Mutant worm strains were kindly provided by the Caenorhabditis Genetics Center. This research was supported by a grant of the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (grant number: HI14C1277).

1.标记探针与地高辛的dUTP通过PCR <ol><li>用含地高辛的dUTP 10X dNTP混合物，如前所述13进行PCR标记。 </li><li>根据制造商的指令用旋转柱纯化纯化PCR产物。 <ol><li>如果探针是短于200碱基对，除去游离的地高辛的dUTP从反应混合物自旋柱色谱法，而不是旋转柱纯化。 </li></ol></li></ol> 2.准备聚赖氨酸涂层幻灯片注意:整个过程大约需要2小时。大多数的步骤都在室温下，除了在干燥步骤完成。 <ol><li>清洁幻灯片<ol><li>放置载玻片在一个塑料容器和漂洗载玻片简要地用蒸馏水​​（DW）。除去水和用含有1％玻璃清洁剂DW填充容器。 </li><li>搅拌15分钟载玻片在室温下（RT）50的转速。用DW洗涤3幻灯片次在RT每次5分钟。 </li><li>洗用70％乙醇的幻灯片搅拌15分钟。丢弃70％的乙醇，然后将玻片65℃的干燥块和风干15分钟上。 </li></ol></li><li>多井载玻片的聚赖氨酸涂层 注:玻璃片聚赖氨酸涂层是重要的一步，因为它提供了在整个染色过程中样品的附着力。差的涂覆的滑动将导致样品的损失。 <ol><li>稀释聚赖氨酸原液〜0.01％（重量/体积）在蒸馏水。添加的稀释0.01％20微升（重量/体积）聚赖氨酸到干净的载玻片上的孔中。 </li><li>在室温下孵育5分钟载玻片。 </li><li>放置在幻灯片的65℃的干体和空气干燥1小时上。 </li><li>存放在无尘箱聚赖氨酸幻灯片。 </li></ol></li></ol> 3.在载玻片上蠕虫固定（图1） <ol><li>准备胚胎FISH 注:嘉实蠕虫所有T之前他细菌食物被看显微镜下生长培养基消耗。饥饿减少产蛋成虫并增加卵孵化。详细方法在14,15进行说明。 <ol><li>根据标准方法14生长的蠕虫在50mM培养皿。 </li><li>所有的细菌食物消耗后，切琼脂培养基在季度锅铲。切割，以防止污染前消毒锅铲。 </li><li>把所有的一块琼脂上100mm的线虫生长介质（NGM）板。打开琼脂片倒挂的蠕虫到达新鲜食品的细菌。 </li><li>经过48〜72小时，收集与M9的缓冲区蠕虫。加入3 - 上NGM板5毫升M9的缓冲区中。 NGM的表面上吸移管的M9缓冲洗蠕虫。 </li><li>收集蠕虫的液体，并添加到15毫升管。 注意:如果在30％的蔗糖溶液的收获，离心后的蠕虫琼脂碎片。虽然碎片成团，虫子浮在的表面上。 </li><li>添加M9的缓冲区，以弥补体积到15ml。沉淀通过离心蠕虫在300×g离心3分钟，并除去大部分的M9缓冲液中。重复此步骤2次以上。 注意:如果蠕虫离心后仍然漂浮，设置离心机的制动电平为值= 1。 </li><li>吸M9的缓冲区。添加漂白解决蠕虫。每0.5ml蠕虫，添加6.5毫升的DW，将2ml次氯酸盐，加入1ml的5M的KOH。 </li><li>在50rpm在RT摇动3分钟孵育蠕虫。 15秒涡虫以机械剪切蠕虫和暴露的蛋。 </li><li>观察漂白过程中解剖显微镜下的管中。确保蠕虫切成两半和鸡蛋被释放。当最成年体的溶解，添加的M9缓冲液补足体积至15ml。 </li><li>离心在300×g离心3分钟。吸大部分的M9和补充新鲜M9的缓冲区，以弥补体积到15ml。重复洗涤步骤3次。 注意:避免使用蠕虫过量，因为它们妨碍漂白过程。保持整个反应时间不超过8分钟，直到洗涤。过度漂白鸡蛋产生强烈的自体荧光。 </li><li>添加具有聚山梨醇酯20（PBST）的磷酸缓冲盐水高达200微升和200微升4％低聚甲醛（PFA），以使2％的PFA。 注意:由于PFA是致癌物质，穿防护服，手套和眼罩使用PFA之前。 </li><li>添加40微升在2％PFA中的鸡蛋到聚赖氨酸涂覆的滑动的良好。 </li><li>放置载玻片在潮湿室中，并在室温下孵育15分钟。关闭潮湿室的幻灯片被放在里面之后。 注意:鸡蛋沉降到滑动件的底部而被固定。 </li></ol></li><li>准备FISH解剖性腺<ol><li>嘉实成虫吹打1毫升M9缓冲生长在50毫米NGM板。细菌性食物前收获蠕虫被耗尽。 </li><li>从任何细菌机智洗净虫体^ h M9的缓冲区，日2次。 注:残余细菌可能粘附到聚赖氨酸包被的载玻片与解剖性腺干扰。 </li><li>颗粒离心蠕虫在300×g的。删除M9和微量转移蠕虫空NGM板。 </li><li>加入30微升含有聚赖氨酸上的处理幻灯片的好2毫米左旋咪唑M9缓冲。 </li><li>加入500微升的M9缓冲液中至1毫升管中。