镜成像软件包单独盖玻片的图像。 </li></ol></li><li>执行解卷积（在这种情况下不使用邻算法）沿Z轴获得的所有的图像栈。 <ol><li>沿获得每个图像栈的总强度投影Z轴。 </li></ol></li><li>在细胞中，其中中心体接近（2中心体之间的距离小于2微米），画一个小方（通常每侧20-30像素）周围两中心体和标记所选区域。 <ol><li>画一个更大的方块（通常每侧24-35像素）围绕第一广场和纪念大广场的选定区域。 </li><li>得到的面积（A）和在每个框中的每个荧光团的总荧光强度（F），（S表示小框且L表示大框）。 </li><li>分析利用豪威尔等 29所描述的公式中的每个荧光背景校正的荧光强度:F = F 的S - [（F L - 五六）×A S /（A L - A S）]。 </li><li>通过计算其FL背景校正的荧光强度的比率获得的利息（此处VDAC3或SAS6）蛋白的标准化荧光强度uorophore根据与用于所选择的内标物（这里γ微管蛋白，SAS6或Cep135）的荧光团。 </li></ol></li><li>在分析单元，其中中心体完全分离的情况下，（2中心体之间的距离大于2微米），分别分析各中心体通过拉伸两个独立的盒套。画一个小方（通常每侧15-25像素）周围的每个中心体和标记所选区域。画一个更大的正方形（每侧20-30像素）周围的第一方阵，并标明所选区域。 <ol><li>得到的面积（A）和在每个框中的每个荧光团的总荧光强度（F）中，并计算每个荧光团的背景校正的荧光强度，分别对每个中心体，如在步骤5.3.3说明。 </li><li>结合各蛋白质的背景校正的荧光强度，从两中心体，以获得在该细胞蛋白质的总中心体水平。 </li><li>获取通过计算其总中心体强度的比率为内标物的总中心体强度归一化强度对感兴趣的蛋白质。 </li></ol></li><li>对于每个实验条件进行分析，至少15-25细胞并绘制图表中的电子表格。 </li></ol> 6.分析总蛋白使用免疫印迹<ol><li>重悬的细胞在放射免疫沉淀测定含有50mM的Tris-HCl pH为8，150 mM氯化钠，1％的Nonidet P-40，1％脱氧胆酸钠，0.1％十二烷基硫酸钠（SDS）（RIPA）缓冲液孵育在冰上10分钟以裂解细胞。 <ol><li>以10,000×g离心离心该混合物10分钟，分离沉淀级分的溶解产物，并用二辛可宁酸（BCA）测定试剂盒测定各裂解物的总蛋白浓度。 </li></ol></li><li>混合40微克裂解物与4倍的SDS-PAGE上样缓冲液（50毫摩尔Tris-CL，pH值6.8，2％SDS，10％甘油，100mM的二硫苏糖醇，0.1％维奇ophenol蓝色）。 <ol><li>煮沸样品10分钟，并运行变性SDS-PAGE上的12.5％的样品在200 V的恒定电压</li></ol></li><li>传送分离在SDS-PAGE上使用蛋白质印迹技术中的硝化纤维素膜的蛋白质（在90 V 1小时在寒冷的恒定电压）。 <ol><li>方框用3％无脂牛奶中的1×PBS的膜，然后孵育稀释的初级抗体溶液的膜（在此情况下的兔抗VDAC3，兔抗γ-tubulin和小鼠抗α微管蛋白）为1小时，在RT。 </li><li>洗膜用PBS-T缓冲三次（每次5分钟温育在摇动下）后（1×PBS和0.2％吐温-20），孵育该膜在近红外（IR）的荧光染料缀合的抗兔的混合物和红外荧光染料缀合的抗小鼠二抗1小时（稀释在PBS-T）的。 </li><li>洗膜三次，然后扫描该膜用红外f以分析频带luorescence成像系统适用于免疫印迹检测。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

