支持JDRF，美国国立卫生研究院（R01 - DK081469），宾夕法尼亚州联邦（卓越再生医学中心授予4100043362），March of Dimes的（罗勒奥康纳入门学者研究奖），和宾夕法尼亚大学DERC试点和可行性补助金（DK19525）。

1。标签组织与CldU和IDU <ol><li>胸苷类似物（CldU IDU）溶解于水。权衡毫克/毫升每一种化学品，并添加到单独的蒸馏水玻璃瓶。我们通常准备的时间每1升。 </li><li>化解不小的轰动板的化学品或其他形式的搅拌器。 CldU会溶解在10分钟。在室温下的躁动。 IDU要求在37小时的搅拌°一个摇床中的C。水源可以影响IDU的溶解度。如果IDU仍然一夜之间晃动后恢复正常运作，请尝试使用清洁的水来源。在4 ° C黑暗环境中存储的解决方案。 </li><li>管理的第一胸苷包含所需的标签内的老鼠的解决方案（CldU或IDU）。不要让小鼠在访问期间的标签未标注水。当指定的标签内完成，开始一个与未标记的水冲洗至少24小时（胸苷含有类似物有几个小时的血清半衰期）。管理的第二个胸苷包含所需的标签内的老鼠的解决方案（CldU或IDU）。定期注水瓶，以防止脱水！ </li><li>此外，小鼠连续腹腔注射的CdlU然后IDU可以标示。准备在10毫克/毫升的浓度CldU或注射毒品。 IDU可能需要明智的下拉除了浓氢氧化钠剧烈摇晃的​​解决方案进入暂停。不添加多余的氢氧化钠。添加氢氧化钠单下降到10 mL的溶液的IDU的一个小时的搅拌，然后在37 °一个摇床中的C。如果不是在溶液中，除了重复的氢氧化钠。每克体重注入腹腔空间100mcg。 </li><li>这一协议的基本控制幻灯片。因此，我们强烈建议调查人员进行了胸苷模拟标签的几个变化，以确保敏感性和特异性。这些应包括：1。小鼠只有CldU标记; 2。小鼠与IDU只标记; 3。小鼠与CldU然后IDU相继被标记; 4。顺序标记以相反的顺序，先与IDU，然后CldU的小鼠5只，只有水标记模拟。学习与CldU和IDU的标签时，调查人员应始终贯彻整个协议从该组织的利益与上述所有5套控制幻灯片。 </li></ol> 2。组织收获，固定，和幻灯片的制备<ol><li>使用适当的麻醉，进行致命的过量，然后通过放血牺牲的鼠标。取出新鲜组织，并与4％paraformalehyde在4 ° C，24小时内修复。转移组织嵌入1:4福尔马林缓冲和储存在4 ° C。 </li><li>另外，进行致命的过量然后通过鼠标左与盐溶液的心脏心室灌注，直到血液从人体中清除，然后立即用30-50毫升的4％多聚甲醛灌注。在4 ° C删除嵌入的兴趣和1:4福尔马林缓冲存储的组织。 </li><li>组织脱水和石蜡包埋后，切成5微米的组织切片和地点将收取的幻灯片。烘烤的幻灯片，在65 ° C下16小时，以确保组织粘连，滑（抗原修复以下步骤至关重要）。 </li></ol> 3。脱水，通透，和抗原修复<ol><li>沉浸在两个二甲苯浴5分钟的幻灯片中删除多余的石蜡。每个。 </li><li>补充水分的组织，通过加水稀释浸泡在乙醇幻灯片：100，95，90，80 70和50％。浸泡3分钟的幻灯片。每个浓度，开始与100％和50％的最终浸泡走向。 </li><li>洗5分钟的幻灯片。在1X PBS。 </li><li>通透细胞浸泡在0.2％的Triton - X为5分钟（每次新）解决方案的幻灯片。 </li><li>微波组织抗原修复准备的幻灯片。放入一个玻璃烧杯的幻灯片足够量0.01M pH值6.0柠檬酸钠缓冲区，这样的解决方案涵盖了5厘米以上的幻灯片的顶部蒸发损失。 </li><li>微波幻灯片，直到溶液沸腾（3-10分钟在满功率）。减少电源，直到解决方案是慢慢沸腾的（10-80％），并继续为一个额外的20分钟。