这项工作是由美国国立卫生研究院赞助授予HL082798和HL111580。

1。肾组织切片<ol><li>福尔马林固定的（10％）的石蜡包埋的肾脏切片使用的是MICROM HM 355 S的幻灯片可以被存储以供分析前几个月切到显微镜载玻片上以5mm的厚度。 </li></ol> 2。溶液的制备<ol><li>准备1升的1×PBS的在DDH 2 O在高压灭菌螺旋盖瓶。填写另一瓶1升双蒸2 O的加入1 ml的DEPC到每一瓶，盖和剧烈震荡。让该溶液静置过夜，在室温下，以破坏RNA酶，然后高压灭菌。添加400毫升吐温20，以1升1×PBS中，使PBS-T。 </li><li>制备的蛋白酶K缓冲液（50 mMTris-盐酸，pH值8.0和1mM的CaCl 2在DEPC处理的水）。 </li><li>制备在PBS-T中的0.2％的甘氨酸溶液。 </li></ol> 3。组织准备<ol><li>准备大量的杂交缓冲液（50-100毫升）50％甲酰胺组成的，5×SSC，0.1％吐温，9.2mM柠檬酸调整pH至6，50微克/毫升肝素和500μg/ ml的酵母于DEPC处理过的RNA双蒸2 O。划分为1毫升分装并储存于-80℃。 </li><li>在新鲜的二甲苯清洗3次，每次5分钟从组织中取出石蜡。 </li><li>通过5分钟的孵育在减少浓度的乙醇（100％，75％，50％和25％）在DDH 2 O再水合的组织切片</li><li>治疗载玻片用DEPC足够完​​全覆盖组织切片（约0.4-0.5毫升），持续1分钟。确保组织不干燥。 </li><li>漂洗DEPC幻灯片处理PBS-T两次5分钟，收集废物妥善处置含DEPC。所有试剂应DEPC处理，以消除RNA酶从这点上。 </li><li> Prewarm蛋白酶K缓冲液和蛋白酶K加至10微克/毫升的最终浓度。涵盖组织切片用蛋白酶K溶液和孵化在37℃下5分钟。 </li><li>快速去除proteinasËK溶液，并加入PBS-T 0.2％的甘氨酸，持续30秒。 </li><li>于DEPC冲洗玻片处理PBS-T两次，每次30秒。 </li><li>固定在新鲜配制的4％多聚甲醛固定切片10分钟。 </li></ol> 4。杂交<ol><li>加50微升杂交缓冲液（50％甲酰胺，5×SSC，0.1％吐温，9.2 mM柠檬酸调整pH至6，50微克/毫升肝素，500微克/毫升酵母RNA）每组织切片，并盖上无RNA酶的HybriSlips切只是比组织切片大。 </li><li> Prehybridize部分在湿度受控杂交烘箱中2小时，在杂交温度下测定的3&#39;地高辛标记的探针（探针通常是DNA的Tm -21℃（ 即 54℃下的miR-382，探针DNA的Tm = 75℃ ），采用浸泡在50％formamide/50％5×SSC组织实验室擦拭，以保持腔室加湿。 </li><li>淡化miRNA的探针，阳性对照组（U6，小核RNA）和阴性对照（炒序列），以40nm的在杂交缓冲液（75微升缓冲液中的每部分需要的话）。 </li><li>热探针在杂交缓冲液中于65℃维持5分钟。 </li><li>从杂交烤箱中取出的幻灯片，并从预杂交取出盖玻片和多余的杂交缓冲液。 </li><li>加75微升每组织切片探针含有缓冲液，直接给组织。涵盖组织与HybriSlips只是大到足以覆盖的组织切片。 </li><li>保持滑架的水平，回归杂交炉过夜孵育温度从步骤4.2（约16小时）。 </li></ol> 5。严格洗涤<ol><li>加入200μl2×SSC，加热到杂交温度，刚下盖玻片的一边，以帮助从组织中释放盖玻片。通过倾斜滑动取下盖玻片。 </li><li>在杂交温度3倍于200微升50％甲酰胺，50％的2倍SSC洗玻片各30分钟。 </li><li>冲洗载玻片5倍的PBS-T在室温下每5分钟对低速轨道摇床。 </li></ol> 6。发现<ol><li>在封闭缓冲液（2％山羊或马血清，2毫克/毫升BSA的PBS-T）1小时，在室温下对低速轨道摇床孵育滑动。 </li><li>稀释的抗DIG-AP Fab片段在封闭缓冲液以1:100。添加每组织切片100微升，盖上HybriSlips切只是大到足以遮盖上述部位。 </li><li>孵育抗体在湿盒中4℃过夜（约16小时）。 </li><li>在PBS-T洗7倍的幻灯片，对于每5分钟在轨道摇床中于低速。 </li><li>在AP缓冲液冲洗载玻片（100mM的TrisHCl pH为9.5，50毫摩尔MgCl 2，100mM的氯化钠，0.1％吐温20）3倍，每次5分钟。 </li><li>加入NBT / BCIP解决AP缓冲液（200毫升μl/10缓冲区） </li><li>加入400-500毫升稀释后NBT / BCIP解决方案，以每张幻灯片完全覆盖所有的组织切片。建立在一个黑暗的，水平，humidif灭蝇灯腔4-5小时。 </li></ol> 7。安装滑轨<ol><li>在每个5分钟递增浓度的乙醇（25％，50％，75％，100％）脱水部分。 </li><li>在孵育二甲苯5倍，以清除部分。 </li><li>山滑入Permount安装介质和干燥过夜。 </li></ol>

