We thank Zhaohua Irene Tang in the W. M. Keck Science Department for the use of her epifluorescence microscope and the Werren lab for donating Nasonia for dissections. This work was supported in part by an NIH-NRSA fellowship (5F32GM105317-02) to AML.

1.组织解剖和固定（60分钟） <ol><li>对果蝇的大脑，放在第三龄幼虫在下降的1×PBS（磷酸盐缓冲盐水）中。选择大的3 龄幼虫正在积极从小瓶或瓶子是不是人满为患爬行。 <ol><li>用一个镊子超细对抓牢口钩，另一镊子对抢2/3下来身体（ 图1A，B）的长度。上嘴钩轻轻一拉，露出大脑，腹侧神经节，唾液腺及部分幼虫消化道。使用镊子分离脑和腹侧神经节（ 图1A，B）中从其他组织中，并装在1倍的PBT（磷酸盐缓冲盐水与吐温）上的塑料培养皿的液滴。 </li></ol></li><li>对于金小蜂睾丸的解剖，选择男3日龄蛹（黄体红色的眼睛）。男金小蜂有小翼片的长度相对到f在蛹期（ 图1C）emales。 <ol><li>握住蛹在与一个镊子对，并使用其他镊子对胸部区域附近的腹部的上，抓住腹部的很远尖端和拉出泪滴形睾丸（它们将被脂肪包围体，可以轻轻摇动走; 图1D）。断开任何外部车身零件的睾丸，这将妨碍正常的挤压，并将其放置在1X PBT上的塑料培养皿液滴。 </li></ol></li><li>对于每一个幻灯片，解剖一共有四五个组织样本（ 即 ， 果蝇幼虫大脑和睾丸金小蜂 ）。 注：以上五个样本会导致过度拥挤的细胞核和染色体。 </li><li>可选地，为了实现在有丝分裂的染色体常染色质区域姐妹染色单体的一些分离，治疗用低渗溶液的组织：从1倍的PBT（传送到大脑的下降为0.5％的柠檬酸钠5-10不超过10）分钟。 注：秋水仙碱（有丝分裂抑制剂）可以增加核分裂象数量在中期11有益的。但是，它的使用是不必要的，如果非常大的健康的幼虫被使用，并且甚至不希望的，因为它可以在染色体分辨率产生负面影响，传播和形态。 </li><li>放置的固定液（45％乙酸的2.5％多聚甲醛）倒在干净Sigmacote处理的盖玻片的表面上的液滴（约20微升）。 注意：请固定液新鲜使用的每一天。固色剂解决方案，从1.8-3.7％多聚甲醛在45％的醋酸产量FISH最好的结果。用面巾纸比大脑和睾丸等，或者此适配协议免疫FISH可能需要与不同的固定剂试验（针对不同的固定剂的列表，请参阅12）。 </li><li>小心地从解剖缓冲液（1×PBT）每一组织样品与超细镊子，米尼米转移到固定剂液滴查懋解剖缓冲器入固定液的转移。定位的组织样本，使得它们被均匀地彼此间隔开的固定剂液滴内。孵育在固定液组织在室温下4分钟。 </li><li>小心地将聚赖氨酸涂覆的滑动面向下到组织和盖玻片。不按下在这一点上，而是允许两个接触轻轻使盖玻片粘到滑动的下侧。倒置滑动，使得盖片是在顶部。 </li><li>夹心折叠一张滤纸内部幻灯片/组织/盖玻片。在一个稳定的表面，用拇指，按非常坚定地直下到盖玻片上方的位置。小心避免横向盖玻片滑动（这将导致涂抹组织）。 </li><li>淹没在滑动/组织/盖玻片入液氮（ 见图1E，女装置），并且静置直至氮停止沸腾（长为fINE）。取出载玻片，并立即通过轻弹盖玻片的角在向上方向上（避免刮伤固定的组织与剃刀刀片）折断盖玻片用新鲜刀片。 <ol><li>预冷幻灯片/组织/盖干冰块上滑，盖滑起来，1-2分钟浸入液态氮将有助于防止开裂的幻灯片之前。 </li></ol></li><li>立即用纸巾滑动放入一个科普林缸填充100％的乙醇在室温下，静置至少5分钟（如果需要，这个时间可以更长）。 注：冷100％乙醇也可以使用。 </li><li>删除与组织滑动，毛细作用带走用Kimwipe过量乙醇（而不触及固定的组织），并离开该滑动风干1小时。 <ol><li>直接进入第2步或保存幻灯片干燥低湿度空气中或在干燥室周来进行杂交之前甚至几个月。 </li></ol></li></ol> 图1：与在那里抢口钩显示位置（A）A 3 路龄幼虫果蝇 （右一），（表示带*号）和下幼虫解剖大脑的方式2/3; （左）的概略幼虫一个相同发育阶段的，描绘了幼虫头部内脑的相对位置。（B）中的脑和腹侧神经节从一个第三龄果蝇幼虫（右）和本示意解剖组织（左）。（C）3日龄蛹金小蜂在黄体红眼阶段。（D）睾丸对从3日龄金小蜂的蛹男（右）与解剖位置，表示从哪里抢蛹在腹部的后（标以*）和中途上的主体; （左）示意描绘男性和女性是P蛹;雄性蛹可以通过翼不延伸超过矢状轮廓（黑色箭头），而相比之下，雌性，其具有翼延伸过去的信息来区分;睾丸对的相对位置示于阳蛹。纸夹-和（F）的方法，用于浸泡在液氮的容器幻灯片（E）的装置， -一个字符串。可用于对多张幻灯片同时浸没多个回形针字符串。 2. 在原位杂交（30月1日分; 1小时，2.5小时长探头，第2天） <ol><li>加入1μl每种探针的（100毫微克），以20微升1.1倍的杂交缓冲液中。吸管探针/杂交缓冲液到固定组织的表面（避免触及组织）。 </li><li>小心直接置于盖玻片上的探针/杂交缓冲液中，并确保盖玻片工作在组织直接居中。该缓冲区应该迁移到THË盖玻片的外缘，不留气泡。 注：小气泡不接触组织不会造成任何问题的程序。通过仔细抬起盖玻片的一个角落里，小心地放弃它返回到幻灯片中删除大量气泡。 </li><li>将幻灯片/组织/盖玻片到块预加热到95°C（盖滑起来）的表面。有一大块铝箔盖，以防止曝光。让滑动孵育在95℃下5分钟。 注意：有孔典型热块管可以翻转，提供在其上放置幻灯片/组织/盖玻片一个平面上。 </li><li>去除幻灯片，让它稍微冷却，直到它是温暖的触感。精心包装了一块拉伸封口膜周围的盖玻片密封它下面的液体。 </li><li>放置一个湿度室内部的密封滑动，并把该室到预热至30℃的培养箱。孵育在30℃下进行4小时至过夜。<ol><li>创建湿度室从空尖框或带盖百惠容器放置在底部阻尼的Kimwipes或纸巾。 注：单链DNA寡核苷酸探针被设计为28-33个碱基，达到45-47℃的理论上的熔解温度（T M）。这些长度和T 米范围反映的事实是，我们已经研究了很多重复序列是富含AT的，因此具有非常低的GC含量。较长的探针将可能有较高的Tm 值值;这可能会导致在30℃的标准杂交温度更高的背景杂交。因此，随着杂交温度一些故障，可能需要以达到最佳的效果。找到最好的杂交温度，增加（或减少）由5℃的温度下，增量。 </li></ol></li><li>仔细从幻灯片中删除封口膜，然后慢慢抬起一个角落里小心地取出盖玻片。华SH幻灯片三次15分钟的0.1X SSC缓冲每次洗。在洗涤覆盖科普林缸用铝箔，以减少曝光。 </li><li>如果不使用长生物素化的探针，继续执行步骤2.8。 <ol><li>如果使用的是长的生物素化的探针，干周围用Kimwipe组织的区域，小心不要触碰组织本身。放置100微升封闭溶液在组织中并用盖玻片覆盖轻轻，注意避免俘获气泡。