NIH / NIDCD和施密特基金会到RP的赠款支持的工作。

该协议是基于标准的放射性和非放射性原位杂交方法，以前我们和其他人开发的检测在脑组织1-7中的一个或两个成绩单物种的发展和完善。下面所描述的协议有2或3天的总长度，这取决于由最终用户选择的程序中断的数量。下面详细的所有步骤都riboprobe杂交步骤和杂交后洗外，在室温下进行。在此方法中涉及的所有步骤所需的解决方案和缓冲区，可以发现在此协议结束。 1。组织准备和切片<ol><li>斩首，并迅速提取受试者的大脑，并将其放置到一个适当大小的塑料模具。 </li><li>盖脑组织TEK包埋剂，并迅速在dry-ice/alcohol浴快速冷冻的塑料模具。冰冻组织可存放于-80 ° C，直到使用。 </li><li>使用低温恒温器，收集到的收费Superfrost加幻灯片幻灯片2或3％的大脑部分。部分的厚度为10-12微米。幻灯片可存放于-80 ° C，直到使用。 </li></ol> 2。探针纯化制备葡聚糖G50列 “可以从商业来源购买葡聚糖列，但是，我们在下面提供一个低成本的替代列的一代，将探头净化。 <ol><li>水合物无RNase，DEPC处理过的水（例如，2克的粉末，在DEPC处理过的水100毫升），混合的解决方案简要和在室温下存储足够量的葡聚糖G50粉沉淀多余的葡聚糖G50。 </li><li>去除上清液（水上层）以下葡聚糖G50降水。 </li><li>重复过程中，上述3 - 5倍。 </li><li>最后一次洗涤后，再暂停使用葡聚糖G50在TE缓冲液（1:1比例）的解决方案，并存储在4 ° C。 </li><li>蒸压成无菌的1 ml注射器玻璃棉和压缩柱塞在底部的致密层，然后放入15毫升猎鹰管的注射器。 </li><li>充分混匀，在TE溶液葡聚糖G50。该解决方案的填充注射器/列。 </li><li>在1000 rpm离心30秒列。 </li><li>重复此过程，直到列的几乎是完全充满葡聚糖G50珠。 </li><li>将200μL洗涤液的列列，并在1000 rpm 2分钟离心。流丢弃。 </li><li>将200μL封闭液列列，并在1000 rpm 2分钟旋转。重复此步骤的4-5倍，达到平衡列。列可以存放在4℃直至使用，如果用封口膜密封。 </li></ol> 3。标记和纯化的Riboprobes 下面我们详细介绍单一riboprobe的产生和净化。 dFISH，每个探头的准备，将涉及同样的方法，除了一个探头将被标记，而其他与生物素标记的双绞线与地高辛（DIG）标记的UTP。 <ol><li>准备集中（&gt; 150毫微克/微升）和纯化线性利益cDNA的解决方案，以生成感（对照组）或反义riboprobes。 </li><li>在1.5 ml离心管中，加入0.5-1微克纯化的cDNA为模板，2 5X探针标记缓冲液，1μL10X辛（或生物素）标签混合，0.5μLRNasin 1μL相应的RNA聚合酶，并带来终体积10μL无RNase的水（DEPC处理）的解决方案。 </li><li>在37℃水浴中孵育的解决方案° C为2小时。 </li><li>加入1μL的tRNA（股票; 20微克/微升），列封闭液和39μL解决方案。 </li><li>准备探针纯化的葡聚糖G50列。为此，添加50μL封闭液列，并在1000 rpm旋转10秒。 重复此步骤2-3次，以平衡列（即，50μL应用于列离心周期后收回）。 </li><li>应用葡聚糖G50列，位置在列的底部的一个新的离心管的探头解决方案，和自旋为3分钟1000转，获得纯化的50μLriboprobe解决方案。 </li><li>评估标记riboprobe的质量和产量，使用一个标准的甲醛琼脂糖RNA凝胶，或分光光度计。 </li></ol> 4。固定后，乙酰化和杂交<ol><li>从-80 ° C冰箱中取出的部分，并允许为室温平衡。随后，冷，新鲜的3％为5分多聚甲醛溶液中孵育部分。 </li><li>简单冲洗在P部分hosphate缓冲液（PBS）的两倍。 </li><li>脱水部分通过一个标准的酒精系列（70，95，和100％; 2分钟），并让它们风干。 </li><li>孵育10分钟的乙酰化解决方案的部分。 </li><li>冲洗2X SSPE的第3次。 </li><li>再次通过上面所述的标准酒精系列脱水部分，并允许他们风干。 </li><li>准备足量的杂交液，并添加两个riboprobes的解决方案（探针浓度为1 ng /μL，每个探头）。总成交量为杂交液杂交的节（每节杂交液16μL）的基础上确定。 </li><li>应用足量的杂交液，以确保没有空气泡沫覆盖组织的组织和盖玻片幻灯片。 </li><li>放置在一个金属滑动支架幻灯片。在矿物油浴中浸泡持有人，在65 ° C过夜。确保盖玻片是朝上（即幻灯片持有人的侧面应与矿物油的容器底部接触）。 </li></ol> 5。后杂交洗<ol><li>翌日，小心地取出幻灯片的持有人，并从矿物油浴简单冲洗氯仿它从幻灯片中删除多余的油。 </li><li>放置幻灯片在2X SSPE的解决方案，为5-10分钟。盖玻片应分离，而在溶液中的幻灯片。 </li><li>转移到一个新的2X SSPE的解决方案的幻灯片，并保持在室温下1小时。 </li><li>转移到含2X SSPE的加50％甲酰胺溶液中的部分。这种解决方案的温度应该在一夜之间杂交过程中使用的温度相匹配。在此解决方案保持1.5小时的幻灯片。 </li><li>转让部分杂交步骤相同的温度预热到0.1X SSPE的解决方案。在这部分孵育30分钟的解决方案。重复这一步额外的30分钟。 </li></ol> 6。检测和可视化的Riboprobes <ol><li> TNT的缓冲区传输幻灯片，其中0.3％的过氧化氢已经增加了10分钟，。 </li><li> TNT缓冲液，3次（每次10分钟）部分洗净。 </li><li>使用DAKO笔，良好的周围绘制包含大脑部分区域。重要的是，应谨慎行事，以确保该路段不燥。 </li><li>每张幻灯片中应用150 TNB的缓冲液，孵育在此解决方案中的部分为30分钟，在潮湿的腔。 </li><li>通过倾斜的幻灯片中删除多余的TNB的解决方案。 </li><li>国能在2小时湿热试验箱含有过氧化物酶标记的抗地高辛抗体，并存储幻灯片的解决方案的应用150μL。抗体浓度应分别确定为各的利益来开展dFISH前谈话。 </li><li>在TNT的缓冲区部分洗净; 3次，每次10分钟。 </li><li>申请150μLAlexa的594 -共轭酪胺工作的解决方案，以每张幻灯片，并储存在1小时的湿热试验箱。该解决方案应根据制造商的指示准备。 </li><li>在TNT的缓冲区部分洗净; 3次，每次10分钟。 </li><li>在TNT的缓冲孵育部分，已添加0.3％的过氧化氢为10 - 30分钟。 </li><li>在TNT的缓冲区部分洗净; 3次，每次10分钟。 </li><li>适用于150μL的每张幻灯片的TNB的缓冲区，并保持在一个30分钟的湿热试验箱。 </li><li>通过倾斜的幻灯片中删除多余的TNB的解决方案。 </li><li>加入150μL国能在2小时湿热试验箱含有过氧化物酶标记的抗生物素的抗体，并存储幻灯片的解决方案。 </li><li>在TNT的缓冲区部分洗净; 3次，每次10分钟。 </li><li>套用一个Alexa 488 - 150μL共轭酪胺的工作方案的幻灯片和1小时的孵化部分。该解决方案应根据制造商的建议准备。 </li><li>在TNT的缓冲区部分洗净; 3次，每次10分钟。 </li><li>加入150μL赫斯特溶液（1:1000在TNT的缓冲区）的部分，并保持在2分钟湿热试验箱。 </li><li>在TNT的缓冲液清洗的部分; 3次，每次5 min。 </li><li>盖玻片用荧光兼容的安装介质（例如，Vectashield或延长抗淬灭）的部分。 </li></ol> 7。代表性的成果<img src="/files/ftp_upload/2102/2102fig1.jpg" alt="图1" /> 图1，我们在这里展示有代表性的dFISH，在斑马雀脑取得的成果。从caudomedial nidopallium（NCM），哺乳动物的听觉皮层的鸣禽模拟获得的显微照片。脑切片进行杂交与生物素标记的riboprobe针对白蛋白（A）的抑制性神经元亚群的标志物，掏标记核糖探头针对活动依赖的基因zenk（二），为歌曲驱动的神经元的可靠标记。 C）（A）和（B）显示的听觉体验激活的抑制性神经元的人口覆盖。箭头和箭头指示两个riboprobes专门标记的细胞，星号显示代表神经元，表达共同的利益都成绩单。比例尺= 25微米。