在细胞生物学定量显微镜通常与活细胞成像试验，如荧光共振能量转移（FRET），荧光恢复漂白相关（FRAP） 等。然而，后有细胞生物学家的例子越来越多开发不同的定量检测显微镜对固定细胞近年来27,34-36。重要的是，了解中心体生物学的进步往往需要的蛋白质，其中心体池可能会受到不同的管制比其他池的中心体特异功能的理解。因此，我们开发了一个定量的IIF分析特异性分析的蛋白质的两个不同处理的样品中的相对中心体水平。因为这需要细胞固定并在单独的盖玻片处理，因此，我们引入了归一化，以用于可能的工件更好的控制的目的，内标由于处理两个experime的必要性ntal细胞分开的人口，并增加了测定的准确性。只有已经使用这样的内标来测量一个测试蛋白的相对荧光强度的几个以前的研究37。 而选择的内标是为我们的测定法的准确性是至关重要的，它可以说是最有挑战性的方面。理想情况下，在S相的中心粒对重​​复，重复的中心粒然后积累更多的PCM组件，包括G2期间γ微管蛋白使两个成熟中心体可以成为有丝分裂纺锤体的两极。因此，我们假设γ微管蛋白的水平中心体不会在细胞都严格S相显著变化。这似乎是在RPE1细胞，其中S期细胞的标志是一个简短的BrdU标记的情况。然而，内部标准的有效性必须为每个细胞类型和实验条件进行试验。例如，当背景CORRE反恐执行局VDAC3荧光的增长超过两倍蛋白酶体抑制阿非迪霉素治疗（S相逮捕）U2OS细胞，也有一个小，但统计学显著（0.001 &lt;p值&lt;0.05），中心体γ微管蛋白的增加（补充如图）。因此，γ微管蛋白并不总是适当的内部控制，即使对于那些在细胞周期停滞细胞。 类似于所有其它IIF显微镜测定法，该测定法的缺点之一是，抗体在测试蛋白和内标，必须在不同的宿主物种得到提升。而我们的数据表明，Cep135比SAS6一个更好的内标，我们无法正常化VDAC3水平针对Cep135作为对这些蛋白质在兔子都提出了可用的抗体。因此，我们来代替针对SAS6，由此引起的后期S相和G2之间的减少VDAC3水平的过高估计归一化。在一个情况下的英特RNAL标准适度变化，可以引入另一个正常化的因素占了变化。例如，以校正在SAS6的变化，我们乘以Cep135-归SAS6强度SAS6标准化VDAC3强度。 在情况下，选择合适的内标是困难的，荧光染料涂层微球（0.02-0.04微米直径）可以提供另一种策略。一个荧光微球存在于同一字段作为感兴趣的小区的信号可以用于标准化对应于感兴趣的蛋白质的荧光信号。另一方面，如果测试和对照细胞进行成像在一起，可以不需要对正常化这样的内标。之前我们已经使用了版本检测细胞中过表达的两种细胞周期蛋白A 19或20抗酶，这无论是预防19或20推广的DEGR MPS1的中心体水平进行比较MPS1的adation在中心体，分别以在成像的同时相邻的未转染细胞。 为了检查中心体VDAC3的经蛋白酶体抑制的效果，我们使用BrdU标记，以鉴定和比较细胞处于S期。而MG115处理可以抑制细胞周期进程，这种抑制需要一个显著较高浓度38比，在此研究中使用。在这里使用MG115可阻断细胞周期关卡和转换，但不阻断细胞周期行进的浓度。然而，必须为每个细胞类型进行验证，因为我们没有在这里通过表明MG115并未改变的BrdU阳性细胞相比的DMSO的百分比。然而，使用阿非迪霉素或羟基脲（HU），或使用有丝分裂"摇断&#39;和释放或双胸苷块和释放可作为替代的方法来生成等效地区人口同步细胞在S期阻止细胞细胞在S期的系统蒸发散。 在BrdU的脉冲追踪策略研究细胞周期的时间点会在需要的泰然自若的细胞周期的许多功能分析非常有用。这是可能能够取代使用各种细胞周期抑制剂的细胞周期停滞的技术的生物更显著测定。使用这些抑制剂的可能经常引起导致细胞周期表型异常变化的细胞不同种类的生理应力。使用脉冲追踪方法使我们能够证明Cetn2那枯竭只是推迟中心粒组装39，而不是造成一个完整的块所建议的以前的研究23。检测BrdU阳性细胞，需要样品的酸水解。我们和许多其他研究人员使用的技术重新修复中小学染色检测蛋白质，之前酸水解后的细胞，用染色强度显着损失differen吨中心体蛋白40。然而，一个潜在的替代可以是使用5-乙炔基-2&#39;-脱氧尿苷（EDU），另一个胸苷类似物即，类似的BrdU，在复制的DNA 41有效地并入。的EdU使用"点击化学的可视化"并不需要酸水解42，并且因此更适合一些抗体。 此外，该测定中可以很容易地被用于测量在中心体中的特定蛋白质的翻译后修饰的改变。例如，磷酸化的中心体水平可以归针对该蛋白质本身的总含量，其前提是磷酸化位点特异性抗体存在。该测定可以立即应用到评估实验条件或细胞周期阶段之间的磷酸化的相对水平，但可能被适于评估磷酸化的总水平，如果抗体艾芬的关键参数伊蒂埃斯是已知的或可确定的。该测定也应适用于在细胞中的任何其他位点的蛋白质的研究中，虽然关心区域不应太大。该测定法将是特别有用的以测量被定位于多个蜂窝站点的蛋白质的特定位置的调制。 有少数的技术参数，例如差动光漂白，数字成像的效果，荧光团的荧光强度的线性度，背景噪声等 ，必须采取考虑最大限度地测量43的精度。例如，一个可以切换与每个次级抗体相关，以评估漂白的贡献的荧光染料。许多其它参数可以很好地控制若用相同的摄像条件（曝光时间，增益，步长大小，的Z-堆栈等数），并在每个成像运行相同的成像软件。虽然我们使用在我们的分析进行图像反卷积的无邻居算法，此法可以与其他反卷积算法，如迭代约束相结合。我们还注意到，这应该是很容易适应该测定以任何数目的不同的成像软件包，因为大多数通常可用的图像分析软件具有工具荧光强度与距离的分析。 该测定中应适用于任何类型的细胞，和我们的选择的细胞在这里是纯粹基于它们在中心体生物学研究的相关性，以及它们在我们以前的中心体VDAC3函数16,28考试中使用。然而，内部标准和相应的时间脉冲追踪的有效性，以确定细胞在S期，G2或有丝分裂，必须针对每个小区类型来确定。总体而言，在与BrdU的脉冲追踪实验组合我们的新开发的定量荧光技术将是解决几个复杂的一种非常有用的方法机制在中心体生物学和在许多更广泛的细胞生物学的各个方面。