包住滑过盖玻片用Parafilm和地点，在37℃下进行30分钟。 </li><li>小心地取出盖玻片和周围涂抹用Kimwipe组织。吸取100μl的若丹明​​ - 亲和素稀释1：1000在SBT过组织并覆盖轻轻用盖玻片，注意避免俘获气泡。包住滑过盖玻片用Parafilm和地点，在37℃下进行30分钟。 </li><li>小心地取出盖玻片和5分钟洗滑3次，每次在4倍SSCT然后3次，每次5分钟，在0.1×SSC。 注：幻灯片可水洗的时间较长。 </li></ol></li><li>去除幻灯片及吸干组织周围用干的Kimwipe以去除多余的缓冲液（避免接触的组织）。放置在滑动组织朝上，在暗处10-15分钟或直至水分完全消散。 </li><li>吸管11微升的Vectashield封固剂（有4&#39;，6-二脒基-2-苯基 - DAPI）到组织上。小心地将一个干净的盖玻片（不与Sigmacote处理）的正上方的安装介质和组织的核心。安装介质应当向外迁移缓慢地向盖玻片的边缘。 注：如果在安装介质无法到达所有各方盖玻片的边缘，再追加1-2微升封固剂可以施加到一个位置，在该盖片的边缘，以填补在所需体积。在这种情况下，可以肯定之前擦去从滑动表面上的任何多余的介质密封。 </li><li>密封盖玻片指甲油的边缘。避免涂抹指甲油在组织样本。 </li><li>放置在滑动直立在暗处，并让指甲油干燥直至完全硬（通常为30分钟以上）。在这一点上，图像中的组织或储存在-20℃下长达1周后成像。 </li></ol>缓冲/解决方案食谱 10X PBS <ul><li> 80克氯化钠</li><li> 2.0克氯化钾</li><li> 14.4克的Na 2 HPO 4 </li><li> 2.4克KH 2 PO 4的</li><li> pH值至7.4，H 2 O到1升</li></ul> 1X PBT <ul><li> 5毫升10X PBS </li><li> 45毫升H 2Ø </li><li> 0.1％吐温20 </li></ul> 20X SSC <ul><li> 175.3克氯化钠</li><li> 88.2克柠檬酸钠</li><li> 800毫升水2 O </li><li> pH值7，H 2 O到1升</li></ul> 4X SSCT <ul><li> 200毫升20X SSC </li><li> 799毫升的H 2 </子&gt; 0 </li><li> 0.1％吐温20 </li></ul> 0.1X SSC <ul><li> 5毫升20X SSC </li><li> 995毫升H 2Ø </li></ul>杂交混合物（20微升;从11修改） <ul><li> 10微升甲酰胺</li><li> 4微升50％硫酸葡聚糖</li><li> 2微升20X SSC </li><li> 4微升水中2 O </li></ul> SBT 8（10毫升）中<ul><li> 2毫升20X SSC </li><li> 0.01克牛血清白蛋白（BSA）的</li><li> 10微升吐温20 </li><li> 7.9毫升H 2Ø </li></ul>封闭溶液8（10毫升）中<ul><li> 0.3克BSA </li><li> 10微升吐温20 </li><li> 2毫升20X SSC </li><li> 8毫升H 2Ø </li></ul>用多聚甲醛固定液（1毫升）中<ul><li> 393.75微升H 2 O（先加水） </li><li> 450微升冰醋酸</li><li> 156.25微升16％多聚甲醛</li></ul>