<ol>
	<li>Jin, L., Lloyd, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization:+methods+and+applications.">In situ hybridization: methods and applications.</a> J Clin Lab Anal. 11, 2-9 (1997).</li><li>Stoler, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization.">In situ hybridization.</a> Clin Lab Med. 10, 215-236 (1990).</li><li>Komminoth, P., Werner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+and+signal+amplification:+approaches+to+increase+the+sensitivity+of+in+situ+hybridization.">Target and signal amplification: approaches to increase the sensitivity of in situ hybridization.</a> Histochem Cell Biol. 108, 325-3233 (1997).</li><li>Kessler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+digoxigenin:anti-digoxigenin+(DIG)+technology--a+survey+on+the+concept+and+realization+of+a+novel+bioanalytical+indicator+system.">The digoxigenin:anti-digoxigenin (DIG) technology--a survey on the concept and realization of a novel bioanalytical indicator system.</a> Mol Cell Probes. 5, 161-205 (1991).</li><li>Panoskaltsis-Mortari, A., Bucy, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+with+digoxigenin-labeled+RNA+probes:+facts+and+artifacts.">In situ hybridization with digoxigenin-labeled RNA probes: facts and artifacts.</a> Biotechniques. 18, 300-307 (1995).</li><li>Qian, X., Lloyd, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+signal+amplification+methods+for+in+situ+hybridization.">Recent developments in signal amplification methods for in situ hybridization.</a> Diagn Mol Pathol. 12, 1-13 (2003).</li><li>Mello, C. V., Jarvis, E. D., Denisenko, N., Rivas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+song-regulated+genes+in+the+brain+of+songbirds.">Isolation of song-regulated genes in the brain of songbirds.</a> Methods Mol Biol. 85, 205-217 (1997).</li><li>Pinaud, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+neurons+participate+in+the+brain's+response+to+birdsong+auditory+stimulation.">GABAergic neurons participate in the brain's response to birdsong auditory stimulation.</a> Eur J Neurosci. 20, 1318-1330 (2004).</li><li>Velho, T. A., Pinaud, R., Rodrigues, P. V., Mello, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-induction+of+activity-dependent+genes+in+songbirds.">Co-induction of activity-dependent genes in songbirds.</a> Eur J Neurosci. 22, 1667-1678 (2005).</li><li>Tremere, L. A., Jeong, J. K., Pinaud, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estradiol+shapes+auditory+processing+in+the+adult+brain+by+regulating+inhibitory+transmission+and+plasticity-associated+gene+expression.">Estradiol shapes auditory processing in the adult brain by regulating inhibitory transmission and plasticity-associated gene expression.</a> J Neurosci. 29, 5949-5963 (2009).</li><li>Pinaud, R., Mello, C. V., Velho, T. A., Wynne, R. D., Tremere, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+two+mRNA+species+at+single-cell+resolution+by+double-fluorescence+in+situ+hybridization.">Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization.</a> Nat Protoc. 3, 1370-1379 (2008).</li><li>Pinaud, R., Jeong, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplex+fluorescence+in+situ+hybridization+in+the+study+of+gene+co-regulation+in+the+vertebrate+brain.+Methods+Mol+Biol.">Duplex fluorescence in situ hybridization in the study of gene co-regulation in the vertebrate brain. Methods Mol Biol.</a> 611, 115-129 (2010).</li></ol>

