我们承认艾米托马森为她准备这篇稿子帮助和弗雷德里克Skarstedt和Ryan克里斯蒂他们在数字的准备工作。

随着机构审查委员会的批准，进行了搜索的电子实验室的数据库来识别福尔马林固定，石蜡包埋（FFPE）代表巨细胞病毒（CMV）感染的胃肠道病例（GI）的诊断经病理组织学活检（苏木块和伊红（H＆E）和/或免疫组织化学（IHC））。 1，从FFPE胃肠道活检组织组织处理<ol><li>收集FFPE胃肠道活检组织块巨细胞病毒（CMV）定量聚合酶链反应（定量PCR）。这些块被存储在室温下。 </li><li>预标签1.7毫升离心管与块的唯一标识号。 </li><li>锁定切片机手轮。 </li><li>座椅的组织块装入纸盒夹。 </li><li>使用新鲜的，未使用的刀片，对准块在切片机刀角。 </li><li>调整切片机切成10微米厚的切片。 </li><li>解锁日Ë手轮和走向刀片块。 </li><li>切5节块在10微米厚的间隔，使每个切滚进组织的画卷。确保每个滚动包含所需的活检组织有足够的代表性。 </li><li>锁定切片机手轮。 </li><li>用钳子，将所有5滚动组织到预标记的离心管中，小心仅由石蜡操纵滚动，以减少潜在的交叉污染。被固定在管帽，并把它放回架上。 </li><li>从切片取出组织块。 </li><li>取下并扔掉刀片。 </li><li>净化可能已经走在与以往组织接触的任何表面或仪器。 </li><li>将一个新鲜的，未使用的刀片插入刀架。 </li><li>对于要处理的每个额外的块重复步骤1.4-1.14。可以存储包含组织滚动离心管在室温下直到添ËDNA提取。 </li></ol>从卷轴2。提取DNA FFPE胃肠道活检组织中注:此协议是改编自FFPE组织DNA提取试剂盒（注意:请参阅材料安全数据表（MSDS））的包装说明书。 <ol><li>加入1 ml二甲苯（注意:请参照MSDS），每个样品的离心管。盖上盖子，涡旋剧烈，持续10秒。 </li><li>继续进行DNA提取在制造商的产品插页中具体概述的进行了以下修改。 </li><li>先加6μL内部控制（IC）的每个离心管中。 <ol><li>继ATL /蛋白酶K裂解液孵育温度56℃，1小时，孵育99℃30分钟，而不是90℃，1小时。 </li><li>在最后的DNA洗脱步骤中，小心地打开DNA纯化柱的盖和应用50μl的蒸馏水DNA酶/ RNA酶的水在膜的中心，而不是buffeřATE。 注:请参考说明书的相应的存储和准备试剂。应的DNA提取所有5 FFPE滚动。执行所有离心步骤在室温（15-25℃）。 </li></ol></li></ol> 3，定量PCR巨细胞病毒<ol><li>聚合酶链反应（PCR）程序<ol><li>生成一个Excel工作表中的PCR为巨细胞病毒检测运行。这个Excel工作表计算所需弥补主结构与镁每种试剂的总量。它乘以每个样品所需的每种试剂的用量（如下所列）样品和对照的次数加一（以补偿损失，由于移液）。 <ol><li>包括在量每个PCR反应如下试剂所示: 试剂0;每个样品量 巨细胞病毒LC预混液小瓶12.5微升 镁溶液黄色小瓶2.5微升 总成交量15.0微升</li></ol></li><li>包括在每一个实验中，运行水或无靶对照（NTC），阴性对照组，低阳性对照，高阳性对照，要么QS3或QS4，所有患者样本。毛细管应设置在此顺序。 </li><li>得到冷却块具体针对被存储在4℃的实时PCR仪器置于适当的细管中的冷却块。请勿触摸毛细管的表面。处理的毛细血管时，请务必使用手套。 </li><li>设置所有聚合酶链反应在清洁油烟机不沾放大生产TS。 <ol><li>从PCR试剂盒中移除主小瓶的正确数目，以测试所有患者样品，加5控制。让主人小瓶，镁溶液，PCR级水解冻的冷却块。每个主管含有足够的试剂进行12项测试。 </li><li>轻轻涡旋主小瓶，并在最大速度快速旋转它们在离心机。 </li><li>结合每个主小瓶的内容到一个小瓶中，并再次轻轻旋涡​​。 </li><li>结合起来，在工作表中的PCR表示成一个单一的无菌离心管的金额母液和Mg的解决方案。 </li><li>轻轻混匀，并等分15μl反应混合液用Mg到每个毛细管，除了在这对定量标准（QS）中第5位的毛细管。 </li></ol></li><li>移动包含毛细管和主结构与镁从清洁油烟机的油烟机加工冷却块。 </li><li>加入2μl内部控制（IC）的其余30μl反应的拌上镁。轻轻混匀后分装15微升至毛细管中的位置5（位置为QS）。 </li><li>移液管加入10μl水，对照DNA或样品DNA插入适当的毛细管。帽用封端工具的毛细管。 </li><li>采取冷却块/样品的实时PCR仪。 </li></ol></li><li>程序的实时PCR仪（ 见表1） </li><li>扩增和检测的实时PCR仪进行<ol><li>登录到计算机</li><li>双击仪器图标，然后登录到软件。 </li><li>打开的实时PCR仪器</li><li>点击从正面屏幕"巨细胞病毒/ EB病毒"。 </li><li>按照宏观向导的提示进行操作。 </li><li>选择框运行自检时，软件提示。自检必须进行每天一次的仪器在使用中。 </li><li>命名运行。 </li><li>调整位于上部来氟米特样本计数包含在运行页面吨角落的对照和患者的总和，并输入每个患者样本的识别号码。 </li><li>点击"绝对定量"选项卡上。 </li><li>根据"样品类型"，指定为标准适当的样品类型。 </li><li>在为定量标准（QS）的预期浓度的类型。 </li><li>从冷却器块取出毛细管并将其放置在转盘中的相应位置（冷却块样品＃1应放置在1号位置上的旋转木马等 ）。 </li><li>将转盘到仪器的离心机，并按下启动。 </li><li>打开离心机除去转盘。 </li><li>放置在传送带进入实时PCR仪。合上盖子。 </li><li>按"开始→运行"。 </li><li>实时PCR仪会检查每个位置的样本。不要走开从仪器上，直到这个检查已经完成，尤其cially如果毛细管的一个不是在传送带上。该仪器会注意到这一点，并询问是否运行将继续进行。临走回答是。 </li><li>一旦运行完成后，关闭生成的报告。 </li><li>点击"绝对定量"。 </li><li>点击旁边的箭头颜色补偿。 </li><li>从下拉框中点击"选择颜色补偿"。 </li><li>选择最新的色彩补偿文件适用于运行。 </li><li>单击OK（确定）时，框弹出。 </li><li>点击箭头由标准曲线。 </li><li>选择"使用外部"，从下拉菜单中。 </li><li>选择最当前的外部标准曲线文件，然后单击确定。 </li><li>再看下的绝对定量菜单中每条曲线以保证曲线的存在，而不是平线，如果浓度分配。的绝对定量为705/Back 530自动分析。 </li></ol></li><li>曲先进而精湛的控制<ol><li>产生每当收到试剂的新批号一个新的标准曲线，或每半年一次，取其更频繁。 </li><li>加入10微升每个标准对母液与镁。考虑包含在套件中先前提取的DNA的标准。 </li><li>加入1μl内部控制的每个标准的主混合物用Mg用于产生标准曲线，以保证准确的定量。不要添加IC到主结构与镁的无模板对照（NTC）。 </li><li>准备一个NTC和定量的标准QS 4，3，2，1，和1:10稀释QS4的运行，以产生标准曲线3次重复，共16个数据点。 </li><li>选择标准重复"重复"中的下拉菜单</li><li>选择通道530/back没有和分析时，运行打开颜色补偿， </li><li>在"标准曲线（在运行）"，选择"另存为前TERNAL"并命名该曲线。保存时输入注释"为标准曲线应用"。 </li><li>格拉夫结果。由此产生的线性相关系数必须≥0.81。 </li><li>日常运行，包括QS3或QS4的一个副本在运行控制。在分析阶段当前外部标准曲线可导入运行。 </li><li>运行控制:无DNA对照，阴性对照，低，高的阳性对照。预计病毒载量是确定和调整各厂商和批号。 </li><li>加的IC到每个样品的DNA提取过程中控制。 </li><li>如果任何控件都属于超出范围，记录不能接受的结果，并参考化验排除故障。 </li></ol></li><li>的频率控制<ol><li>包括在每次运行:一个NTC，阴性，弱阳性，强阳性，和标准的控制。 </li></ol></li></ol>注:可接受的限度控制。无峰应遵守d表示无模板（NTC）和阴性对照。峰值应该出现在530路和预期的病毒载量被确定并调整每个厂商和批号为低和高阳性对照。