微波定期检查，以确保解决方案仍然在慢熬。 </li><li>置于烧杯中含有一条长凳上的幻灯片，并允许轻轻地冷却到室温（约2小时）。 </li><li>确认幻灯片在室温下使用温度计。 </li><li>在1.5N盐酸中浸泡40分钟的幻灯片。在室温下。 </li><li>洗两次幻灯片1X PBS，5分钟。每洗。 </li></ol> 4。小学及中学抗体<ol><li>重要的是，幻灯片通过整个协议仍然非常湿润。干组织将有更多的汽车荧光，使所产生的图像无法使用。为了防止死亡，在时间的过程一张幻灯片。 </li><li>圈每个部分阻塞的液体（即蜡）笔在幻灯片上。到1X PBS的容器放置在幻灯片。 </li><li>放置湿纸巾潮湿的孵化室平放不透气的塑料容器的底部。 </li><li> 1X PBS稀释10％的驴血清拦截的解决方案。重要的是要完全覆盖与解决方案的每个部分。对于组织切片，1 × 1.5厘米，解决方案，每节50微升量就足够了。 </li><li>封闭液添加的幻灯片。从1X PBS中取出的幻灯片，并消除多余的液体，由上一摞干纸巾轻轻拍打幻灯片边缘。在孵化室中放置幻灯片，并添加50微升10％块​​解决方案的每个部分。当完成所有的幻灯片关闭容器在室温下孵育1小时。 </li><li>准备第一抗体稀释，以检测IDU。 1:25 1X PBS浓度在5％驴血清稀释的小鼠抗BrdU。从幻灯片塔块的解决方案，并重新放入孵化室。每节添加上述主要抗体，4℃过夜孵育° C。 </li><li>准备高紧缩洗（最大限度地减少鼠抗BrdU抗血清CldU约束力）。彩妆新鲜低盐TBST缓冲液（三，36MM，50MM氯化钠0.5％Tween - 20的pH值8.0）。您将需要40毫升每对缓冲区的幻灯片。添加40毫升缓冲每次50 mL锥形管。添加幻灯片之前，解决方案预热至37 ° C。 </li><li>两个时间，从孵化室中删除幻灯片，将它们放置到（使组织样本面对彼此远离），然后点击关闭多余的抗体。充满低盐TBST和覆盖的管放置在幻灯片。搅拌20分钟管。在一个惊天随温度的细菌培养培养箱在37 ° C，并设置到225转的速度。 </li><li>洗净的幻灯片，在新鲜的1X PBS的2倍。 5分钟。每洗。 </li><li>准备下一个主要的抗体溶液CldU和组织抗原的检测，如果需要的话。 1:25 1X PBS浓度在5％驴血清稀释的鼠抗BrdU。在工作​​纳入初级抗体溶液浓度稀释对组织抗原的主要抗血清。 </li><li>点击多余的1X PBS的幻灯片，并将其放置在孵化室。各节及加入一抗孵育4℃过夜° C。 </li><li>洗净的幻灯片，在新鲜的1X PBS的2倍。 5分钟。每洗。 </li><li>准备二次抗体溶液。稀CY2驴抗大鼠，以1:25和Cy5驴抗小鼠在1X PBS含有5％驴血清的解决方案1:500。加入Cy3标记辅助检测组织抗原的抗血清的主要物种。同时，淡化了的DAPI 0.4mg / mL到1:150的股票在二级抗体溶液的解决方案。 </li><li>在孵化室，塔多余的1X PBS关闭幻灯片和地点。加入二级抗体溶液，每节1小时在室温下孵育。 </li><li>洗净的幻灯片，在新鲜的1X PBS的2倍。 5分钟。每洗。 </li><li>塔多余的1X PBS幻灯片和延长黄金防褪色安装媒体（不要使用带有dyanine染料Vectashield！）坚持用玻璃盖。按盖玻片消除气泡。保存在4 ° C。 </li></ol> 5。成像和分析<ol><li>配备高能量光源，计划复消色差的目标，和高效率的显微镜摄像头（如滨松ORCA二）荧光显微镜图像的利益的组织。 AMCA（DAPI），CY2（CldU），Cy3标记（组织抗原），和Cy5（IDU）渠道捕捉图像。合并图像来创建一个单一的多层图像文件。 </li><li>如果正确执行，DAPI染色细胞核，将均匀明亮。组织抗原（如胰岛素），揭示细胞的利益。如果用DAPI或组织抗原染色弱或不一致的，沉浸在1X PBS过夜幻灯片，删除盖玻片和重复二次血清中的应用。 </li><li>重点组织抗原在Cy3标记。检查CldU和IDU染色CY2和Cy5通道，通道之间切换。寻找潜在的交叉反应过度IDU和CldU合作阳性标记，之间。寻找只包含CldU或注射毒品的组织。缺乏CldU或注射毒品阳性细胞核可能表明一个标签或染色的技术问题。在这种情况下，寻找一个高度复制的组织（如淋巴结）。 </li><li>分析图像。显示层含有的DAPI和组织抗原。白板使用中的主导和在非优势手的细胞计数，每个细胞核标记与干擦除标记（又名白板）的对比标志。的DAPI阳性细胞计数组织内的利益。记录总细胞计数。使用干燥的橡皮擦，以消除痕迹。显示层小号包含的DAPI，组织抗原，以及CldU。 CldU阳性细胞计数组织内的利益。记录CldU细胞计数，但不清除标记。更改显示可视化层包含组织抗原，CldU，IDU。计数CldU IDU双阳性细胞。记录的值，清除所有标志，并显示层包含的DAPI，组织抗原，IDU。总数IDU阳性细胞计数范围内的利益的组织。记录IDU细胞计数。 </li></ol> 6。代表性的成果我们按顺序标记，一个星期，然后IDU 6周龄雌性小鼠两个星期，立即牺牲CldU。胰腺染色检测CldU，IDU，胰岛素（组织抗原），以及DAPI。与胰岛素的胰岛均匀染色。 CldU染细胞核IDU染细胞核（图2）不同。胰岛外的许多核CldU IDU合作阳性（图2，右下角，白色箭头）。一个单一的胰岛素阳性细胞为CldU和IDU（图2，右下角，洋红箭头）核染色。 小鼠。根据费城儿童医院的IACUC委员会的指引，所有实验用小鼠。泰康利（Germantown的纽约），在实验动物设施设在费城儿童医院，和美联储鼠标饮食5015 PMI国际营养（Richmond.印第安纳州）均购自女B6.129 F1野生型小鼠。 标签策略。 IDU（红色标记在这项计划）标记的第一个单元格划分与CldU事件（标记在这个计划中的绿色）和第二次细胞分裂，细胞在合作与CldU和IDU（绿色/红色）标记的细胞分工的结果顺序。 胸苷内小鼠的胰腺细胞的新陈代谢标签代表的结果。小鼠胰腺胰岛胰岛中心与免疫荧光图像。鼠标注入CldU为1周，然后立即牺牲前2周在饮水IDU。胰腺样品处理，在协议中所述。 40X的客观物象，不同的层，显示适合用于计数。合并后的图像包含的DAPI（白）CldU（绿色），IDU（红色），胰岛素（黄色）。 （上，左）的DAPI和胰岛素。 （上，中，右）CldU和胰岛素。 （下，左）IDU和胰岛素。 （低，右）CldU，IDU和胰岛素。比例尺：100μm的。 <img src="/files/ftp_upload/2166/2166fig1.jpg" alt="图1" /> 图1标签策略。 IDU（红色标记在这项计划）标记的第一个单元格划分与CldU事件（标记在这个计划中的绿色）和第二次细胞分裂，细胞在合作与CldU和IDU（绿色/红色）标记的细胞分工的结果顺序。 <img src="/files/ftp_upload/2166/2166fig2.jpg" alt="图2" /> 图2。胸苷代表标签内小鼠的胰腺细胞的新陈代谢结果。小鼠胰腺胰岛胰岛中心与免疫荧光图像。鼠标注入CldU为1周，然后立即牺牲前2周在饮水IDU。胰腺样品处理，在协议中所述。 40X的客观物象，不同的层，显示适合用于计数。合并后的图像包含的DAPI（白）CldU（绿色），IDU（红色），胰岛素（黄色）。 （上，左）的DAPI和胰岛素。 （上，中，右）CldU和胰岛素。 （下，左）IDU和胰岛素。 （低，右）CldU，IDU和胰岛素。比例尺：100μm的。

