该项目的资金支助是由加拿大卫生研究所的 HMK 赠款提供的 (赠款拖把 133473)。

<ol>
	<li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nature Rev Genet. 10, 57-63 (2009).</li><li>Tautz, D., Pfeifle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-radioactive+in+situ+hybridization+method+for+the+localization+of+specific+RNAs+in+Drosophila+embryos+reveals+translational+control+of+the+segmentation+gene+hunchback.">A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback.</a> Chromosoma. 98, 81-85 (1989).</li><li>Hughes, S. C., Krause, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+labeling+with+fluorescence+in+situ+hybridization+in+Drosophila+whole-mount+embryos.">Double labeling with fluorescence in situ hybridization in Drosophila whole-mount embryos.</a> Biotechniques. 24, 530-532 (1998).</li><li>Lecuyer, E., 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=Global+analysis+of+mRNA+localization+reveals+a+prominent+role+in+organizing+cellular+architecture+and+function.">Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.</a> Cell. 131, 174-187 (2007).</li><li>Kosman, D., 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=Multiplex+detection+of+RNA+expression+in+Drosophila+embryos.">Multiplex detection of RNA expression in Drosophila embryos.</a> Science. 305, 846-846 (2004).</li><li>Raap, A. K., 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=Ultra-sensitive+FISH+using+peroxidase-mediated+deposition+of+biotin-+or+fluorochrome+tyramides.">Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides.</a> Human Mol Genet. 4, 529-534 (1995).</li><li>Lecuyer, E., Parthasarathy, N., Krause, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+in+situ+hybridization+protocols+in+Drosophila+embryos+and+tissues.">Fluorescent in situ hybridization protocols in Drosophila embryos and tissues.</a> Methods Mol Biol. 420, 289-302 (2008).</li><li>Wilk, R., Murthy, S. U. M., Yan, H., Krause, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols Essential Laboratory Techniques. , 1-9 (2010).</li><li>Holt, C. E., Bullock, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+mRNA+Localization+in+Animal+Cells+and+Why+It+Matters.">Subcellular mRNA Localization in Animal Cells and Why It Matters.</a> Science. 326, 1212-1216 (2009).</li><li>Martin, K. C., Ephrussi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+Localization:+Gene+Expression+in+the+Spatial+Dimension.">mRNA Localization: Gene Expression in the Spatial Dimension.</a> Cell. , 719-730 (2009).</li><li>Wilk, R., Hu, J., Blotsky, D., Krause, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+and+pervasive+subcellular+distributions+for+both+coding+and+long+noncoding+RNAs.">Diverse and pervasive subcellular distributions for both coding and long noncoding RNAs.</a> Genes Dev. 30, 594-609 (2016).</li><li>Zhou, X. L., Vize, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximo-distal+specialization+of+epithelial+transport+processes+within+the+Xenopus+pronephric+kidney+tubules.">Proximo-distal specialization of epithelial transport processes within the Xenopus pronephric kidney tubules.</a> Dev Biol. 271, 322-338 (2004).</li></ol>