<ol>
	<li>Hodinka, 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=.">.</a> Manual of Clnical Microbiology. 2, 1558-1574 (2011).</li><li>Lasry, 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=Interstrain+variations+in+the+cytomegalovirus+(CMV)+glycoprotein+B+gene+sequence+among+CMV-infected+children+attending+six+day+care+centers.">Interstrain variations in the cytomegalovirus (CMV) glycoprotein B gene sequence among CMV-infected children attending six day care centers.</a> The Journal of infectious diseases. 174, 606-609 (1996).</li><li>Murph, J. R., Baron, J. C., Brown, C. K., Ebelhack, C. L., Bale Jr, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+occupational+risk+of+cytomegalovirus+infection+among+day-care+providers.">The occupational risk of cytomegalovirus infection among day-care providers.</a> JAMA : the journal of the American Medical Association. 265, 603-608 (1991).</li><li>Pass, R. F., 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=Vaccine+prevention+of+maternal+cytomegalovirus+infection.">Vaccine prevention of maternal cytomegalovirus infection.</a> The New England journal of medicine. 360, 1191-1199 (2009).</li><li>McCoy, M. H., 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=qPCR+increases+sensitivity+to+detect+cytomegalovirus+in+paraffin-embedded,+formalin-fixed+tissue+of+gastrointestinal+biopsies.">qPCR increases sensitivity to detect cytomegalovirus in paraffin-embedded, formalin-fixed tissue of gastrointestinal biopsies.</a> Human Pathology. 45, 48-53 (2014).</li><li>Mills, A. M., Guo, F. P., Copland, A. P., Pai, R. K., Pinsky, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+CMV+detection+in+gastrointestinal+mucosal+biopsies+using+immunohistochemistry+and+PCR+performed+on+formalin-fixed,+paraffin-embedded+tissue.">A comparison of CMV detection in gastrointestinal mucosal biopsies using immunohistochemistry and PCR performed on formalin-fixed, paraffin-embedded tissue.</a> The American journal of surgical pathology. 37, 995-1000 (2013).</li><li>Krech, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement-fixing+antibodies+against+cytomegalovirus+in+different+parts+of+the+world.">Complement-fixing antibodies against cytomegalovirus in different parts of the world.</a> Bulletin of the World Health Organization. 49, 103-106 (1973).</li><li>Chou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newer+methods+for+diagnosis+of+cytomegalovirus+infection.">Newer methods for diagnosis of cytomegalovirus infection.</a> Reviews of infectious diseases. 12 Suppl 7, (1990).</li><li>Brytting, M., Xu, W., Wahren, B., Sundqvist, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytomegalovirus+DNA+detection+in+sera+from+patients+with+active+cytomegalovirus+infections.">Cytomegalovirus DNA detection in sera from patients with active cytomegalovirus infections.</a> Journal of clinical microbiology. 30, 1937-1941 (1992).</li><li>Revello, M. 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=Human+cytomegalovirus+in+blood+of+immunocompetent+persons+during+primary+infection:+prognostic+implications+for+pregnancy.">Human cytomegalovirus in blood of immunocompetent persons during primary infection: prognostic implications for pregnancy.</a> The Journal of infectious diseases. 177, 1170-1175 (1998).</li><li>Shinkai, M., Bozzette, S. A., Powderly, W., Frame, P., Spector, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+urine+and+leukocyte+cultures+and+plasma+DNA+polymerase+chain+reaction+for+identification+of+AIDS+patients+at+risk+for+developing+human+cytomegalovirus+disease.">Utility of urine and leukocyte cultures and plasma DNA polymerase chain reaction for identification of AIDS patients at risk for developing human cytomegalovirus disease.</a> The Journal of infectious diseases. 175, 302-308 (1997).</li></ol>