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ordered+architecture+of+murine+ear+epidermis+is+maintained+by+progenitor+cells+with+random+fate.">The ordered architecture of murine ear epidermis is maintained by progenitor cells with random fate.</a> Dev Cell. 18 (2), 317-317 (2010).</li></ol>

与小鼠的所有实验均在费城儿童医院的体制动物护理和使用委员会（IACUC）的指导方针，在动物设施。

我们的做法胸苷为基础的方法，标记细胞的营业额已在生物医学研究中的许多潜在的应用。迄今为止，我们集中对胰腺，但我们也采用这一战略，研究皮肤细胞的新陈代谢，肠，肝，肾上腺，肾，睾丸，卵巢，甲状腺，淋巴结，造血，和脑1。由于细胞的营业额变化，从组织到组织，应当自定义标签的战略获得利益的机构，从最引人注目的信息。莫里森和他的同事们使用CldU和IDU为1天，每个标签造血2。库恩和他的同事们使用，每次4天CldU和IDU检测细胞分裂再生心肌3。相比之下，我们已经使用了长达9个月共标签基底细胞胰岛内的营业额，组织与动物年龄 1,4-6最小的细胞周转CldU和IDU 。最明显的潜在的应用是我们的标签策略的研究，​​以确定细胞组织内稳态营业额。但我们的技术有一些其他潜在的应用。例如，我们最近还应用这种技术来识别致癌基因改造，在组织内，增加复制的重点领域，可以很容易地增加纳入 CldU或IDU 5确定。莫里森和他的同事们使用CldU和IDU为1天，每一个测试标签保留造血干细胞的行为。我们也适用于连续胸苷类似物的标签，以确定是否过境扩增细胞有助于增长或组织 1续期。胸苷类似物在此应用序贯给药的代表参与组织的动态平衡细胞研究的起源和生存的一个新方法。 胸苷类似物不应该使用不小心。合成胸苷类似物有细胞分裂的潜在毒性，其使用，理论上可损害细胞的营业额在不断增长的动物。胸苷类似物已被用来作为为癌症病人的放射增敏剂，并可能会减缓细胞的营业额。因此，我们呼吁审慎调查，在其利益的组织的潜在毒性的调查。例如，Trumpp和他的同事们发现，全身BrdU可以强制造血干细胞进入细胞周期7。举例来说，鲁特和他的同事观察到高剂量胸苷类似物是有毒的发展胰腺8。最近在KineMed Hellerstein和他的同事，公司发现BrdU管理减慢16-25％的胰岛内使用一个专有的重水标记技术的扩散。 Hellerstein和他的同事们使用的胰岛整个筹备工作中许多其他内分泌和非内分泌细胞类型，这可能包括多达50％的总胰岛制剂。但是，我们发现，长时间输注的BrdU不影响在年轻的成年小鼠的β-细胞的增殖，Ki67表达4。同样，长期CldU和IDU的管理似乎并不损害β细胞的大规模扩张 1 。此外，β细胞增殖率相当于之间长期的时间，只为几个小时（相当于时段的利率正常化）标记的小鼠与BrdU标记的小鼠。总之，这些结果表明，小鼠可以安全地忍受长期低剂量胸苷类似物的管理。尽管如此，我们不能排除胸苷类似物在胰腺β细胞的生长的潜在毒性。因此，我们将继续标签胸苷类似物小鼠时执行控制，并敦促其他研究人员也这样做。 我们的顺序胸苷模拟标签技术也不是没有缺陷和技术挑战。因此，控制幻灯片尤为重要。我们已经生成控制各种组织组成的幻灯片，1）小鼠只有CldU标记;。2）小鼠只与IDU标记; 3）小鼠CldU和然后IDU顺序标记; 4）小鼠顺序标记以相反的顺序，先与IDU，然后CldU; 5）小鼠，只有水标记模拟。学习与CldU和IDU的标签时，调查人员应随身携带上述所有5套幻灯片，从组织利益与整个协议。 轻微CldU和IDU之间的交叉反应，是一个不幸的，但不可避免的缺点，两个胸腺嘧啶核苷类似物的标记。 CldU和IDU技术基础上的亲疏差异之间的抗血清在不同的物种（小鼠和大鼠）原先对BrdU派生。我们的协议，而繁琐的，是为了尽量减少这种交叉反应。因此，我们敦促谨慎考虑跳过步骤的协议（S）之间的调查。特别是，我们有二次血清中遇到的问题，在小鼠和大鼠之间的交叉反应。这可以最小化杰克逊的抗血清的使用，完全在小鼠和大鼠之间相互吸附。 我们偶尔会无法充分检测胸腺嘧啶掺入。在这种情况下，至关重要的是使用阳性对照幻灯片，以确定试剂或步骤不能正常工作。困难通常是由于抗血清，干燥的幻灯片，或核通透不足的不良等分。我们未能成功标签发展与IDU胚胎。因此，并非所有的组织可能会渗透到IDU。 双胸苷模拟标签CldU和IDU的替代品在地平线上。 EDU（5 -乙炔- 2尿苷）可基材为铜催化点击化学检测10,11。 EDU检测方法不要求分离的DNA链，因此非常适合进行分析流式细胞仪检测12。 EDU是兼容的，但不交叉反应用BrdU 10。 EDU已用于量化各种小鼠组织的营业额，包括胰腺β细胞13，肠道L细胞14和15表皮细胞。 EDU是相当昂贵的时刻（$ 1000美国每500毫克）。尽管如此，埃杜的检测，似乎更为敏感比BrdU检测10。因此，它可能是经济可行的结合，而不是CldU和IDU的埃杜和BrdU。这种方法也可能会允许三个不同的细胞分裂埃杜，CldU和IDU三重标记的小鼠轮检测。 总之，我们的顺序胸苷模拟标签技术是一种新的方法来检测细胞的营业额。我们希望我们的做法，让其他科学家更准确地量化细胞分裂，并期望它会在组织动态平衡打开新的范例。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>5-chloro-2-deoxyuridine (CldU)</td>