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没有什么可以透露。

本文的目的是描述了miRNA的原位杂交协议，在福尔马林固定的肾组织效果很好。虽然制定这个协议染色神器的几个重要来源已经确定。仔细注意这几点可以帮助避免染色神器，增加成功的ISH执行的可能性。 可以发生一个染色神器的最可避免的原因时，组织切片成为在漫长的孵化干涸。当此发生时变成干燥的组织部分将NBT / BCIP发育（ 图1）中染色很暗。在本协议是很关键的组织切片保持在所有的孵育和该载片保持完全水平完全覆盖液体。此外，该组织切片应该保持完全在长时间的孵化如杂交步骤涵盖HybriSlips和<html>该抗体孵育，以帮助防止液体流失。如果组织切片部分或完全干燥，观察它是不可能的样品将产生最佳的染色，这将不允许miRNA表达的那些样品中被解释。 在协议中，其中液体的覆盖是必需的其他步骤是蛋白酶K消化和多聚甲醛在治疗过程中。此消化需要，以允许所述miRNA探针的访问进入细胞。如果组织的覆盖面这短暂的孵育过程中是不完整的，未暴露在酶消化方面不会允许尽可能多的探针进入和绑定，并最终不会弄脏的黑暗其他的组织的其他区域。当应用该协议向其他组织类型，蛋白酶K消化的持续时间可能需要进行优化。随后的多聚甲醛固定也是必不可少的，以防止miRNA的损失以下通透性。 NT"&gt; NBT / BCIP发展的持续时间也控制的一个重要变量。我们发现，超过4或5个小时更长NBT / BCIP孵育显著背景工件开始在探测与加扰-MIR对照探针（组织发展图2A）。四小时NBT / BCIP发展的足够用于检测诸如U6小RNA阳性对照组（ 图2B）表示的相对高丰度探测目标。停止孵化不出来，以允许额外的低水平表达，以被检测到，而这可能只是导致增加的背景神器。 这导致了较低的表达的miRNA的可探测性的重要课题。虽然我们已经找到了5&#39;地高辛标记的探针来为我们提供我们的肾脏中的应用足够的检测灵敏度。碱性磷酸酶为主色调的发展使得被测量的EAC多个信号颗粒探头h的分子，但一些低级别的miRNA仍可能无法检测到。 Exiqon开发了miRNA的探针被标记的5既&#39;和3&#39;末端上，允许为每个微小RNA分子的色度发展两次的可能性。虽然我们的实验室还没有测试过这些探针，其在肾组织有类似协议的使用已报道8。 这个基本的协议，可以通过优化蛋白酶K消化时间可以适用于其他类型的组织和miRNA探针，miRNA的探针浓度，杂交温度和NBT / BCIP开发时间。另外，也可以在与在其中的组织的自发荧光是适当的低荧光抗体组合使用的协议的第一部分。