作者什么都没有透露。

许多实验室方法可用于巨细胞病毒的诊断病毒（CMV）感染，包括血清学，病毒培养，分子血症测定法，活组织检查材料的组织学，并且，最近表明，分子的福尔马林固定，石蜡包埋（FFPE）的活检测定组织5,6。血清学检查，一旦确诊的中流砥柱，只有可靠地识别风险，并不会与急性CMV感染7很好的相关性。病毒培养，常规和早期抗原壳小瓶中，通过长时间的测定时间的阻碍，在后者<...

的巨细胞病毒（CMV）感染在临床标本中的解释是非常重要的。巨细胞病毒是疱疹病毒的betaherpesvirinae亚家族的成员。类似于其他疱疹病毒，巨细胞病毒必须建立持续性或隐性感染者的能力特征是缺乏病毒复制活跃1。在压力或其他免疫抑制的时间可能会发生病毒的再活化，导致病毒复制活跃其特征在于，病毒体释放，这可以伴随疾病表现2-4。胃肠道（GI），巨细胞病毒病的症状通常包括腹部疼痛和/或血便1。 巨细胞病毒性肠胃炎的组织学诊断采用苏木精伊红（H＆E），以确定巨细胞病毒包涵体。在临床上怀疑是高还没有明显的包裹体观察病例，免疫组织化学（IHC）经常作为一种辅助的测试方法。然而，IHC也可以通过稀有非经典出现细胞染色模式（模棱两可）的阻碍，使得难以解释（ 图2）5。它试图利用DNA从福尔马林固定，石蜡包埋（FFPE）的GI活检组织和定量聚合酶链反应（定量PCR）来检测CMV胃肠活检萃取。该技术已被证明是有价值的巨细胞病毒在胃肠活检的检测，并且特别有用的模棱两可的IHC染色例5,6。此外，定量PCR也显示出额外的巨细胞病毒临床试验数据以及相关可用时6。在检测巨细胞病毒使用的qPCR可能会导致早期诊断和治疗的情况下，临床怀疑为巨细胞病毒是很高，但H＆E和CMV免疫组化是否定的。

<table border="0" cellpadding="0" cellspacing="0" width="745">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">1.7 mL microfuge tubes</td>
			<td style="width:195px;">Costar</td>
			<td align="right" style="width:160px;">3620</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">serrated forceps</td>
			<td style="width:195px;">Electron Microscopy Services</td>
			<td style="width:160px;">62086-1S</td>
			<td>Micro Forceps MF-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Tabletop centrifuges (microfuge)</td>
			<td style="width:195px;">Eppendorf</td>
			<td align="right" style="width:160px;">5424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Xylene</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:160px;">Fisher Scientific: X3P</td>
			<td>1 gallon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Ethanol</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:160px;">Fisher Scientific: ET108</td>
			<td>96-100%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">microtome</td>
			<td style="width:195px;">Leica</td>
			<td style="width:160px;">RM2255</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">QIAamp DNA FFPE Tissue Kit</td>
			<td style="width:195px;">Qiagen</td>
			<td align="right" style="width:160px;">56404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">artus CMV LightCycler PCR ASR</td>
			<td style="width:195px;">Qiagen</td>
			<td align="right" style="width:160px;">4500025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">CMV LC PCR Supplement Kit</td>
			<td style="width:195px;">Qiagen</td>
			<td align="right" style="width:160px;">1031873</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">LightCycler centrifuge</td>
			<td style="width:195px;">Roche</td>
			<td align="right" style="width:160px;">75005087</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">LightCycler Cooling Block</td>
			<td style="width:195px;">Roche</td>
			<td align="right" style="width:160px;">10800058001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">LightCycler Capping Tool</td>
			<td style="width:195px;">Roche</td>
			<td align="right" style="width:160px;">3357317</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Color Compensation Kit</td>
			<td style="width:195px;">Roche</td>
			<td align="right" style="width:160px;">2158850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">LightCycler 20 &#956;L Capillaries</td>
			<td style="width:195px;">Roche</td>
			<td style="width:160px;">1 909 339</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">blades</td>
			<td style="width:195px;">Sakura</td>
			<td style="width:160px;">Fisher Scientific: 4689</td>
			<td>Accu-Edge low profile</td>
		</tr>
	</tbody>
</table>