中心体由一对中心粒由pericentriolar材料（PCM）所包围的。为在哺乳动物细胞中主要的微管组织中心（MTOCs），中心体作为有丝分裂纺锤体的两极在分裂的细胞，从而有助于维持基因组完整性1。在静止期细胞（ 例如 ，在G0期）时，中心体，即母中心粒的两个中心粒中的一个，被转换成一个基体组装在主纤毛，感官细胞器从细胞表面2突出出来。一旦细胞重新进入细胞周期，原发性纤毛被拆卸并且每个中心粒指挥一个procentriole在其近端逐渐伸长，以形成一个成熟的中心粒3的组装。在S期的开始，一个侧手翻状结构，它提供了9倍对称性的中心粒形成每个现有中心粒的表面上，将成为各procentriole的基础。 SAS6吨帽子是必不可少的中心粒组件被招募来形成手翻4-6的中心。其他中心粒蛋白，然后组装到侧手翻在一个高度管制，近端远端的方式7。经过精确完成中心粒的复制，细胞聚集更多的pericentriolar材料由G2期8月底前建立两个功能中心体。在除了核心部件中心粒9-11，其它几种蛋白质，包括蛋白激酶，磷酸酶，蛋白伴侣，脚手架元件，膜相关蛋白和降解机械与中心粒，基体和PCM在细胞周期12-16的不同的时间相关联。它经常被注意的是，许多蛋白质​​的中心体的水平在时间上由中心体定位的机制和/或蛋白酶体降解在中心体调节。重要的是，在几个蛋白质如PLK4，MPS1，SAS6和CP110 a的中心体水平的波动吨的不同点的细胞周期似乎是至关重要的调节中心粒装配5,17-22，和在MPS1的防止这个中心体的降解的情况下会导致过量中心粒19的形成。另一方面，相对于细胞溶质池几种蛋白质的中心体部分较少不稳定。例如的siRNA介导的下调中金2（Cetn2）导致了在中心粒蛋白水平仅温和下降，尽管大大降低了整个细胞水平的23。因此，关键是要测量在中心体的变化中心体蛋白的水平，而不是评估其中心体特定的功能时，测定全细胞蛋白质的水平。 在这项研究中，我们开发了使用间接免疫荧光（IIF）量化蛋白质在中心体的相对水平的测定法。该测定被显影尤其来分析细胞，来自不同样品因此不能在同一时间被成像。这些样品可以是用不同的试剂（ 即 ，药物与对照）中，在不同的时间点收集经处理的细胞（ 即 ，脉搏与追），或者是在细胞周期的不同阶段。这个测定的原理在于测量对应于蛋白质在一个小区域和正常化针对同一该值对于另一种蛋白质，其水平所选择的实验条件下不发生变化的背景校正的荧光强度。在中心体生物学几项研究最近使用的各种定量显微技术，在这两种活的或固定细胞，以确定候选蛋白24-27的中心体特定的函数。类似测定法，本发明的技术还可以测量测试蛋白的背景校正的荧光强度。然而，在该测定法使用内标列入正常化将可能提供更多的准确性和信心在分析两种不同的样本是在两个不同的盖玻片。此外，除了在研究中心体蛋白质水平，用小的调整这种方法可以应用到的实验条件多样化或其它细胞位点。 在这里，我们结合定量检测显微镜用的BrdU脉冲追踪策略，比较不同的细胞周期阶段的细胞。代替使用标准的细胞周期停滞和释放技术，研究各种细胞周期的时间点，以异步方式生长的细胞的孵育用BrdU在S期标记细胞，标记的细胞追逐不同​​的时间（通常为4-6小时）。大多数标记的细胞将在脉冲后立即处于S期。追逐的长度被选择为使得追后，标记细胞将在晚S期，G2或有丝分裂，其可以通过形态特征中心体相对于NUC如 - 位置进行验证雷，中心体之间的距离，染色体等的缩合因此，追逐的长度取决于为S，G2和特定细胞类型的M期的平均持续时间。由于这种方法避免了细胞周期抑制剂，如羟基脲，阿非迪霉素，诺考达唑等 ，其允许更生理学相关细胞周期分析。 因此，我们在这里表明，单独的定量荧光显微术测定中，或在用BrdU脉冲追踪试验组合，是一种简单而功能强大的技术中的未扰动的细胞周期，以精确地测量在候选蛋白质的中心体水平的相对变化。我们测量VDAC3，我们在中心体最近确定除了线粒体16,28的蛋白质的中心体的水平，使用这些测定。在这里所得到的结果验证我们之前的看法，即VDAC3的中心体游泳池被降解调节，也各有不同的细胞周期dependen吨方式16中 ，还证实了该方法的适用性。