微RNA是小（约22个核苷酸长），非编码所产生的内源性的RNA。他们一般通过功能翻译抑制或mRNA降解，抑制蛋白的表达。的miRNA结合到靶mRNA不完全互补，从而有可能为一个单一的miRNA抑制多个目标。 了解哪些类型的细胞和结构表达的miRNA是理解机制的重要组成部分，通过它在miRNA的表达影响细胞和组织表型的改变。而方法如miRNA的测序，定量PCR和Northern杂交可用于检测在整个组织中的miRNA，这种方法不允许之一，以确定哪个特定的细胞类型，他们从一个给定组织中来了。使用这些方法和蜂窝结构成分分析之前夹层可以是非常困难的，以获得足够的隔离度所需的条件能导致人terations在基因表达或RNA的降解。微小RNA 原位杂交是用于可视化组织中微小RNA的位置和表达水平的方法。这种技术是在组织异质结构，如肾组成的特别有价值。 小分子RNA已被证明发挥肾脏发育2,3和生理学4的调节作用。微小RNA表达的改变也被证明参与与肾脏病理如纤维化5-10，糖尿病肾病7，肾癌11,12，和急性肾损伤13。在我们的研究中，我们已经发现， 在对肾组织原位杂交优化微小RNA是用于确定miRNA的表达在健康和疾病14的确切结构的位置有价值。不同的microRNA的管状和细胞表达的测定是很重要的，因为TA自己调节rgets可能依赖于细胞功能。在疾病状态也很重要，以确定如何在miRNA表达的改变可能影响功能。 这里描述的方法的目标是建立在由Kloosterman 等 15，其他研究者16,17开发现有ISH方法，以及那些建议的Exiqon 1和优化方法对福尔马林固定的肾组织。我们已经成功地使用这种方法来确定单侧输尿管梗阻18在肾小分子RNA的miR-382的表达明显的地区差异。这种方法可以与其他组织可以用作良好，额外的优化。

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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Paraformaldehyde</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">P6148</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PBS</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:137px;">70011044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Diethyl Pyrocarbonate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">D5758</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Tween-20</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">P1379</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Xylenes</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">534056</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Ethanol</td>
			<td style="width:71px;">Ultra Pure</td>
			<td style="width:137px;">200CSPTP</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Tris-HCl</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">15506-017</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CaCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">C2536</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Glycine</td>
			<td style="width:71px;">J.T. Baker</td>
			<td style="width:137px;">4059-00</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Citric Acid</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">C2404</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Formamide</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">F9037</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">20x SSC Buffer</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">15557-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Heparin</td>
			<td style="width:71px;">SAGENT</td>
			<td style="width:137px;">25021-400-30</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Yeast RNA</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">AM7118</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">MgCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">M8266-1004</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">NaCl</td>
			<td style="width:71px;">J.T. Baker</td>
			<td style="width:137px;">4058-01</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Proteinase K</td>
			<td style="width:71px;">Fermentas</td>
			<td style="width:137px;">EO0491</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">3&#8217; DIG- miRNA probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">miR dependent-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">3&#8217; DIG- Scrambled miR probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">99004-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">3&#8217; DIG- U6 miR probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">99002-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Anti-Digoxigenin-Ab Fab</td>
			<td rowspan="2" style="width:71px;">Roche</td>
			<td rowspan="2" style="width:137px;">11093274910</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Fragments</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NBT/BCIP</td>
			<td style="width:71px;">Roche</td>
			<td style="width:137px;">11681451001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Permount</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">SP15-500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Coverslips</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">Fit to tissue size</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Microtom HM 355 S</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:137px;">23-900-672</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">In-slide Out Hybridization Oven</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">241000</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Aluminum Tray Assembly</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">C2403973</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Stainless Steel Rack Insert</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">C2403754</td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

microrna in situ hybridization for formalin fixed kidney tissues

在本文中，我们通过与地高辛原位杂交描述了在肾脏比色法检测miRNA的方法标记的小分子RNA探针。这个协议中，最初是由Kloosterman和同事们用Exiqon miRNA探针1广泛使用而开发的，已被修改以克服固有的miRNA分析在肾组织中的挑战。这些包括诸如结构识别和难以除去残留的探针和抗体。使用比较薄的，厚5毫米，可清晰肾脏结构的可视化，同时强大的探测信号被保留在细胞组织切片。此外，探针浓度和培养条件进行了优化，以方便与低背景和非特异信号的microRNA表达谱的可视化。此处，优化的协议进行了说明，通过幻灯片在该过程结束时，安装覆盖所述初始组织收集和制备。其基本组件管理此协议的NTS可以改变应用到其它组织和细胞培养模型。