<td>&nbsp;</td>
<td>MP Biomedical</td>
<td>0210547891</td>
<td>&nbsp;</td>
<tr>
<td>5-Iodo-2-deoxyuridine (IdU)</td>
<td>&nbsp;</td>
<td>MP Biomedical</td>
<td>0210035701</td>
<td>&nbsp;</td>
<tr>
<td>4% Paraformaldehyde</td>
<td>&nbsp;</td>
<td>USB</td>
<td>19943</td>
<td>&nbsp;</td>
<tr>
<td>10% Buffered Formalin</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>23-427-098</td>
<td>&nbsp;</td>
<tr>
<td>XYLENES</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>EM-XX0060-4</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol, Anhydrous (Histological)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>A405P-4</td>
<td>&nbsp;</td>
<tr>
<td>PBS, 10X Powder Concentrate</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP665-1</td>
<td>&nbsp;</td>
<tr>
<td>Triton &#x2013;X-100</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP151-500</td>
<td>&nbsp;</td>
<tr>
<td>Hydrochloric Acid</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>BDH3028-2.5LG</td>
<td>&nbsp;</td>
<tr>
<td>Normal Donkey Serum</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
<td>017-000-121</td>
<td>&nbsp;</td>
<tr>
<td>Mouse Anti BrdU</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>347580</td>
<td>&nbsp;</td>
<tr>
<td>Rat mab anti BrdU</td>
<td>&nbsp;</td>
<td>Accurate Chemical</td>
<td>OBT0030</td>
<td>&nbsp;</td>
<tr>
<td>Cy2 donkey anti-rat</td>
<td>&nbsp;</td>
<td>Jackson Immuno</td>
<td>712-225-153</td>
<td>Fully adsorbed secondaries from Jackson (minimal cross reactivity).</td>
</tr>
<tr>
<td>Cy5 donkey anti-mouse</td>
<td>&nbsp;</td>
<td>Jackson immuno</td>
<td>715-175-151</td>
<td>See above</td>
</tr>
<tr>
<td>Glass Cover Slips</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C9056-1CS</td>
<td>&nbsp;</td>
<tr>
<td>YFP Filter</td>
<td>&nbsp;</td>
<td>Chroma</td>
<td>49003 ET EYFP</td>
<td>Reduces auto-fluorescence in Cy2 channel</td>
</tr>
<tr>
<td>Cy3 Filter</td>
<td>&nbsp;</td>
<td>Chroma</td>
<td>49004 ET Cy3</td>
<td>&nbsp;</td>
<tr>
<td>Cy5 Filter</td>
<td>&nbsp;</td>
<td>Chroma</td>
<td>49006 ET Cy5</td>
<td>&nbsp;</td>
<tr>
<td>ORCA ER Microscope Camera</td>
<td>&nbsp;</td>
<td>Hamamatsu</td>
<td>C4742-95-12ER</td>
<td>Excellent detection of fluorophores</td>
</tr>
<tr>
<td>ProLong Gold antifade reagent</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P36930</td>
<td>Do not use Vectashield (toxic to cyanine dyes).</td>
</tr>		</tbody>
		</table>
		</div>