使用该缓冲区口吸管转移的蠕虫。 </li><li>通过将毛细管在含有M9的缓冲区1毫升管填充M9缓冲嘴吸管尖。 </li><li>在解剖显微镜，把口吸管尖刚刚在成虫并拖动口吸管的头前，使蠕虫的头进入口腔吸管。一旦蠕虫的头部进入尖端，蠕虫的整个主体将被吸引到吸头。 </li><li>转移蠕虫使用吸管口聚赖氨酸涂层幻灯片。 </li><li>使用剃刀，切F中的头部或幻灯片上蠕虫的尾部的尖端。当蠕虫被切断，性腺就会弹出。性腺会坚持到幻灯片。 <ol><li>在一口井至少准备30蠕虫。多个孔可以用于另外30蠕虫。 </li></ol></li><li>把幻灯片在潮湿的室内和吸掉用口吸液管M9的缓冲区。 </li><li>通过在湿润室中在室温下加入20微升2％的PFA的15分钟固定的样品。 </li></ol></li></ol> 4.固定和透<ol><li>放置在干冰上的铝块，并将其存储在深冷冻机（-80℃）。商店甲醇和丙酮在-20℃。 </li><li> PFA固定步骤3.1.15或3.2.11后，用微量留下〜5微升固定液除去固定液。 </li><li>放在样品载玻片另一聚赖氨酸涂层幻灯片。除去用纸巾固定液如果溶液是过分的。一旦它们粘在一起，不要移动幻灯片。 </li><li>冻结幻灯片在铝块至少15分钟。 注:样品可以贮存至少2 - 3天。 </li><li>虽然被冻结的幻灯片，放冷含有甲醇和丙酮冰的罐子。 </li><li>就拿滑出并扭转他们冻结破解样本。丢弃上滑动。立即浸泡滑入冰冷甲醇5分钟。 </li><li>转移的幻灯片到冰冷的丙酮5分钟。 </li><li>洗玻片用PBST 3次，每次5分钟，以去除残留的固定剂。继续进行下一步骤，或在100％乙醇的幻灯片储存于4℃。 注:样品可以贮存至少2 - 3天。 </li></ol> 5.修正了杂交细胞<ol><li>添加的RNA酶溶液20微升（含10微克/毫升RNA酶A PBST）。孵育在潮湿室中的滑动，在37℃1小时。 </li><li>在2×盐水和柠檬酸钠与聚山梨酯20（2×SSCT），每次15分钟，洗涤载玻片两次。 </li><li>添加20杂交液微升，把湿热试验箱在37℃培养箱。 1小时后，除去通过移液杂交溶液。 </li><li>去除杂交液之前，准备探头。如果探针双链DNA，通过加热在95℃的干块上5分钟变性探针。加热后，在冰上冷却简要探头。 </li><li>加入10微升含探针与样品的杂交溶液。对于PNA探针，使用浓度以1:1的比例:2,000和DIG标记的探针，使用浓度以1:200的比例。盖上盖玻片样品。 </li><li>放与水的热块（80℃）上浸湿的纸巾。把热块的塑料盒盖以保持湿度和温度。 </li><li>加热块的温度稳定（80℃）后，放置在加热的纸巾样品载玻片，盖上塑料盒盖的样品。变性样品3分钟。 </li><li>公司乌瓦特在潮湿室中的载玻片在37℃下过夜。 </li></ol> 6.清洗和免疫<ol><li>预热杂交洗涤溶液（2×SSC，50％甲酰胺）至37℃。 </li><li>在RT洗在PBST样品两次5分钟。取下玻璃盖。 </li><li>在37℃下洗杂交洗涤溶液中的样品30分钟。 </li><li>洗在PBST中的样品玻片3次在RT。注:在室温下进行的湿热试验箱所有的后续步骤。 </li><li>加入20μl阻断溶液并在室温下孵育在潮湿的腔室1小时。 </li><li>除去封闭溶液，加入FITC缀合的抗洋地黄毒苷抗体溶液（1:200），3小时在RT或过夜，在4℃。 </li></ol> 7.安装和观测<ol><li>用PBST洗样品玻片2次，每次15分钟。 </li><li>加10μl用DAPI安装的解决方案。把玻璃盖，并轻轻按下。删除多余的解决方案用纸巾。 </li><li>为了防止安装溶液的蒸发，密封玻璃盖指甲油的边缘。 </li><li>共聚焦显微镜下观察。排除与高背景胚胎。注重与4场 - 20核。 </li><li>根据制造商的使用100X物镜指令拍摄图像。 注:激发样品与405 nm激光为DAPI，555纳米激光的Cy3，与488纳米激光FITC。 </li></ol> 8.端粒信号的量化注意:定量分析如先前16描述的进行。所有要被比较的图像，也可以采取包括曝光时间和光源相同的设置。 <ol><li>在.TIF格式导出图像。 </li><li>下载并安装图像分析软件。 </li><li>执行图像分析软件，点击同意按钮。 </li><li>单击打开按钮。通过双击图像文件打开端粒FISH图像。 </li><li>点击[编辑] - [选择加工区域]，通过左键单击并拖动选择感兴趣的区域。排除所有的非特异性染色。 </li><li>点击[措施] - [现货光密度]，选择端​​粒信号通道，输入文件名保存在.txt文件的结果。 注:结果列在顺序如下:现货荧光，现货和现货领域的背景强度。 </li><li>复制值和斑点的荧光斑点减去背景强度。值现在可以进行统计学分析。 </li></ol>