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The authors declare that they have no competing financial or any other conflict of interest.

DNA的原位杂交是经常用于映射特定序列的染色体。我们描述了一个简单的方法DNA ISH高拷贝数，异色序列优化。而不是使用洗涤的甲酰胺溶液中，这是在其他现有的DNA ISH协议的要求，我们直接放置在预加热块组织安装滑动到使DNA变性。这种方法绕过了使用大量的甲酰胺。用于产生脆的杂交信号的一个关键步骤是使用新鲜制备的固定溶液;如果不这样做（或者使用多聚甲醛是已经开放超过一个月新溶...

原位杂交（ISH的DNA）的DNA是一种常用的方法进行映射的序列，以特定的染色体区域。可以通过少数的方法，包括缺口转译或长DNA产物1,2-末端标记和deoxygenin（DIG）的掺入-attached核苷酸及其识别通过各种各样的产生内常染色质探针，以单拷贝区基的缀合抗体1-3。在几个或单拷贝数常染色体序列的可视化需要使用任何单一的大型探测器具有高比活或多个较小的探针共同增强信号的鸡尾酒。 与此相反，在异发现高度重复序列，诸如卫星的DNA，是用于DNA ISH更容易的目标，因为它们通常作为几十到重复聚集在被称为块的单染色体区域的数千存在。转座元件也可以是在不同的时染色体位点2个高拷贝数发现。在这些情况下，单个探针具有低比活性可以有效标记异色序列由于在多个位点的杂交。探针的重复序列可被合成市售的短的寡核苷酸（30-50碱基对）和化学缀合与任何多种不同的荧光团。通过基因组测序技术绘制内异染色质重复序列是困难的，因为在高度重复的卫星模块4-6,7建筑脚手架遇到的挑战。目前，ISH站作为在子染色体水平映射这些序列的最有效的方法。这种策略是映射大批正在通过发现正在进行的基因组和转录组测序研究的重复序列的重要。 映射重复序列上滑动安装染色体的效率和易于将选取gre通过对DNA ISH简化协议atly增强。例如，对于DNA的ISH现有的协议涉及杂交的组织中的甲酰胺溶液2,8-多次洗涤，从而增加大致映射序列所需的时间，也产生这种昂贵试剂的大量化学废物。在这里，我们描述了一个修订后的DNA ISH方法规避，需要甲酰胺清洗，并利用基本的实验室设备和试剂。该方法最初是通过使用缀合有荧光染料商业合成的寡核苷酸设计为在果蝇幼虫的神经母细胞异染色质区域高度重复的DNA序列的快速映射。但是，这种方法也适用于通过使用通过其他方式9,10和跨多个不同的组织和染色体类型合成更大的探针映射重复序列。此外，此方法可用于通过使用较长或MULTIP映射常染色质的序列乐，感兴趣的常染色体序列中短探针。