作者没有竞争的金融利益。

所描述的协议为检测固定的果蝇胚胎或组织中的大多数 rna 提供了一种高度重现性、灵敏和经济的方法。虽然比传统的碱性磷酸酶探针检测方法稍微复杂一些, 但鱼获得的分辨率要大得多, 灵敏度是可比较或改进的7,8。通过使用整体或部分孔板, 该协议可用于 large-scale 或有限分析的兴趣基因。应该注意的是, 所生成的探针可能检测到所有或多个成绩单拼接形式, 除非探头更具体地设计 (即单显)。尽管我们已经测试了小到200核苷酸的探针, 但有合理的成功, 较小的探针往往不太有效, 而且可能不太具体。显然, 此协议不适用于检测小内含子、显或处理的 rna。
虽然这种方法使用酶的步骤, 以提高信号的强度, 信号强度似乎成正比的目标基因表达在一个相对线性和广泛的表达水平。在高倍率下, 信号被视为细胞和组织内的点或斑点。为相对地罕见的抄本仅少数微小的点被看见。随着成绩单的增多, 这些增长的数量和规模。与单分子鱼 (smFISH) 一样, 单个的小点也可以对应于单一的 rna, 并且还应该使用成像和信息学软件进行量化。使用 smFISH 与我们为同一目标所策划的图像进行对比, 表明这两种方法的整体性、灵敏度和分辨率是相对相似的, 尽管这应该通过更严格的比较来测试。鉴于 smFISH 的费用要高得多, 这里提供的方法应该以成本的一小部分给出类似的结果。然而, 如果目标是检测小的成绩单, 或不同部分的成绩单, 建议 smFISH。
由于一些鱼探头的体积较小, 这种方法在穿透组织大或有明显障碍的情况下也会更好。然而, 我们使用更高的洗涤剂水平和组织固定和性调整似乎解决了这个问题。最后, 虽然开发了为果蝇组织, 这里描述的高洗涤剂缓冲的组织也应该为果蝇胚胎以及大多数其他有机体和组织工作。

新的全基因组 rna 检测方法, 如 rna 序列, 极大地扩展了我们对何时何地表达基因的认识, 以及在什么级别的1。然而, 这些提供了相对较差的时间和空间分辨率。RNA 原位杂交允许在固定组织中视觉上观察转录物的空间分布, 从而揭示细胞和亚型本地化2的细节。使用荧光原位杂交 (fish) 允许更多的空间分辨率, 因为它允许使用强大的显微镜技术, 如共焦和光片显微镜3。荧光标记 (如 DAPI) 也可用于建立亚细胞关系4。鱼类还有助于观察多个 rna 和蛋白质同时有明确的重叠在亚细胞水平的迹象。double-labeling 所需的独特试剂和步骤可在以下参考资料3,5中找到。
这里描述的程序利用96井孔板的所有步骤, 包括 cDNA 模板生产 (菌落 PCR), RNA 探针生产 (使用 T7, T3 或 SP6 聚合酶依赖性径流转录),原位杂交,荧光信号的发展。在96井板的每个井中加入足够数量的胚胎或组织, 以便评估一致性和变异性。每一个井然后接受不同的探针。信号发育后, 每个井中的胚胎或组织都在各自的显微镜切片上排列, 进行显微分析。
使用 tyramide 信号放大 (TSA) 进行探针检测产生了具有特殊的亚细胞分辨率5,6,7,8的鲁棒信号。rna 的亚细胞定位是一个重要的调节机制9 , 它出现在几乎所有 rna 的4、10、11中。这些分析还显示, 许多未被 RNA 序列检测到的记录被鱼8轻易检测到。RNA 的适应原位杂交到96井板允许分析最多96基因的兴趣 (如果更多的板块被使用), 使得分析大数据集成为可能。通过将板块切割成更小的部分, 这种方法也很容易适应少数样品。所提供的协议适用于大多数类型组织中 RNA 分布的分析。虽然没有显示, 它也适用于非果蝇组织。