我们使用这个协议来研究脊椎动物的大脑是如何neurochemically和功能组织，并确定如何行为相关的感官刺激的影响在成人大脑 8-10元基因组机械。我们已经成功地使用这种方法在小鼠，大鼠和鸣禽脑组织，但预计此协议将很容易适应从脊椎动物物种的阵列，或许非神经组织得到的脑切片。需要获得可靠的结果和最佳的信号的关键控制已经详细广泛，本集团其他地方11，12 。

解决方案<ol><li>列洗涤缓冲液：10毫米的Tris - HCl，0.15 M氯化钠（NaCl），0.05毫米EDTA，50微克/μL的tRNA，0.1％SDS于50 ml DEPC处理过的水。 </li><li>列封闭液：10毫米的Tris - HCl 0.1毫米，50毫米氯化钠，EDTA于50 ml DEPC处理过的水。 </li><li> TNT缓冲液：60毫升的Tris - HCl，1米18毫升5 M氯化钠和1.8毫升的Triton X - 100的DEPC处理过的水在600毫升。 </li><li> TNB的缓冲区：100毫米的Tris - HCl，8.3微克/μLBSA，0.15 M氯化钠和3％的Triton X - 100的DEPC处理过的水。 </li><li> TE缓冲液：10毫米的Tris - HCl，pH值7.5加1 mM的EDTA，pH值7.5。 </li><li>乙酰化的解决方案：三乙醇胺加DEPC处理过的水200毫升醋酸酐0.5毫升2.7毫升。 </li><li>杂交液：50％甲酰胺，SSPE的2倍，2微克/μL的tRNA，1μg/μL的BSA和1微克/微升聚在DEPC处理的水一个。它需要每节16升。 </li></ol>

double fluorescence in situ hybridization in fresh brain sections

在这里，我们描述了一个双荧光修改后的版本<em&gt;原位</em优化杂交（dFISH）方法检测mRNA的新鲜冰冻脑切片的兴趣。我们的小组已经成功地使用这种方法，研究基因的共同监管。更具体地说，我们使用的dFISH方法，探讨组织的解剖，神经化学物质的属性，和感官体验在中央感官电路的影响，在单细胞分辨率。在小鼠，大鼠和鸣禽脑组织，但该协议已经得到验证，预计将很容易适应其他脊椎动物物种，以及非神经组织的数组。在这部影片中，我们提供详细演示了此过程的主要步骤。

Watch this Scientific Journal Video about Double Fluorescence in situ Hybridization in Fresh Brain Sections at JoVE.com

Double Fluorescence in situ Hybridization in Fresh Brain Sections

该协议涉及非放射性<em&gt;原位</em&gt;杂交程序，使在脊椎动物的大脑薄片，同时识别两个种成绩单，在一个单细胞的决议。

双荧光原位杂交食脑切片

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

double-fluorescence-in-situ-hybridization-in-fresh-brain-sections

Research

JoVE Journal

Neuroscience

Double Fluorescence in situ Hybridization in Fresh Brain Sections

16.0K 次观看.  University of Rochester. 该协议涉及非放射性原位杂交程序，使在脊椎动物的大脑薄片，同时识别两个种成绩单，在一个单细胞的决议。

视频: Double Fluorescence in situ Hybridization in Fresh Brain Sections

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

结合亲脂性染料，原位杂交，免疫组织化学和组织学

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

科研

神经科学

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

设计和组装的超轻型电动微型硬盘（Microdrive）慢性神经录音的小动物

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

在脑切片检测轴突本地化mRNAs的高空间分辨率<em&gt;原位</em&gt;杂交

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

快速<em&gt;原位</em使用寡核苷酸探针对大鼠多聚甲醛为前缀的大脑5-羟色胺综合征&gt;杂交

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

结合双荧光<em&gt;原位</em&gt;杂交免疫标记为三个基因在小鼠大脑切片中表达的检测

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

通过RNA定位蝗虫天线中的气味受体基因<em&gt;原位</em&gt;杂交

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

将多重荧光 原 位杂交与荧光免疫组化相结合，对新鲜冷冻或固定的小鼠脑切片进行研究

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

生态HIV脑感染的老鼠模型

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.