<table border="0" cellpadding="0" cellspacing="0" width="542">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:285px;"></td>
			<td style="width:257px;">Company</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Materials&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Poly-L-lysine coated slides (regular slides also can be used)</td>
			<td>Sigma Aldrich</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ultrafine tweezers (5 gauge)</td>
			<td>Dumont</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">22mmx22mm cover slips, Sigmacote-treated by immersion for 15 seconds, blotting dry, and wiping away all traces of Sigmacote so that cover slip is clear</td>
			<td>Fisher</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sigmacote</td>
			<td>Sigma</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Filter paper (75-150mm)</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Paraffin wax paper</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heat block with thermometer</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dry incubator</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Razor blades</td>
			<td></td>
		</tr>
		<tr height="17">
			<td colspan="2" height="17" style="height:17px;">Humidity chamber (empty pipette tip box or Tupperware, lined with moistened paper towels or Kimwipes)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Coplin jars (with slide grooves)</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Aluminum foil</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pasteur pipettes</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1.5 mL microfuge tubes</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Nail polish (clear or colored)</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">2-20 &#956;L micropipette and plastic tips</td>
			<td></td>
		</tr>
		<tr height="17">
			<td colspan="2" height="17" style="height:17px;">20-25 standard metal paperclips linked to form a chain</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td>Company/Notes</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Reagents</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">16% EM grade paraformaldehyde</td>
			<td>Electron Microscopy Reagents</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Acetic acid</td>
			<td>Sigma</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Liquid nitrogen</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">100% Ethanol, chemical grade</td>
			<td></td>
		</tr>
		<tr height="17">
			<td colspan="2" height="17" style="height:17px;">Commercially synthesized, fluorescently labeled oligos</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Long biotinylated probe (e.g. nick translated and biotinylated with BioNick from Invitrogen)</td>
			<td>Invitrogen; Alternative steps 2.7.1-2.7.3</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rhodamine-Avidin (for detection of long biotinylated probe)</td>
			<td>Roche; Alternative steps 2.7.1-2.7.3</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hybridization buffer</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">4X SSCT (Saline-sodium citrate + Tween)</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">0.1X SSC (Saline-sodium citrate)</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Blocking solution</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">SBT (SSC, Bovine serum albumin, Tween)</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1X PBT (Phosphate-buffered saline + Tween)</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1X PBS (Phosphate-buffered saline)</td>
			<td></td>
		</tr>
		<tr height="17">
			<td colspan="2" height="17" style="height:17px;">Hypotonic solution (0.5% sodium citrate in H2O)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Formamide</td>
			<td>Sigma Aldrich</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vectashield mounting medium with DAPI</td>
			<td>Vector laboratories</td>
		</tr>
	</tbody>
</table>

simple method for fluorescence dna in situ hybridization to squashed chromosomes

原位杂交（ISH的DNA）的DNA是一种常用的方法进行映射的序列，以特定的染色体区域。这种方法是在映射高度重复序列，以异染色质区域，其中，计算方法面临令人望而却步的挑战特别有效。在这里，我们描述了一个精简的协议，用于DNA ISH是规避洗甲酰胺是在其他DNA ISH协议的标准步骤。我们的协议被优化用于与携带荧光染料，从而有效地标记在多个不同的昆虫组织类型的异色染色体区域内的重复DNA序列的短单链DNA探针杂交。然而，应用程序可被扩展较大探针和单拷贝（非重复）的DNA序列的可视化中使用。我们证明了该方法通过映射几个不同的重复序列从果蝇染色体挤压神经果蝇细胞和金小蜂vitripennis精母细胞。我们表明杂交模式均小，商业合成的探针和用于比较的较大探针。此过程使用简单的实验室耗材和试剂，是理想的谁与执行DNA ISH一点经验调查。

为了证明这种方法，我们杂交的一组小，以致被化学修饰的荧光偶联物（ 图2）和一个较长的生物素化的探针商业合成的寡核苷酸（通过PCR产物的切口平移制成; 图2B），以染色体从几个不同的组织类型（ 见表1）。目标序列包括位于从D.有丝分裂染色体着丝粒（异）地区的卫星重复果蝇幼虫神经母细胞（ 图2A-C）和减数分裂染色体从?...

Watch this Scientific Journal Video about Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes at JoVE.com

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.

对于荧光DNA的简易方法<em&gt;原位</em&gt;杂交来压扁染色体

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

simple-method-for-fluorescence-dna-situ-hybridization-to-squashed

Research

JoVE Journal

Biology

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

17.9K 次观看.  University of Rochester. 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.

视频: Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

焦磷酸测序：一个准确的基因分型的简单方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

科研

生物学

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 ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

高通量原位杂交方法表征在胎鼠下泌尿生殖道的基因表达模式

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

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 ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

高通量使用自动染色体的物理图谱<em&gt;原位</em&gt;杂交

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

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 ...

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 ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

结合DNA-RNA荧光<em&gt;原位</em&gt;杂交（FISH）研究X染色体失活的分化雌​​性小鼠胚胎干细胞

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

整个山<em&gt;原位</em&gt;对腹足软体动物杂交方法<em&gt;静水椎实螺</em

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

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 ...