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The authors have nothing to disclose.

我们的协议的主要优点是过程的，但无细胞结构的形态明显损坏的简单性。几个步骤C.进行了优化线虫 FISH在此协议。成功鱼的关键步骤包括探针标记，胚胎和渗透固定。地高辛的dUTP标记方法提供了通过PCR或缺口转译一个易于使用的标记方法。要标记长的靶序列，缺口平移是首选。在这种情况下，探头应用适当的限制性酶消化，以促进探针的渗透。不推荐生物素-dUTP标记，因为生物素...

端粒可以防止异常融合和降解染色体末端。哺乳动物端粒是由G-丰富的六聚体重复，TTAGGG和shelterin复合物。线虫的端粒重复序列是相似的哺乳动物（TTAGGC）的。大多数真核生物利用端粒酶端粒重复添加到他们的染色体末端。然而，10 -癌细胞的15％利用端粒酶独立的机制，被称为端粒（ALT）3的替代延长。此前，我们报道了端粒重复序列及其相关序列，命名为TALT，端粒酶的突变系存活的关键不育2端粒扩增。 端粒长度通过定量PCR或通过Southern印迹，其中提供总端粒-4,5,6,7-的平均长度测量。在全基因组测序数据端粒重复的读取次数也总端粒8含量的指标。虽然仙GLE端粒长度分析（STELA）可以提供一个单一端粒的长度，它不能提供端粒9的空间信息。而POT-1 :: mCherry报告蛋白提供在体内的端粒的空间信息，它不能代表双链端粒长度为POT-1是一个单链端粒结合蛋白10。 而上述的方法提供了重复序列的平均信息，原位杂交（FISH）荧光允许观察染色体规模的量和感兴趣的个体的序列的空间图案。代替的DNA的纯化，组织或细胞被固定到保持在FISH本地空间信息。因此，鱼为个别重复序列，如端粒重复序列的观察定量和定性的工具。 这个协议提供了两个TELO同时检测的有效方法基于从先前描述的方法11,12的改进仅仅是与其它重复。C.线虫幼虫或成虫多细胞生物体具有高度分化的细胞。细胞的异质性阻碍对大量端粒斑点的定量分析。为了最大限度地提高了分析的细胞数，胚胎分离并散布在用于FISH聚赖氨酸包被的载玻片。此外，该协议也可以与免疫结合。 作为该协议的工作证明，我们表明，它是可能的观察和量化两个不同的重复序列。针对TALT1 DNA探针，用简单的PCR结合地高辛的dUTP产生。那么这个TALT1探针和荧光标记的端粒PNA探针杂交同时进行。随后，地高辛被规范的免疫荧光法检测。我们在座的地方TALT1在TRT-1共定位与端粒的代表图像</eM&gt;幸存者。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PNA probe</td>
			<td>PANAGENE</td>
			<td>custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin-Fluorescein, Fab fragments</td>
			<td>Roche</td>
			<td>11207741910</td>
			<td>use 1:200 diluted in PBST</td>
		</tr>
		<tr>
			<td>Digoxigenin-dUTP</td>
			<td>Roche</td>
			<td>11573152910</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>SIGMA-ALDRICH</td>
			<td>A-7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>SIGMA-ALDRICH</td>
			<td>P-6148</td>
			<td>prepare 4% paraformaldehyde by heating in DW with few drops of NaOH. add 0.1 volume of 10x PBS.</td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridizaiton solution</td>
			<td></td>
			<td></td>
			<td>3X SSC, 50% formamide, 10% (w/v) dextran sulfate, 50 ug/ml heparin, 100 ug/ml yeast tRNA , 100ug/ml sonicated salmon sperm DNA</td>
		</tr>
		<tr>
			<td>Hybridizaiton wash solution</td>
			<td></td>
			<td></td>
			<td>2X SSC, 50% formamide</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>BIONEER</td>
			<td>C-9012</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carlo Erba</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Carlo Erba</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>SIGMA-ALDRICH</td>
			<td>H3393</td>
			<td>make 10 mg/ml for stock solution</td>
		</tr>
		<tr>
			<td>Dextran sulfate</td>
			<td>SIGMA-ALDRICH</td>
			<td>67578</td>
			<td></td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td></td>
			<td></td>
			<td>For 1 Liter DW : 80 g NaCl, 2.0 g KCl, 27 g Na2HPO4:7H2O, 2.4 g KH2PO</td>
		</tr>
		<tr>
			<td>PBST</td>
			<td></td>
			<td></td>
			<td>1X PBS, 0.1% tween-20</td>
		</tr>
		<tr>
			<td>Polysorbate 20</td>
			<td>SIGMA-ALDRICH</td>
			<td>P-2287</td>