qpcr is a sensitive and rapid method for detection of cytomegaloviral dna in formalin-fixed, paraffin-embedded biopsy tissue

关键的是要确定在胃肠道（GI）的免疫抑制病人的呼吸道巨细胞病毒（CMV）感染，给出了发展为严重感染的风险更大。已经开发了许多实验室方法为巨细胞病毒感染的检测，包括血清学，病毒培养和分子生物学方法。通常情况下，这些方法反映全身受累的巨细胞病毒，不具体确定局部组织的参与。因此，在胃肠道中检测CMV感染是通过活检组织的传统组织学频繁进行。苏木精伊红（H＆E）染色与免疫组织化学（IHC）结合仍然检查这些切片的支柱。 H＆E和免疫组化有时会导致非典型（模棱两可）染色模式，使解释困难。它表明，定量聚合酶链反应（定量PCR）对CMV可以成功地对福尔马林固定，石蜡包埋（FFPE）的活检组织为非常执行高敏感性和特异性。本协议的目的是演示如何在临床实验室的设置进行定量PCR检测巨细胞病毒在FFPE组织活检的检测。这种方法很可能是为病人大有裨益的胃肠活检模棱两可染色巨细胞病毒的病例。

共有228组织块用定量聚合酶链反应（定量PCR），这是由91巨细胞病毒（CMV）阳性例可疑CMV免疫组化的基础上组织学和积极的免疫组织化学（IHC），18，79和阴性对照试验。 如图2，巨细胞病毒阳性病例会表现出典型的巨细胞病毒包涵体（A）和/或阳性IHC染色（B）。模棱两可的情况下会表现出罕见的，非经典的出现染色模式（C），而阴性对照会显示?...

Watch this Scientific Journal Video about qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue at JoVE.com

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

定量PCR是用于检测巨细胞病毒DNA在福尔马林固定，石蜡包埋的活检组织一个灵敏快速的方法

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

qpcr-is-sensitive-rapid-method-for-detection-cytomegaloviral-dna

Research

JoVE Journal

Biology

15.7K 次观看.  Indiana University School of Medicine. This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

视频: qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxy&#64258;uorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.

小学组织两个胸苷类似物的免疫荧光检测（CldU IDU）

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

科研

生物学

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

从石蜡包埋材料的遗传和表观遗传分析的DNA提取

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

修正了福尔马林蛋白质组学样品的制备和石蜡包埋组织

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

微RNA<em&gt;原位</em&gt;杂交的福尔马林固定的肾组织

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

检测现场的<em&gt;大肠杆菌</em&gt; O157:H7细胞的PMA-qPCR的

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

快速分离纯化线粒体移植用组织分离和鉴别过滤

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

The goal of this video is to demonstrate how to perform&#160;automated DNA extraction from formalin-fixed paraffin-embedded (FFPE) reference standard cell lines and digital droplet PCR&#160;(ddPCR) analysis to detect rare mutations in a clinical setting. Detecting mutations in FFPE samples demonstrates the clinical utility of ddPCR in FFPE samples.

采用数字液滴PCR技术检测BRAF V600E突变的福尔马林固定石蜡包埋的参考标准细胞系

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

福尔马林固定石蜡包埋的组织核心的制备RNA和DNA提取

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...