<table><tbody><tr><td>Fibronectin</td><td>Sigma</td><td>F8141</td><td>Stock of 1 mg/ml in water</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>MG115</td><td>Sigma</td><td>SCP0005</td><td>Stock of 10 mM in DMSO</td></tr><tr><td>BrdU</td><td>Sigma</td><td>B5002</td><td>Stock of 10 mM in DMSO</td></tr><tr><td>Anti-&amp;#947;-tubulin (mouse monoclonal, clone GTU 88)</td><td>Sigma</td><td>T 6557</td><td>1:200 in IIF blocking buffer&amp;nbsp;</td></tr><tr><td>Anti-VDAC3 (rabbit polyclonal)&amp;nbsp;</td><td>Aviva Systems Biology</td><td>ARP35180-P050</td><td>1:50 in IIF blocking buffer, 1:1,000 in WB blocking buffer</td></tr><tr><td>Anti-Sas6 (mouse monoclonal)&amp;nbsp;</td><td>Santa cruz biotechnology</td><td>sc-81431</td><td>1:100 in IIF blocking buffer</td></tr><tr><td>Anti-Cep135 (rabbit polyclonal)</td><td>Abcam</td><td>ab-75005</td><td>1:500 in IIF blocking buffer</td></tr><tr><td>Anti-BrdU (rat monoclonal)&amp;nbsp;</td><td>Abcam</td><td>ab6326</td><td>1:250 in IIF blocking buffer</td></tr><tr><td>Alexa Fluor 350 Goat Anti-Rat IgG (H+L)</td><td>Life technologies</td><td>A21093</td><td>1:200 in IIF blocking buffer</td></tr><tr><td>Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L) Antibody</td><td>Life technologies</td><td>A21202</td><td>1:1,000 in IIF blocking buffer</td></tr><tr><td>Alexa Fluor 594 Donkey Anti-Mouse IgG (H+L) Antibody</td><td>Life technologies</td><td>A21203</td><td>1:1,000 in IIF blocking buffer</td></tr><tr><td>Alexa Fluor 488 Donkey Anti-Rabbit IgG (H+L) Antibody</td><td>Life technologies</td><td>A21206</td><td>1:1,000 in IIF blocking buffer</td></tr><tr><td>Alexa Fluor 594 Donkey Anti-Rabbit IgG (H+L) Antibody</td><td>Life technologies</td><td>A21207</td><td>1:1,000 in IIF blocking buffer</td></tr><tr><td>Anti-&amp;#947;-tubulin (rabbit polyclonal)</td><td>Sigma</td><td>T5192</td><td>1:1,000 in WB blocking buffer</td></tr><tr><td>Anti-&amp;#945;-tubulin (mouse monoclonal, DM1A)</td><td>Sigma</td><td>T9026</td><td>1:20,000 in WB blocking buffer</td></tr><tr><td>Alexa Fluor 680 Donkey Anti-Rabbit IgG (H+L)</td><td>Life technologies</td><td>A10043</td><td>1:10,000 in WB blocking buffer</td></tr><tr><td>Mouse IgG (H&amp;amp;L) Antibody IRDye800CW Conjugated</td><td>Rockland antibodies</td><td>610-731-002</td><td>1:10,000 in WB blocking buffer</td></tr><tr><td>SlowFade Gold Antifade Reagent&amp;nbsp;</td><td>Life technologies</td><td>S36936</td><td>Mounting media</td></tr><tr><td>Round coverslips 12CIR.-1</td><td>Fisherbrand</td><td>12-545-80</td><td></td></tr><tr><td>Olympus IX-81 microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Retiga ExiFAST 1394 IR camera</td><td>QImaging&amp;nbsp;</td><td>32-0082B-238</td><td></td></tr><tr><td>100X Plan Apo oil immersion objective</td><td>Olympus</td><td></td><td>1.4 numerical aperture</td></tr><tr><td>Slidebook software package</td><td>Intelligent Imaging Innovations</td><td></td><td></td></tr><tr><td>Odyssey IR Imaging System</td><td>Li-cor Biosciences</td><td></td><td></td></tr><tr><td>Bicinchoninic acid (BCA) assay</td><td>Thermo Scientific</td><td>23227</td><td></td></tr><tr><td>U-MNU2 Narrow UV cube</td><td>Olympus</td><td>U-M622</td><td>Filter</td></tr><tr><td>U-MNU2 Narrow Blue cube</td><td>Olympus</td><td>U-M643</td><td>Filter</td></tr><tr><td>U-MNU2 Narrow Green cube</td><td>Olympus</td><td>U-M663</td><td>Filter</td></tr></tbody></table>