			<td>Commercial name is Tween-20</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine solution (0.1 % w/v)</td>
			<td>SIGMA-ALDRICH</td>
			<td>P-8920</td>
			<td>prepare fresh 0.01 % w/v solution before use</td>
		</tr>
		<tr>
			<td>M9</td>
			<td></td>
			<td></td>
			<td>3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 L</td>
		</tr>
		<tr>
			<td>Bleaching solution</td>
			<td></td>
			<td></td>
			<td>20% sodium hypochlorite, 0.5 M KOH</td>
		</tr>
		<tr>
			<td>Antibody buffer</td>
			<td></td>
			<td></td>
			<td>1X PBST, 1mM EDTA, 0.1% BSA, 0.05% Sodium azide (toxic)</td>
		</tr>
		<tr>
			<td>Blocking solution</td>
			<td></td>
			<td></td>
			<td>Antibody buffer with 5% bovine serum albumin (BSA)</td>
		</tr>
		<tr>
			<td>illustra Microspin G-50</td>
			<td>GE healthcare</td>
			<td>27-53310-01</td>
			<td></td>
		</tr>
		<tr>
			<td>20X SSC</td>
			<td></td>
			<td></td>
			<td>To make 1L, 175.3 g of NaCl, 88.2 g of sodium citrate, H2O to 1 L, adjust pH to 7.0</td>
		</tr>
		<tr>
			<td>2X SSCT</td>
			<td></td>
			<td></td>
			<td>2X SSC, 0.1 % tween-20</td>
		</tr>
		<tr>
			<td>10x digoxigenin-dUTP mix</td>
			<td></td>
			<td></td>
			<td>1 mM dATP, 1 mM dGTP, 1 mM dCTP, 0.65mM dTTP, 0.35mM DIG-11-dUTP</td>
		</tr>
		<tr>
			<td>PCR purification columns</td>
			<td>Cosmo genetech</td>
			<td>CMR0112</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cleaner / ULTRA CLEAN</td>
			<td>Dukssan pure chemicals</td>
			<td>8AV721</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-well glass slide</td>
			<td>MP biomedicals</td>
			<td>96041205</td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode growth media</td>
			<td></td>
			<td></td>
			<td>to make 1 L, 3 g of NaCl, 17 g of agar, 2.5 g of peptone, H2O to 974 mL. Autoclave and cool the flask. Add 1 mL of 1M CaCl2, 1 ml of 4 mg/mL cholesterol in ethanol, 1 ml of 1 M MgSO4, 25 mL of 1 M KPO4.</td>
		</tr>
		<tr>
			<td>Levamisole</td>
			<td>SIGMA-ALDRICH</td>
			<td>196142</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor</td>
			<td>Feather</td>
			<td>blade No. 11</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Enzynomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>SIGMA-ALDRICH</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microsope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td>EC Plan-Neofluar 100x was used as objective lens.</td>
		</tr>
		<tr>
			<td>Dry block / aluminum block</td>
			<td>Labtech</td>
			<td>LBH-T03</td>
			<td>Set temperature to 80&#8451;</td>
		</tr>
		<tr>
			<td>Humid chamber</td>
			<td></td>
			<td></td>
			<td>Plastic box filled with paper towel soaked in DW</td>
		</tr>
		<tr>
			<td>Image Analysis Software&#160;</td>
			<td>Dr. Peter Landsdorp</td>
			<td>TFL-telo</td>
			<td><a href="http://www.flintbox.com/public/project/502">http://www.flintbox.com/public/project/502</a></td>
		</tr>
	</tbody>
</table>