immunofluorescent detection of two thymidine analogues (cldu and idu) in primary tissue

细胞分裂的精确测量，是一个根本性的挑战变得越来越复杂，缓慢分裂的细胞进行分析时，实验生物学。建立的方法检测细胞分裂包括细胞培养，重要的染料，如羧基DI - aetate琥珀酰亚胺酯（CFSE），如丝分裂抗原Ki67的或增殖细胞核抗原免疫检测稀释和胸苷类似物，连续显微镜直接观察。胸苷类似物，可以检测多种方法，包括无线电检测氚胸腺嘧啶，溴脱氧尿苷（BrdU），氯 - 脱氧尿苷（CldU）和碘脱氧尿苷（IDU）的免疫检测，化学检测乙炔 - 脱氧尿苷（EDU）。我们已经得出检测到成年小鼠组织中掺入不同的胸苷类似物的顺序（CldU IDU）的策略。我们的方法允许调查准确量化的细胞分裂的两个连续两轮。通过优化的免疫协议，我们的方法可以检测到非常低的剂量胸苷类似物，通过饮用水，安全管理，以管理为长时间的小鼠。因此，我们的技术可用于检测细胞寿命很长，组织，周转。最佳的免疫荧光染色结果，可以实现在多种组织类型，包括胰腺，皮肤，肠，肝，肾上腺，睾丸，卵巢，甲状腺，淋巴结，和脑。我们也应用这种技术来识别组织内的​​致癌转化。我们必须进一步应用这一技术，以确定是否过境扩增细胞增长或组织重建作出贡献。在这个意义上说，胸苷类似物序贯给药代表参与组织的动态平衡细胞研究的起源和生存的一个新方法。

Watch this Scientific Journal Video about Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue at JoVE.com

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

我们派生的策略，以检测到成年小鼠组织中的胸苷类似物的顺序法团（CldU IDU），以量化的细胞分裂的两个连续两轮。这一战略是有用的检测长寿命的组织细胞的营业额，致癌基因改造，或过境扩增细胞。

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 ...

immunofluorescent-detection-two-thymidine-analogues-cldu-idu-primary

Research

JoVE Journal

Biology

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

30.4K 次观看.  University of Pennsylvania-School of Medicine. 我们派生的策略，以检测到成年小鼠组织中的胸苷类似物的顺序法团（CldU IDU），以量化的细胞分裂的两个连续两轮。这一战略是有用的检测长寿命的组织细胞的营业额，致癌基因改造，或过境扩增细胞。