quantitative immunofluorescence assay to measure the variation in protein levels at centrosomes

中心体是充当有丝分裂纺锤体的两极保持基因组的完整性，或装配初级纤毛以促进感觉功能在细胞中的小但很重要的细胞器。一个蛋白的水平可在中心体比其他.cellular位置来调节不同的，并且在一些蛋白在不同点的细胞周期的中心体水平的变化似乎是中心粒装配的适当调节是至关重要的。我们开发了用于测量在一个蛋白的水平在中心体中固定的细胞来自不同样品的相对变化，在细胞周期的不同阶段，例如，或用各种试剂处理后的定量荧光显微术测定。这个测定的原理在于测量对应于蛋白质在一个小区域的背景校正的荧光强度，和正常化了的测量对相同的对不选择的实验中c而变化的另一蛋白质ondition。利用该测定在用BrdU脉冲和追策略学习未扰动细胞周期的组合，我们已定量具体地说在中心体验证我们最近的观察结果VDAC3的中心体池在细胞周期中被调节在中心体，可能是由蛋白酶体介导的降解。

我们最近的研究中发现VDAC3，线粒体孔蛋白16,28中的一个新的中心体定位和功能。几种哺乳动物细胞，包括使用VDAC3特异性抗体RPE1细胞的免疫染色表明突出中心体染色和较弱线粒体染色。我们还发现，中心体VDAC3优先与母亲中心粒，而无论是内源性的中心体池关联和异位表达VDAC3被降解16规范。为了验证定量的IIF分析我们检查了在S期的变化，中心体相关VDAC3池中的细胞。 这里，异步生长RPE1细胞用两种蛋白酶体抑制剂MG115或对照溶剂的DMSO 4小时。限制我们的分析，以细胞是在S相和接受中心体组装，我们只研究细胞掺入的BrdU杜环相同的4小时，有两个密集的中心体（在2微米彼此间隔）。我们研究了细胞染色的BrdU和细胞核从对照和MG115处理（ 图1A）的多个随机字段断定MG115的简要治疗没有影响BrdU掺入（大约58％）相比，控制处理（大致56％）在RPE1细胞。当我们比较仅细胞是在S期（作为判断两个γ微管蛋白灶的存在下在2微米彼此间隔开），我们考虑γ微管蛋白作为用于标准化的潜在内标。因此，我们首先如果在BrdU阳性细胞的中心体γ微管蛋白水平的变化时抑制蛋白酶体检查。不足为奇的是，出现了相当大的细胞至细胞的变化γ微管蛋白荧光强度从一个小区到另一个，并且我们发现，这种变异是最好的呈现在呈现集为一体的可用数据盒须图。我Ñ​​盒须图，方框代表下和上四分位数，在框中标记指示每个系列的中值，并且晶须表示的最小值和最大值，那是通常（尽管并不总是）离群值。正如图1D所示 ，在中心体γ微管蛋白水平是不与任一MG115或DMSO处理，从而验证γ微管蛋白作为此实验的内标BrdU阳性细胞间显著不同。而相比之下，γ微管蛋白，对应于中心体VDAC3背景校正的荧光强度为大约2.5倍高的中MG115处理的细胞比对照细胞（ 图1C）。当我们归针对该γ微管蛋白的总VDAC3荧光，所述倍增加略微下降至大约2倍（ 图1E）。虽然倍数变化是更大的不正常化，我们更有信心在规范化的数据，这是我们感到BETT的准确性由于来样加工呃控件MG115治疗，以及变化中的任何效果一般。 VDAC3染色在非中心体部位（ 图1B）或VDAC3（ 图1F）的总细胞水平没有受到蛋白酶抑制。因此，我们定量验证我们之前的那个VDAC3的中心体游泳池被蛋白酶体介导的降解调控的观察。 为了在细胞周期中，测量在中心体VDAC3的变化，我们用4小时的BrdU脉冲后跟标记细胞4小时追标记的细胞。由于只有S期的细胞将在脉冲期间掺入的BrdU（2中心体之间的平均距离为1.35±0.16微米），大多数BrdU阳性RPE1细胞后4小时追将在任一晚S期或早期G2期（ 2中心体之间的平均距离为1.55±0.22微米），用较小的人口在后期G2期，其中炫酷一些货币对显著分离30（双中心体&gt; 2微米之间的平均距离）。 γ微管蛋白，是一个重要的组成部分PCM极大地聚集在中心体在中心体成熟，发生在G2期31,32，这一增长使得它成为评估其他中心体蛋白的细胞周期变化的可怜的内部标准。另一方面，SAS6期间早S期招募procentrioles刺激侧手翻4,5,7的装配，并保持在该新形成的中心粒的底座，直至细胞进入有丝分裂时，它开始被降解（ 图2和参考文献5）。