Name: combining double fluorescence in situ hybridization with immunolabelling for detection of the expression of three genes in mouse brain sections
Uploaded: 2016-03-26T15:00:00.000+00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections at JoVE.com

Work in the authors' laboratories was supported by the UK Biotechnology and Biological Sciences Research Council (BB/J006602/1 and BB/L003236/1), the Wellcome Trust (WT100269MA) and the European Research Council (ERC, "Ideas" Programme 293544). SJ was supported by an EMBO long-term fellowship. The authors thank Stephen Grant for his technical assistance.

伦理声明: 鼠标畜牧业和处理都是按照英国内政部规定和UCL伦理委员会的指导方针，与动物（科学程序）英国和它的修订规例2012年1986年法案规定。 注意:所有的解决方案应与焦碳酸二乙酯（DEPC）进行 - 处理后的水破坏任何残留的核糖核酸酶。对于DEPC处理，加入DEPC（1毫升，每公升），用力摇晃直到所有的DEPC球已经消失，然后高压灭菌降解DEPC。 1. RNA探针合成<ol><li>注意:在处理甲酰胺，佩戴个人防护装备（PPE），并使用安全柜。制备杂交缓冲液:DEPC处理，用50％（体积/体积）去离子甲酰胺，200 mM氯化钠，5mM的EDTA，10mM的Tris-盐酸pH为7.5，去离子水的5mM的NaH 2 PO 4，5mM的的 Na 2 HPO 4， 0.01毫克/毫升酵母tRNA，1×Denhardt氏soluti上，和10％w / v的硫酸葡聚糖。 </li><li>选择包含​​感兴趣的基因在与RNA聚合酶启动子的质粒中的cDNA序列的DNA克隆。对于这个例子，使用的pCMV-SPORT6克隆，IRAVp968C0654D（Genbank登录accessionBC024934），用T7和SP6 RNA聚合酶启动子为电力局 （ 补充图1）。 </li><li>制备线性化质粒DNA为模板。 <ol><li>生长的DNA克隆，根据生产商的方案和序列与T7和SP6通用引物与一个小规模的质粒纯化试剂盒提取质粒。 </li><li>消化与切割在cDNA的有义链的5&#39;端的限制性内切酶1020微克质粒DNA。在这个例子中，使用100单元萨尔我消化的pCMV-SPORT6- 电力局质粒。孵育1.5小时在37℃。 </li><li>在琼脂糖凝胶上运行一个小等分，以检查质粒是完全线性化。 </li></ol></li><li>净化线性化质粒用苯酚 - 氯仿。 <ol><li>添加1/10体积的3M乙酸钠，10毫摩尔Tris盐酸（pH 8.0）中的一体积 - 平衡酚和氯仿/异戊醇（IAA）的一个卷（24:1）。 </li><li>离心16,000 xg离心在RT 1分钟（20 - 25℃，RT）和提取上层水相。 </li><li> 16,000 xg离心添加氯仿/ IAA，离心机一体积为1分钟，并提取上层水相。 </li><li>重复步骤1.4.3。 </li><li>加入2倍体积的乙醇，在-20℃下1小时或O / N离开。 </li><li>离心机在4℃，16,000g XG 10分钟。弃去上清液。 </li><li>用70％冷乙醇洗涤沉淀，并在TE的每2.5微升近似1微克的cDNA（10毫米的Tris-HCl，1mM EDTA中pH7.5）中再悬浮。 </li></ol></li><li>合成两个探针，其中一个标与地高辛（DIG）和其他与异硫氰酸荧光素（FITC）。 <ol><li>选择适当的RNA聚合酶，使防本身NSE探针（与靶mRNA互补）。对于这个例子，使用T7 RNA聚合酶使电力局探针。 </li><li>制备体外转录反应:添加线性DNA的1微克，4.0微升5×转录缓冲液，6.0微升100mM的DTT，含有的dNTPs 10倍DIG或FITC RNA标记混合2.0微升，1微升RNA酶抑制剂，和20 - 40 RNA聚合酶的单位;使终体积达20微升用DEPC处理过的水。 </li><li>在37℃下1.5小时。 </li></ol></li><li>在1.0％琼脂糖凝胶的转录反应的RUN1微升，以检查反应工作过。凝胶应显示线性模板和探针的亮带。 </li><li>定容至100微升与杂交缓冲液和储存10微升等分在-80℃。 </li></ol> 2，灌注，固定和组织收集<ol><li>注意:在处理多聚甲醛（PFA），固体和水，穿PPE和使用安全柜。通过使用热（55℃）中溶解11％（重量/体积）的PFA成1×磷酸盐缓冲盐水（PBS）溶液制备甲醛溶液。过滤用滤纸甲醛溶液。 </li><li>通过将20％（重量/体积）在蒸馏水蔗糖制备蔗糖溶液，并添加0.1％（V / V）的DEPC溶液。离开O / N在室温下用一个松散的盖子，然后高压灭菌。 </li><li>由戊巴比妥的腹腔注射（50毫克/千克）末期麻醉鼠标。通过脚趾捏评估麻醉深度和无撤退反射表示深度麻醉。 </li><li>一旦鼠标深度麻醉下，使肋笼下方的切口，并通过在两侧胸廓切开，将其提起，以暴露心脏。 </li><li>插入一个25-G针头插入心脏的左心室。使心脏的右心房一个小切口。 </li><li>灌注用20ml PBS中动物然后再加入40ml甲醛溶液。对于P30和老年人小鼠中，使用12毫升/分的灌注速率，但对于较年轻的动物使用较低的速率（7 - 10ml /分钟）。 </li><li>解剖大脑。 <ol><li>通过从耳朵切后断绝老鼠头部手术剪。使皮肤尾部正中切口，并吻侧工作从颅骨去除皮肤。 </li><li>从尾椎部位开始，沿着穿过中线颅骨顶部和虹膜剪眼睛之间切割。通过倾斜每次骨板的一侧和用镊子捕捉它关闭除去壁层和额骨板。 </li><li>轻轻地用镊子前部向上倾斜大脑并切断视神经和其他颅神经。轻轻提起大脑出了头骨。 </li></ol></li><li>将脑入小鼠大脑冠状矩阵。切片脑入3约4mm件用羽毛刀片。 </li><li>转移切片放入4％PFA解决方案，并在4℃孵育O / N。 </li><li>脑转移切片DEPC-处理20％蔗糖溶液中并在4℃孵育O / N </li><li>每个脑切片放入干cryomould，环绕以最佳的切削温度（OCT）中，冻结的干冰。存储在-80℃。 </li></ol> 3. Cryosectioning <ol><li>切冷冻组织15微米的部分在低温恒温器和收集部分到显微镜载玻片。如果需要的话，润湿部分用DEPC处理过的PBS中，并用细画笔弄平它们。 </li><li>离开载玻片干燥约1小时，以确保该组织附​​着在滑动。 </li></ol> 4.杂交<ol><li>制备65℃洗涤缓冲液:150mM的氯化钠，15mM的钠3 C 3 H 5 O（COO）3（= 1×盐水柠檬酸钠），50％（体积/体积）甲酰胺和0.1％（体积/体积）吐温20。 </li><li>制备MABT缓冲液:100mM的马来酸，150mM的氯化钠，0.1％（体积/体积）吐温20，pH值7.5。 </li><li>稀两个探针（DIG标记和FITC-拉贝里d）在杂交缓冲1 / 1,000预热至65℃，并充分混合。 </li><li>应用约300微升杂交混合到每张幻灯片，coverslipwith烤箱烤制（200℃）和盖玻片在湿盒孵育O / N在65℃。 </li><li>转移滑入含有预热的洗涤缓冲液中的科普林缸。在65℃用洗涤缓冲液洗玻片30分钟两次。 </li><li>在RT洗玻片10分钟以MABT三次。 </li></ol> 5.可视化的FITC探头<ol><li>制备ISH封闭缓冲液:MABT用2％封闭试剂和10％热灭活的羊血清。 </li><li>可选:用疏水性笔在幻灯片上绘制周围的组织切片圈，以减少所需的抗体溶液的体积。 </li><li>用原位杂交在湿润的腔室封闭缓冲液孵育1小时的幻灯片在RT。 </li><li>孵育的幻灯片O / N在4℃用辣根过氧化物酶（POD） - 共轭ð抗FITC抗体稀释1/500（体积/体积）中的ISH封闭缓冲液。 </li><li>放置载玻片到含有PBS中的0.1％（体积/体积）吐温20（PBST）一个科普林缸。洗3次，在PBST 10分钟，并用新鲜的PBST替换各一次。 </li><li>临用前，稀释100倍到扩增稀释剂准备荧光酪胺。对于这个例子，使用FITC标记的酪酰胺。 </li><li>这种添加到幻灯片，并留下在室温下10分钟。 </li><li>放置滑入一个科普林缸和在PBST中洗涤3次，每次10分钟。 </li></ol> 6.可视化的DIG探头<ol><li>准备0.1M的盐酸三 - 8.2 pH值。 </li><li>孵育ISH封闭缓冲液的幻灯片在RT 1小时。 </li><li>用碱性磷酸酶（AP）孵育载玻片O / N在4℃偶联的抗DIG抗体稀释1/1500（V / V）在ISH封闭缓冲液。 </li><li>与MABT洗10分钟三次。 </li><li>用0.1M的Tris-HCl pH值8.2洗两次，在室温5分钟。 </li><li>在使用之前，立即在0.1米的Tris pH值8.2溶解药片准备快速红色溶液。过滤通过0.22微米过滤器的溶液。 </li><li>用在湿润的腔室快速红溶液孵育载玻片在37℃。作为最佳发展的时间变化，定期检查载玻片用荧光显微镜监测沉淀物的形成。对于这个例子，停止后2中的反应 - 3小时。 </li><li>用PBST洗10分钟三次。 </li></ol> 7.免疫组化<ol><li>制备IHC阻断缓冲液:10％（V / V）血清在PBST（不同物种初级抗体主机）。 </li><li>孵育IHC在湿润室中于室温封闭缓冲液1小时幻灯片。 </li><li>准备一抗:稀释一级抗体在PBST中适当的稀释，用5％血清（不同的物种，以初级抗体主机）。对于这个例子，使用兔抗Olig2的抗体以1/400（体积/体积）稀释。 </li><li>孵育在4℃CO / N第一抗体溶液。 </li><li>用PBST洗10分钟三次。 </li><li>制备第二抗体:在PBST适当稀释稀释荧光团共轭次级抗体，用5％（V / V）的血清和0.1％（体积/体积）的Hoechst 33258（一个不同的物种，以初级抗体主机）对于本例使用驴抗兔Alexa647第二抗体以1 / 1,000（体积/体积）稀释。 </li><li>孵育在室温二级抗体溶液1小时。 </li><li>用PBST洗10分钟三次。 </li></ol> 8.安装<ol><li>部分干燥在RT载玻片，并用荧光封固介质装入。离开滑动10X下，在共聚焦显微镜干燥，然后图像，20X和使用4个不同的信道使用以下的激发波长63X目标:用于固红570纳米，FITC 488纳米，Olig2的免疫标记647 nm和Hoechst的350纳米。 </li></ol>