在杂交中，孵育该变得干涸或一般洗涤步骤结束的NBT / BCIP发育过程中染色更暗的组织切片的区域， 图1示出了肾部分，其中HybriSlip滑落的边缘的一部分的组织，使之成为部分脱水。尽管补液和覆盖范围中的其余步骤，在脱水部分的信号是虚高。 控制NBT-BCIP发展的时间的重要性是体现在图2A和2B。比4-5小时，结果明显的非特异性显色的炒-MIR控制探讨组织整个肾（ 图2A）较长的开发周期。肾脏探查高丰度的目标，如U6小核RNA控制，没有表现出改善的信号，比孵育4-5小时延长（ 图<html> URE 2B）。 这里描述的miRNA检测肾组织中的原位杂交方法的适用性是表现在图3中 ，一个身影从KRIEGEL转载等 18在这项研究中，我们发现，单侧输尿管梗阻（UUO）诱导的qPCR近3倍时相比，假手术动物检测匀浆肾组织的miR-382的表达。这一增长阻断静脉提供防的miR-382（数据未显示）。 ISH对这些肾脏组织使用表明，该探测器提供的的miR-382的检测是能够非常敏感地反映了基于定量PCR表达分析量化的结果。此外，ISH使我们能够衡量肾脏内miRNA的表达明显的地区差异。 600px的"/&gt; 图1大鼠肾脏部分的部分脱水的过程中杂交步骤的影响。校准条= 100毫米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50785/50785fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/50785/50785fig2.jpg" width="600px" /> 图2。优化NBT / BCIP发展的持续时间。允许NBT / BCIP发展到持续超过4-5小时的结果显著背景染色在加扰-MIR探测组织中（ 图2A），同时提供了在没有进一步改善在积极控制信号，U6小核RNA探测样品（ 图2B）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50785/50785fig2highres.jpg" target="_blank">点击这里查看</a>更大的图像。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/50785/50785fig3.jpg" width="600px" /> 图3中通过原位杂交获得的小鼠的肾脏的miR-382表达的代表性的图像。校准棒= 50微米。 （B）的miR-382表达为假手术+抗扰码处理的小鼠％的平均IOD。这个数字已经被修改KRIEGEL 等人 ，（ 图5），它描述了治疗组，方法的细节和结果18。反扰频后的n = 10/group;抗的miR-382 N = 8/group; *显著​​不同的抗扰频处理的小鼠与相同手术（P &lt;0.05），＃显著不同假手术动物接受相同的抗-MIR治疗。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50785/50785fig3highres.jpg" target="_blank">点击这里</a>查看大图。

Watch this Scientific Journal Video about MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues at JoVE.com

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

本文介绍了比色法检测福尔马林固定的肾部分microRNA表达谱进行优化的原位杂交协议。

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 ...

microrna-in-situ-hybridization-for-formalin-fixed-kidney-tissues

Nanyang Technological University

Research

JoVE Journal

Biology

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

14.9K 次观看.  Medical College of Wisconsin. 本文介绍了比色法检测福尔马林固定的肾部分microRNA表达谱进行优化的原位杂交协议。 

视频: MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

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 ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

原位杂交技术在植物中的精确定位成绩单

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

放射性的原位杂交检测组织不同的基因表达模式

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

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 ...

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 ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

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

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

修正了福尔马林蛋白质组学样品的制备和石蜡包埋组织

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

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

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

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

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

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

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.

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双整装原位杂交技术在早期鸡胚

荧光原位杂交（FISH）的议定书“在人类精子

原位杂交技术在植物中的精确定位成绩单

放射性的原位杂交检测组织不同的基因表达模式

荧光检测病毒RNA

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

修正了福尔马林蛋白质组学样品的制备和石蜡包埋组织

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

对于荧光DNA的简易方法