然而，在HeLa和U2OS细胞，SAS6的中心粒的水平逐渐增加细胞S期进展到G2期5。因此，我们首先测定了中心体SAS6水平相对于该Cep135，即定位于中心粒的整个近端另一个芯中心粒蛋白细胞周期33。我们没有观察到来自脉冲或追逐，BrdU阳性RPE1细胞Cep135或SAS6的背景校正总荧光强度的任何显著的差异时，中心体相距很近（ 图3A-D;&#39;接近&#39;细胞，其中平均两者之间的距离中心体是小于2微米）。因此，该归一化SAS6水平保持在这些细胞中（ 图3E）类似。但是，我们观察到适度增加SAS6的整体荧光强度值和相同Cep135（双中心体&gt; 2微米之间的距离;"遥远的"细胞）略有增加细胞中心体的地方是很好的分离。因此，在细胞中有两个良好分离的中心体的归一化总中心体SAS6水平略有下降，但比统计学显著高于细胞紧密间隔中心体。这可能是由于SAS6水平机智的内在规定H单元周期进程5。但是，它也有可能是轻微的增加（其中或更低统计学意义）是由于加入了分别进行了分析，相比于分析中的细胞与紧密间隔中心体2中心体在同一时间2中心体的荧光强度。尽管如此，当VDAC3的荧光强度进行归到SAS6单独的，在两个紧密间隔的中心体的VDAC3水平增加大约1.5倍的细胞中是在后期S-/早期G2期，相对于早期的S相细胞（ 图4A-C，F;&#39;接近&#39;细胞）。当这些细胞进展到晚期G2期，在两个中心体的标准化VDAC3水平相比，在晚S期（ 图4C，F），减少了2.4倍。 因为SAS6水平略微增加从晚S期到G2，使用SAS6作为内部标准导致分解的轻微高估rease在VDAC3水平在后期G2期的细胞（中心体由大于2μm隔开）。虽然我们的数据表明，Cep135是一个更好的选择，作为评估细胞周期的变化内部标准，我们不能用它来VDAC3，因为提供给我们的VDAC3和Cep135抗体都来自兔子。然而，通过Cep135-归SAS6的中值乘以用于SAS6标准化VDAC3（F VDAC3 /女SAS6）的中值使我们对Cep135标准化VDAC3〔（F VDAC3 /女SAS6）×近似值（F SAS6 / ˚FCep135）= F VDAC3 /女Cep135]。当我们进行这种分析，迟的S / G2早期和晚期G2之间在VDAC3水平的变化略微下降到2倍。 BrdU阳性细胞，在有丝分裂的任何阶段作为判断稠染色体和弱SAS6染色被排除在我们的分析中，由于在SAS6水平的急剧下降。然而，我们注意到，中心体VDAC3级别重新有丝分裂期间mained低（数据未显示）。因为Cep135水平有丝分裂期间不降（数据未显示），我们可以在原则上重复进行重新归一化过程，以估计Cep135标准化VDAC3水平的有丝分裂。然而，在现实中心体SAS6水平在有丝分裂太变量以允许准确测定在首位的SAS6标准化VDAC3水平。无论如何，总的来说，我们的数据证实VDAC3的中心体池是在细胞周期中控制在中心体。 <img alt="图1" src="/files/ftp_upload/52030/52030fig1highres.jpg" /> 图1.异步生长RPE1孵育细胞用BrdU和MG115（MG）或DMSO（DM）4小时。（A）中所示的是DMSO或MG115的细胞用抗BrdU抗体（红色）和Hoechst公司（随机字段处理DNA;蓝色）。在这里和在所有其它图像的条代表5微米。（BE）DMSO或MG115处理的细胞用抗VDAC3，γ微管蛋白和BrdU。 BrdU阳性细胞在相同的摄像条件拍摄（曝光时间─400毫秒为蓝色荧光团/尿嘧啶; 500毫秒为绿色荧光团/ VDAC3; 300毫秒用于红色荧光团/γ微管蛋白），和背景校正VDAC3的荧光强度和γ微管蛋白进行了测定。示VDAC3（VD3;绿色）的代表图像，γ微管蛋白（γ-桶;红色）和BrdU（蓝色）显示在（B）中 。在这里和在所有其它图像，插图显示数字变倍中心体由正方形表示。背景校正的荧光强度（F;以任意单位）是由25细胞对应VDAC3和γ微管蛋白分别标绘在（C）中盒须图，（D）中 。在这里，并在所有其他情况下，框表示上下四分，马rker在框中（ - ）表示各系列的中值，并且晶须表示最小值和最大值。 VDAC3从这些25细胞标准化荧光强度值标绘在（E）。为（CE）中，p的值从非配对t检验得到的。 ***表示P &lt;10 -5，和NS表示P&gt; 0.05。 （F）免疫印迹显示VDAC3（包括VDAC3a和VDAC3b 16）和γ微管蛋白（γ-桶），其中α微管蛋白（α-桶）加载控制的全细胞水平。 <a href="https://www.jove.com/files/ftp_upload/52030/52030fig1large.jpg" target="_blank">请点击此处查看本一个更大的版本数字。</a> <img alt="图2" fo:content-width="3in" src="/files/ftp_upload/52030/52030fig2highres.jpg" width="300" /> 图2.被标记为4小时溴异步增长RPE1细胞dU的脉冲和追为在 ​​不存在尿嘧啶的另外4个小时进行染色用抗SAS6（绿色）和γ微管蛋白（γ-桶;红色）。代表性的图像中（A） 的 S相显示SAS6染色（用两个靠近SAS6灶），（B），中期（两个弱SAS6灶在两极）和（C）末期（SAS6灶是从两极丢失）。 <a href="https://www.jove.com/files/ftp_upload/52030/52030fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52030/52030fig3highres.jpg" /> 染色Cep135和SAS6 图3.异步生长RPE1细胞用一个4小时的BrdU脉冲和追为在 ​​不存在尿嘧啶的另外4小时。BrdU阳性细胞在相同的摄像条件进行成像（曝光时间─400毫秒蓝色荧光/尿嘧啶; 400毫秒绿色荧光/ Cep135; 500毫秒为红色的荧光团/ SAS6），和SAS6和Cep135的背景校正的荧光强度进行了测定。 2中心体之间的距离，也测量在所有细胞中。一种细胞，其中两个中心体之间的距离小于2μm被视为"关闭"和其中的值是大于2μm被归类为"遥远"。 （AB）表示Cep135（C135;绿色）代表的图像，SAS6（红色）和BrdU（蓝色）被示出。在（B）中 ，C1和C2是代表&#39;遥远&#39;细胞的中心体2。 （CD）的背景校正的荧光强度（F;以任意单位）从15细胞每个类别对应于SAS6（C）和Cep135（D）的标绘在盒须图。 （E）SAS6的标准化强度来回回米的那些细胞被绘制在盒须图。为（CE）中，p的值从非配对t检验得到的。 *表示0.001 &lt;P &lt;0.05，与NS表示P&gt; 0.05。 <a href="https://www.jove.com/files/ftp_upload/52030/52030fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52030/52030fig4highres.jpg" /> 图从上述的BrdU脉冲追踪试验4. BrdU阳性细胞（图3）染色VDAC3和SAS6分别在相同的摄像条件拍摄（曝光时间─400毫秒为蓝色荧光团/尿嘧啶; 500毫秒为绿色荧光团/ SAS6;测定1000毫秒为红色的荧光团/ VDAC3），和SAS6和VDAC3的背景校正的荧光强度。2中心体之间的距离，测定各细胞中，并将细胞分类为&#39;C输&#39;（2中心体&lt;2微米之间的距离）&#39;&#39;（AC）的代表性图像，如在图3（2微米的中心体2之间的距离）。关闭"细胞中的BrdU脉冲（A）中遥远&gt;&#39;，在BrdU的追逐（B）中 ，并在BrdU的追逐（C）的 "遥远"细胞染色VDAC3（VD3;绿色），SAS6（红色）和BrdU（蓝色）被示出。在（C）中 ，C1和C2是在两个中心体。 [DE]背景校正的荧光强度（F;以任意单位）从15细胞每个类别对应于VDAC3（D）和SAS6（E）的标绘在盒须图。 VDAC3从那些细胞（F）的归一化强度绘制在盒须图。为（DF）中，p的值从非配对t检验得到的。 ***表示P &lt;10 -5，**表明10 -5 &lt;P &lt;0.001，而NS表示P&gt; 0.05。 <a href="https://www.jove.com/files/ftp_upload/52030/52030fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes at JoVE.com