<ol>
	<li>Lobsiger, C. S., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cells+as+intrinsic+components+of+non-cell-autonomous+neurodegenerative+disease.">Glial cells as intrinsic components of non-cell-autonomous neurodegenerative disease.</a> Nat Neurosci. 10 (11), 1355-1360 (2007).</li><li>Baslow, 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=N-acetylaspartate+in+the+vertebrate+brain:+metabolism+and+function.">N-acetylaspartate in the vertebrate brain: metabolism and function.</a> Neurochem Res. 28 (6), 941-953 (2003).</li><li>Hoshino, H., Kubota, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canavan+disease:+clinical+features+and+recent+advances+in+research.">Canavan disease: clinical features and recent advances in research.</a> Pediatr Int. 56 (4), 477-483 (2014).</li><li>Kaul, R., Gao, G. P., Balamurugan, K., Matalon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+the+human+aspartoacylase+cDNA+and+a+common+missense+mutation+in+Canavan+disease.">Cloning of the human aspartoacylase cDNA and a common missense mutation in Canavan disease.</a> Nat Genet. 5 (2), 118-123 (1993).</li><li>Divry, P., Mathieu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspartoacylase+deficiency+and+N-acetylaspartic+aciduria+in+patients+with+Canavan+disease.">Aspartoacylase deficiency and N-acetylaspartic aciduria in patients with Canavan disease.</a> Am J Med Genet. 32 (4), 550-551 (1989).</li><li>Bartalini, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+diagnosis+of+Canavan+disease.">Biochemical diagnosis of Canavan disease.</a> Childs Nerv Syst. 8 (8), 468-470 (1992).</li><li>Moffett, J. R., Ross, B., Arun, P., Madhavarao, C. N., Namboodiri, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Acetylaspartate+in+the+CNS:+from+neurodiagnostics+to+neurobiology.">N-Acetylaspartate in the CNS: from neurodiagnostics to neurobiology.</a> Prog Neurobiol. 81 (2), 89-131 (2007).</li><li>D'Adamo, A. F., Smith, J. C., Woiler, 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+occurrence+of+N-acetylaspartate+amidohydrolase+(aminoacylase+II)+in+the+developing+rat.">The occurrence of N-acetylaspartate amidohydrolase (aminoacylase II) in the developing rat.</a> J Neurochem. 20 (4), 1275-1278 (1973).</li><li>Bhakoo, K. K., Craig, T. J., Styles, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+and+regional+distribution+of+aspartoacylase+in+rat+brain+tissue.">Developmental and regional distribution of aspartoacylase in rat brain tissue.</a> J Neurochem. 79 (1), 211-220 (2001).</li><li>Sommer, A., Sass, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+aspartoacylase+(ASPA)+and+Canavan.">Expression of aspartoacylase (ASPA) and Canavan.</a> Gene. 505 (2), 206-210 (2012).</li><li>Klugmann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+distribution+of+aspartoacylase+in+the+postnatal+rat+brain.">Identification and distribution of aspartoacylase in the postnatal rat brain.</a> Neuroreport. 14 (14), 1837-1840 (2003).</li><li>Madhavarao, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+localization+of+aspartoacylase+in+the+rat+central+nervous+system.">Immunohistochemical localization of aspartoacylase in the rat central nervous system.</a> J Comp Neurol. 472 (3), 318-329 (2004).</li><li>Hershfield, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspartoacylase+is+a+regulated+nuclear-cytoplasmic+enzyme.">Aspartoacylase is a regulated nuclear-cytoplasmic enzyme.</a> Faseb J. 20 (12), 2139-2141 (2006).</li><li>Moffett, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+aspartoacylase+expression+in+the+rat+central+nervous+system.">Extensive aspartoacylase expression in the rat central nervous system.</a> Glia. 59 (10), 1414-1434 (2011).</li><li>Kirmani, B. F., Jacobowitz, D. M., Kallarakal, A. T., Namboodiri, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspartoacylase+is+restricted+primarily+to+myelin+synthesizing+cells+in+the+CNS:+therapeutic+implications+for+Canavan+disease.">Aspartoacylase is restricted primarily to myelin synthesizing cells in the CNS: therapeutic implications for Canavan disease.</a> Brain Res Mol Brain Res. 107 (2), 176-182 (2002).</li><li>Kirmani, B. F., Jacobowitz, D. M., Namboodiri, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+increase+of+aspartoacylase+in+oligodendrocytes+parallels+CNS+myelination.">Developmental increase of aspartoacylase in oligodendrocytes parallels CNS myelination.</a> Brain Res Dev Brain Res. 140 (1), 105-115 (2003).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RNA-sequencing+transcriptome+and+splicing+database+of+glia,+neurons,+and+vascular+cells+of+the+cerebral+cortex.">An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex.</a> J Neurosci. 34 (36), 11929-11947 (2014).</li></ol>