observation and quantification of telomere and repetitive sequences using fluorescence in situ hybridization (fish) with pna probes in caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

据报道，此前ALT幸存者可以从端粒酶缺陷的突变通过复制内部本地化&#39;ALT的模板"（TALT）的端粒维持2序列出现，TRT-1（ok410），在低频。使用PNA探针，我们能够想象在解剖性腺（ 图2A）的端粒。微弱的信号端粒检测无论是在TRT-1（ok410）和ALT幸存者。模糊信号被重叠只用DAPI，这表明它们可能不是自发荧光。间质性端粒样重复（ITR）的TRF分?...

Watch this Scientific Journal Video about Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans at

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

观察与端粒的定量和重复序列用荧光<em&gt;原位</em&gt;杂交（FISH）与PNA探针<em&gt;秀丽隐杆线虫</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

observation-quantification-telomere-repetitive-sequences-using

Research

JoVE Journal

Biology

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

10.3K 次观看.  Seoul National University. We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT). 

视频: Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

荧光原位杂交（FISH）的议定书“在人类精子

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

科研

生物学

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的<em&gt;线虫</em&gt;方法:同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

单细胞基因转录的RNA荧光分析<em在原位</em&gt;杂交（FISH）

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

结合荧光染色体复制时序<em在原位</em&gt;杂交

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

前列腺素的提取与分析<em&gt;线虫</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

强大的3D直接标记的探针DNA鱼

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

观察与端粒的定量和重复序列用荧光

摘要

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此视频中的章节

荧光原位杂交（FISH）的议定书“在人类精子

时间推移显微镜早期胚胎发育线虫

荧光检测病毒RNA

基本的

单细胞基因转录的RNA荧光分析

结合荧光染色体复制时序

可视化和分析的mRNA分子，使用荧光

前列腺素的提取与分析

强大的3D直接标记的探针DNA鱼

对于荧光DNA的简易方法