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

定量免疫检测，以衡量在中心体的变异蛋白水平

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

quantitative-immunofluorescence-assay-to-measure-variation-protein

Nanyang Technological University

Research

JoVE Journal

Biology

15.0K 次观看.  The Ohio State University. Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein’s fluorescence intensity to that of an appropriate internal standard. 

视频: Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

定量荧光测量全球本地化翻译

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

科研

生物化学

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

突触荧光自动定量<em&gt; C。线虫</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

神经科学

人原代细胞中核蛋白染色的分析工作流程

A Versatile Pipeline for Analyzing Dynamic Changes in Nuclear Bodies in a Variety of Cell Types

Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the immunofluorescence technique. Investigating the dynamics of these foci under diverse cellular conditions via microscopy yields valuable insights into the molecular mechanisms governing cellular identity and functions. However, performing immunofluorescence assays across different cell types and assessing alterations in the assembly, diffusion, and distribution of these foci present numerous challenges. These challenges encompass complexities in sample preparation, determination of parameters for analyzing imaging data, and management of substantial data volumes. Moreover, existing imaging workflows are often tailored for proficient users, thereby limiting accessibility to a broader audience.
In this study, we introduce an optimized immunofluorescence protocol tailored for investigating nuclear proteins in different human primary T cell types that can be customized for any protein of interest and cell type. Furthermore, we present a method for unbiasedly quantifying protein staining, whether they form distinct foci or exhibit a diffuse nuclear distribution.
Our proposed method offers a comprehensive guide, from cellular staining to analysis, leveraging a semi-automated pipeline developed in Jython and executable in Fiji. Furthermore, we provide a user-friendly Python script to streamline data management, publicly accessible on a Google Colab notebook. Our approach has demonstrated efficacy in yielding highly informative immunofluorescence analyses for proteins with diverse patterns of nuclear organization across different&#160;contexts.

作者聚焦：使用免疫荧光检测研究人类细胞可塑性和环境适应性的综合表观遗传学分析

This method describes an immunofluorescence protocol and quantification pipeline for evaluating protein distribution with varied nuclear organization patterns in human T lymphocytes. This protocol provides step-by-step guidance, starting from sample preparation and continuing through the execution of semi-automated analysis in Fiji, concluding with data handling by a Google Colab notebook.

用于分析各种细胞类型中核体动态变化的多功能管道

Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the ...

遗传学

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

免疫荧光定量小鼠脑突触蛋白的异种分布

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.

Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.

在飞睾丸成像中心体

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

生物学

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators.
Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software1&#160;to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.

工作流的高含量，荧光标记的万能工具显微镜数据，支持开源软件的单个细胞定量

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

指南现代荧光定量免疫印迹与故障排除策略

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

定量免疫检测，以衡量在中心体的变异蛋白水平

摘要

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此视频中的章节

定量荧光测量全球本地化翻译

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

突触荧光自动定量

用于分析各种细胞类型中核体动态变化的多功能管道

用于分析各种细胞类型中核体动态变化的多功能管道

用于分析各种细胞类型中核体动态变化的多功能管道

免疫荧光定量小鼠脑突触蛋白的异种分布

在飞睾丸成像中心体

工作流的高含量，荧光标记的万能工具显微镜数据，支持开源软件的单个细胞定量

指南现代荧光定量免疫印迹与故障排除策略