The authors declare that they have no competing financial interests.

该协议提供了一个一步一步的过程原位杂交双RNA后免疫。我们已经用这种协议来确认电力局在几个脑区表达成熟的少突胶质细胞。 这个多步过程有可能影响灵敏度，应该避免了许多潜在的隐患。首先，用于转录反应的所有解决方案和存储缓冲器需要是无RNase。第二，cDNA的模板的选择是重要的，我们在长度大约1kb的偏爱模板。 体外转录之前，有必要进行清?...

神经胶质细胞，这是最丰富的细胞在中枢神经系统（CNS），包括少突胶质细胞（CNS的髓鞘形成细胞），少突胶质细胞前体（OP的，也被称为"NG2细胞"），星形胶质细胞和小胶质细胞。有在神经胶质细胞和他们的潜在作用的功能越来越大的兴趣在神经性疾病1。例如，卡纳万病（CD）是一种遗传性神经退行性疾病与海绵状脑白质营养不良和神经元的逐渐丧失起步阶段起步早，一般10年之前的2,3岁时导致死亡。在Aspartoacyclase（ASPA）基因突变导致急剧减少ASPA活动4 CD已经确定。 ASPA是催化N-乙酰（NAA）的脱乙酰酶，高度集中在脑的分子，产生乙酸和天冬氨酸5-7。许多CD患者表现出由于缺乏交流ASPA更高水平的NAA拉了一。一些研究推测NAA衍生醋酸可以是脂肪酸/在大脑中脂质的主要来源发育期间和CD可能导致引起NAA未能发展被分解-3,5,6-期间减少髓鞘合成。 ASPA在大脑的肾脏，肝脏和白质主要存在，并给予ASPA的在光盘中的重要作用，在大脑中的这种酶的细胞表达，研究了几个实验室。通过查看在大脑ASPA酶活性，以前的研究发现，脑发育过程中的增加ASPA活性平行髓鞘8-10的时间过程。在细胞水平，对酶的活性，以及原位杂交（ISH）和免疫组织化学（IHC）测定分析表明，ASPA主要表达在大脑，但不是在神经元或胶质细胞11-16少突胶质细胞。一些研究发现，ASPA也说不定在小神经胶质细胞在CNS 12,14来表示。在有机磷农药对ASPA表达到目前为止的数据是有限的。根据最近的研究，其中在小鼠大脑皮层包括神经元，星形胶质细胞，有机磷农药，新形成的少突胶质细胞，髓鞘少突胶质细胞，小胶质细胞，内皮细胞和周细胞不同的细胞类型的转录由RNA测序17进行了分析，ASPA专门在少突胶质细胞表达，特别是在髓鞘少突胶质细胞（http://web.stanford.edu/group/barres_lab/brain_rnaseq.html）。尽管在大脑ASPA表达模式这些研究中，一些不确定因素仍然存在。 不同的技术可用于研究基因表达模式。 IHC是用于组织切片检测基因表达的功能的产品（ 即，蛋白质）通常使用的方法。尽管它的巨大效用，这种技术具有局限性，因为它的应用和特异性对象TØ所需的抗体的可用性和特异性。通过比较，ISH具有能够在mRNA水平发现任何基因的表达的优点。然而，它可以是在技术上具有挑战性的，以便定位一个基因的表达，以特定的细胞类型，同时使用几种探针。在这篇文章中，我们描述了一个协议， 原位杂交蛋白质的免疫标记荧光双结合的荧光RNA。我们使用这组技术来检查电力局在小鼠大脑的表达模式。此方法允许使用共聚焦显微镜的基因表达的精确研究。