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

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

科研

生物学

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

瑞士改进轧制技术肠组织准备免疫组化和免疫分析

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

肌肉干细胞中自噬的原位免疫荧光染色

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of these methods to non-model organisms. A series of protocols to optimize tissue collection from bats has been developed for a series of high-throughput sequencing approaches. Outlined here are protocols for the capture of bats, desired demographics to be collected for each bat, and optimized methods to minimize stress on a bat during tissue collection. Specifically outlined are methods for collecting and treating tissue to obtain (i) DNA for high molecular weight genomic analyses, (ii) RNA for tissue-specific transcriptomes, and (iii) proteins for proteomic-level analyses. Lastly, also outlined is a method to avoid lethal sampling by creating viable primary cell cultures from wing clips. A central motivation of these methods is to maximize the amount of potential molecular and morphological data for each bat and suggest optimal ways to preserve tissues so they retain their value as new methods develop in the future. This standardization has become particularly important as initiatives to sequence chromosome-level, error-free genomes of species across the world have emerged, in which multiple scientific parties are spearheading the sequencing of different taxonomic groups. The protocols outlined herein define the ideal tissue collection and tissue preservation methods for Bat1K, the consortium that is sequencing the genomes of every species of bat.

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

蝙蝠组织收集 -细胞分析和原细胞培养

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological substances or characterization of specific biological activities. However, due to the lack of universally applicable cell culture media and protocols, well described cell culture methods for marine organisms are still limited. Meanwhile, the commonly occurring microbial contamination and polytropic properties of marine invertebrate cells further impede the establishment of an effective cell culture strategy for marine invertebrates. Here, we describe an easy-to-handle method for culturing intestinal cells from sea cucumber Apostichopus japonicus; additionally, we provide an example of in vitro apoptosis induction and detection in primary cultured intestinal cells. Moreover, this experiment provides details about the appropriate culture medium and cell collection method. The described protocol is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea, and it can provide sufficient cells for multiple in vitro experimental applications. This technique would enable researchers to efficiently manipulate primary cell cultures from marine invertebrates and to facilitate the functional evaluation of targeted biological materials on cells.

This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

海参肠细胞原培养的凋亡诱导与检测

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological ...

Simple Detection of Primary Cilia by Immunofluorescence

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to mitosis. Primary cilia can be visualized with highly sophisticated methods, including transmission electron microscopy, 3D imaging, or using software for the automatic detection of primary cilia. However, immunofluorescent staining of primary cilia is needed to perform these methods. This publication describes a protocol for the easy detection of primary cilia in vitro by staining acetylated alpha tubulin (axoneme) and gamma tubulin (basal body). This immunofluorescent staining protocol is relatively simple and results in high-quality images. The present protocol describes how four cell lines (C2C12, MEF, NHLF, and skin fibroblasts) expressing primary cilia were fixed, immunostained, and imaged with a fluorescent or confocal microscope.

Primary cilia are extracellular structures associated with the centriole. Primary cilia detection by immunofluorescent staining is a relatively simple procedure that results in extremely high-quality images. In this protocol, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope.

通过免疫荧光简单检测原发性西莉亚

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.

使用基于多反应监测的人类脑组织蛋白质的多反应监测工作流程定量蛋白质组学工作流程

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related ...

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are highly efficient at virus transmission plays a key role in virus epidemics in fields. Studying virus-insect vector interactions can advance our understanding of virus transmission and epidemics with the aim of designing novel strategies to control plant viruses and their vector insects. Immunofluorescence labeling has been widely used to analyze interactions between pathogens and hosts and is used here in the white-backed planthopper (WBPH, Sogatella furcifera), which efficiently transmits the southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), to locate the virions and insect proteins in the midgut epithelial cells. Using laser scanning confocal microscopy, we studied the morphological characteristics of midgut epithelial cells, cellular localization of insect proteins, and the colocalization of virions and an insect protein. This protocol can be used to study virus activities in insects, functions of insect proteins, and interactions between virus and vector insect.

This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission.

半翅目肠道中植物病毒和昆虫载体蛋白的免疫荧光标记

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are ...