<table><tbody><tr><td>QIAprep Miniprep</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>Deionized formamide</td><td>Sigma</td><td>F9037</td><td>for ISH blocking buffer</td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S3014</td><td></td></tr><tr><td>Trizma Base</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>Hydrochloric acid</td><td>VWR International</td><td>20252.290</td><td></td></tr><tr><td>Sodium phosphate monobasic anhydrous</td><td>Sigma</td><td>S8282</td><td></td></tr><tr><td>Sodium phosphate dibasic dihydrate</td><td>Sigma</td><td>30435</td><td></td></tr><tr><td>Yeast tRNA</td><td>Roche</td><td>10109495001</td><td></td></tr><tr><td>50x Denhardt&#039;s solution</td><td>Life Technologies</td><td>750018</td><td></td></tr><tr><td>Dextran sulfate</td><td>Sigma</td><td>D8906</td><td></td></tr><tr><td>Aspa cDNA clone</td><td>Source Bioscience</td><td>IRAVp968C0654D</td><td></td></tr><tr><td>SalI</td><td>New England Biolabs</td><td>R0138</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma</td><td>S2889</td><td></td></tr><tr><td>Equilibrated phenol</td><td>Sigma</td><td>P4557</td><td></td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>C2432</td><td></td></tr><tr><td>Isoamyl alcohol</td><td>Aldrich</td><td>496200</td><td></td></tr><tr><td>Ethanol</td><td>VWR International</td><td>20821.321</td><td></td></tr><tr><td>T7 RNA polymerase</td><td>Promega</td><td>P4074</td><td></td></tr><tr><td>Transcription buffer</td><td>Promega</td><td>P118B</td><td></td></tr><tr><td>100 mM DTT</td><td>Promega</td><td>P117B</td><td></td></tr><tr><td>UTP-DIG NTP mix</td><td>Roche</td><td>11277073910</td><td></td></tr><tr><td>Rnasin</td><td>Promega</td><td>N251B</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Filter paper</td><td>Fisher scientific</td><td>005479470</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>59378</td><td></td></tr><tr><td>Diethyl pyrocarbonate</td><td>Sigma</td><td>D5758</td><td></td></tr><tr><td>Pentobarbitone</td><td>Animalcare Ltd</td><td>BN43054</td><td></td></tr><tr><td>Dissecting scissors</td><td>World Precision Instruments</td><td>15922</td><td></td></tr><tr><td>25 gauge needle</td><td>Terumo</td><td>300600</td><td></td></tr><tr><td>Peristaltic pump</td><td>Cole-Parmer Instrument Co. Ltd</td><td>WZ-07522-30</td><td></td></tr><tr><td>Iris scissors</td><td>Weiss</td><td>103227</td><td></td></tr><tr><td>No.2 tweezers</td><td>World Precision Instruments</td><td>500230</td><td></td></tr><tr><td>Coronal Brain Matrix</td><td>World Precision Instruments</td><td>RBMS-200C</td><td></td></tr><tr><td>Razor blade</td><td>Personna Medical</td><td>PERS60-0138</td><td></td></tr><tr><td>OCT medium</td><td>Tissue tek</td><td>4583</td><td></td></tr><tr><td>Cryostat/microtome</td><td>Bright</td><td></td><td></td></tr><tr><td>Superfrost plus slides</td><td>Thermo Scientific</td><td>J1800AMNZ</td><td></td></tr><tr><td>Sodium citrate</td><td>Sigma</td><td>S4641</td><td>for 65 &amp;deg;C wash buffer</td></tr><tr><td>Formamide</td><td>Sigma-Aldrich</td><td>F7503</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>Coverslips</td><td>VWR International</td><td>631-0146</td><td></td></tr><tr><td>Coplin Jar</td><td>Smith Scientific Ltd</td><td>2959</td><td></td></tr><tr><td>Blocking reagent</td><td>Roche</td><td>11096176001</td><td></td></tr><tr><td>Heat-inactivated sheep serum</td><td>Sigma</td><td>S2263</td><td></td></tr><tr><td>Hydrophobic pen</td><td>Cosmo Bio</td><td>DAI-PAP-S</td><td>1:500</td></tr><tr><td>&amp;alpha;-FITC POD-conjugated antibody</td><td>Roche</td><td>11426346910</td><td></td></tr><tr><td>TSA&amp;trade; Plus Fluorescein System</td><td>Perkin Elmer</td><td>NEL741001KT</td><td>1:1,500</td></tr><tr><td>&amp;alpha;-DIG AP-conjugated</td><td>Roche</td><td>11093274910</td><td></td></tr><tr><td>Fast red tablets</td><td>Roche</td><td>11496549001</td><td></td></tr><tr><td>.22 &amp;micro;M filter</td><td>Millex</td><td>SLGP033RS</td><td></td></tr><tr><td>&amp;alpha;-Olig2 Rabbit antbody</td><td>Millipore</td><td>AB9610</td><td></td></tr><tr><td>Alexa Fluor&amp;reg; 647-conjugated &amp;alpha;-rabbit antibody</td><td>Life technologies</td><td>A-31573</td><td>1:1,000</td></tr><tr><td>bisBenzimide H 33258</td><td>sigma</td><td>B2883</td><td></td></tr><tr><td>Mounting medium</td><td>Dako</td><td>S3023</td><td></td></tr><tr><td>Leica SP2 confocal microscope</td><td>Leica</td><td></td><td></td></tr></tbody></table>

combining double fluorescence in situ hybridization with immunolabelling for detection of the expression of three genes in mouse brain sections

在不同类型的脑细胞如神经元，星形胶质细胞，少突胶质细胞，少突胶质细胞前体和小胶质细胞的基因表达的检测，可通过缺乏免疫染色特定一次或二次抗体的受到阻碍。这里，我们描述一个协议，以检测在同一脑切片三种不同的基因使用原位杂交双荧光表达两个基因特异性探针，接着用高特异性针对由第三基因编码的蛋白质的抗体的免疫染色。所述Aspartoacyclase（ASPA）基因，其中突变可导致一种罕见的人类白质疾病-卡纳疾病-被认为在少突胶质细胞和小胶质细胞，但不是在星形胶质细胞和神经元中表达。然而，在大脑ASPA的精确表达模式还没有被确立。该协议使我们能够确定ASPA在成熟的少突胶质细胞的一个子集表示第二，可以普遍适用于广泛的基因表达模式的研究。

本文介绍了一个双荧光原位杂交，然后在鼠脑切片免疫标记的方法。此协议的简要描述在图1中示出的第一步骤是合成特定电力局和MBP（髓鞘碱性蛋白 ）探针。检查该探针已经合成，每个反应中的一小等份在琼脂糖凝胶上运行。微弱的线性模板和大量的RNA探针可以看出（ 图2）。 为 电力局和MBP双荧光原位杂交，其...

Watch this Scientific Journal Video about Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections at JoVE.com

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 ...

combining-double-fluorescence-situ-hybridization-with-immunolabelling

Research

JoVE Journal

Neuroscience

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

13.1K 次观看.  University College London. 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.

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

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

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 ...

科研

神经科学

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 ...

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

多目标显色全安装<em&gt;原位</em&gt;在比较基因表达域杂交<em&gt;果蝇</em&gt;胚胎

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

发育生物学

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

引物对免疫冰冻切片大鼠脑组织:例为染色小胶质细胞和神经元

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

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 ...

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 ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

成年小鼠大脑中诱导荧光标记干细胞的谱系示踪

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ within a given tissue remain limited. The EASI-FISH protocol described here has been adapted to detect the expression of dozens of genes in the intact adult Drosophila central nervous system (CNS) using commercially available reagents. This protocol includes a new gel formulation that enhances gel robustness, enabling multiple rounds of hybridization and allowing the embedding of multiple brains per gel. This improvement increases throughput, facilitates optimal comparison of experimental conditions, and reduces reagent costs. Additionally, by employing the GAL4-UAS system for co-detection of green fluorescent protein (GFP), gene expression can be visualized in specific neuronal or glial cell types. Notably, the high resolution achieved through expansion microscopy, combined with the sensitivity of the method, allows for the detection of single RNA transcripts. This approach effectively integrates high image quality with high throughput, making it a powerful tool for studying gene expression throughout the intact fly brain.

Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) is a robust technique that combines expansion microscopy with fluorescence in situ hybridization (FISH) to detect the expression of multiple genes in thick tissue. This protocol outlines recent advancements in the method and its adaptation for labeling the intact Drosophila central nervous system..

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ ...

Histological-Based Stainings Using Free-Floating Tissue Sections

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative approaches are widely used to handle the tissue sections during the staining procedure, one approach consists of mounting the sections directly on glass slides, while a second approach, the free-floating, allows for fixed sections to be maintained and stained while suspended in solution. Although slide-mounted and free-floating approaches may yield similar results, the free-floating technique allows for better antibody penetration and thus should be the method of choice when thicker sections are to be used for 3D reconstruction of the tissues, for example when the focus of the experiment is to gain information on dendritic and axonal projections in brain regions. In addition, since the sections are kept in solution, a single aliquot can easily accommodate 30 to 40 sections, handling of which is less laborious, particularly in large-scale biomedical studies. Here, we illustrate how to apply the free-floating method to fluorescent immunohistochemistry staining, with a major focus on brain sections. We will also discuss how the free-floating technique can easily be modified to fit the individual needs of researchers and adapted to other tissues as well as other histochemical-based stainings, such as hematoxylin and eosin and cresyl violet, as long as tissue samples are properly fixed, typically with paraformaldehyde or formalin.

The free-floating technique allows researchers to perform histological-based stainings including immunohistochemistry on fixed tissue sections to visualize biological structures, cell type, and protein expression and localization. This is an efficient and reliable histochemical technique that can be useful for investigating a multitude of tissues, such as brain, heart, and liver.

使用自由浮动组织切片进行基于组织学的染色

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative ...

生物学

 Performing Double Fluorescence In Situ Hybridization to Detect Gene Expression in Mouse Brain Sections

<a href='https://app.jove.com/v/53976/combining-double-fluorescence-situ-hybridization-with-immunolabelling'>Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections. J. Vis. Exp. (2016).</a>This video demonstrates the use of double fluorescence in situ hybridization to visualize the expression of two RNA targets in mouse brain sections. The protocol involves sequential detection of RNA using probes labeled with fluorescein isothiocyanate and digoxigenin, followed by enzymatic signal amplification to enhance sensitivity. The resulting fluorescence enables co-localization analysis of the target RNAs within the tissue.

Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse ...

结合双荧光

摘要

探索更多视频

此视频中的章节

双荧光原位杂交食脑切片

结合亲脂性染料，原位杂交，免疫组织化学和组织学

多目标显色全安装

引物对免疫冰冻切片大鼠脑组织:例为染色小胶质细胞和神经元

在脑切片检测轴突本地化mRNAs的高空间分辨率

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

成年小鼠大脑中诱导荧光标记干细胞的谱系示踪

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

使用自由浮动组织切片进行基于组织学的染色

Performing Double Fluorescence In Situ Hybridization to Detect Gene Expression in Mouse Brain Sections

结合双荧光

摘要

探索更多视频

此视频中的章节

双荧光原位杂交食脑切片

结合亲脂性染料，原位杂交，免疫组织化学和组织学

多目标显色全安装

引物对免疫冰冻切片大鼠脑组织:例为染色小胶质细胞和神经元

在脑切片检测轴突本地化mRNAs的高空间分辨率

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

成年小鼠大脑中诱导荧光标记干细胞的谱系示踪

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

使用自由浮动组织切片进行基于组织学的染色

Performing Double Fluorescence In Situ Hybridization to Detect Gene Expression in Mouse Brain Sections

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