<table><tbody><tr><td>15 ml Polypropylene conical centrifuge tubes</td><td>Frogga Bio</td><td>TB15-500</td><td>Certified DNase and RNase free</td></tr><tr><td>2x PCR Master Mix with dye(Ambiogene)</td><td>Biomart (Canada)</td><td>141106-1K</td><td>Certified DNase and RNase free</td></tr><tr><td>50 ml Polypropylene conical centrifuge tubes</td><td>Frogga Bio</td><td>TB50-500</td><td>Certified DNase and RNase free</td></tr><tr><td>96-well filteration plate (1.2 um, hydrophilic PVDF membrane</td><td>EMD MILLIPORE</td><td>MSBVS1210</td><td>Used to remove liguid from bottom of the plate with floting tissues</td></tr><tr><td>96-well PCR plate (200 &amp;mu;l)</td><td>Denville</td><td>C18096-10</td><td></td></tr><tr><td>Acrodisc syringe filter (0.2 u)</td><td>PALL</td><td>PN4612</td><td></td></tr><tr><td>Acrodisc syringe filter (0.45 u)</td><td>PALL</td><td>PN4614</td><td></td></tr><tr><td>Agarose</td><td>Bioshop</td><td>AGA002.500</td><td></td></tr><tr><td>AXYGEN 96-well assay storage plates</td><td>UltiDent Scientific</td><td>24-P96-450V-C</td><td>To store bacterial glycerol stocks</td></tr><tr><td>AXYGEN Aluminum Plate Seals</td><td>UltiDent Scientific</td><td>24-PCR-AS-200</td><td>To seal plates</td></tr><tr><td>AXYGEN Wide-bore tips (200 ul)</td><td>UltiDent Scientific</td><td>24-T205-WB-C</td><td>To transfer samples</td></tr><tr><td>Biotin-SP (long spacer) IgG Fraction Monoclonal Mouse Anti-Digoxin</td><td>Jackson ImmunoResearch Lab</td><td>200062156</td><td>Primary antibody for in situ (stock conc. 1 mg/ml, working conc. 2.5 &amp;mu;g/ml or 1/400 dilution.</td></tr><tr><td>BRAND 8-channel manifold (autoclavable)</td><td>BrandTech Scientific Inc.</td><td>704526</td><td>To remove liquids from the top of 96-well PCR plates</td></tr><tr><td>Chloramphenicol</td><td>Sigma-aldrich</td><td>C1919</td><td>Stock concentration 34 mg/ml dissolved and 0.2&amp;mu;m filtered in 100 % EtOH (use as 1/1000)</td></tr><tr><td>Cyanine 3 Tyramide (Cy3-TSA)</td><td>Home made</td><td></td><td>Could be ordered from Perkin-Elmer Cat# SAT704A001EA</td></tr><tr><td>DABCO (1,4-Diazabicyclo[2.2.2]octane)</td><td>Sigma-aldrich</td><td>D2522</td><td>Antifade reagent in mounting media</td></tr><tr><td>Digoxigenin-11-uridine-5&amp;rsquo;-triphosphate</td><td>Roche</td><td>11-209-256-910</td><td></td></tr><tr><td>Embryo collection cage</td><td>Home made</td><td>N/A</td><td>Size: 75 cm X 75 cm X 75 cm made with Polymethyl methacrylate</td></tr><tr><td>Eppendorf centrifuge 5804</td><td>Eppendorf</td><td>Model 5804</td><td>Centrifuge for 96-well plates on A-Z-DWP rotor</td></tr><tr><td>Formamide</td><td>Bioshop</td><td>FOR001.500</td><td></td></tr><tr><td>Glycerol</td><td>Bioshop</td><td>GLY002.4</td><td></td></tr><tr><td>Glycine</td><td>Bioshop</td><td>GLN001.500</td><td></td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H4784-250MG</td><td>Stock: 50 mg/ml. Working concentration 1 mg/ml in Hybridization solution</td></tr><tr><td>Lab Armor Beads</td><td>DiaMed</td><td>LAA42370-001</td><td>Fill in heating unit</td></tr><tr><td>Labnet Gyro M Mini Nutating Mixer</td><td>Fisher Scientific</td><td>50-998-347</td><td>Sample mixing</td></tr><tr><td>Mesh - 100 micons</td><td>FlyStuff</td><td>57-103</td><td>For collecting embryos</td></tr><tr><td>Mesh - 800 microns</td><td>SEFAR - Nytex</td><td>06-780/53</td><td>For catching loose tissue / may be used for special tube in figure 4.</td></tr><tr><td>Micro Cover Glasses</td><td>VWR</td><td>48366-067</td><td>Size: 22 X 22 mm</td></tr><tr><td>Micro slides,frstd</td><td>VWR</td><td>48312-003</td><td>Size: 25 X 75 mm</td></tr><tr><td>Multi-Well Plate Vacuum Manifold</td><td>Pall Corporation</td><td>P/N 5017</td><td>Remove liquid from bottom of 96-well filter plate</td></tr><tr><td>Paraformaldehyd (PFA)</td><td>Bioshop</td><td>PAR070.500</td><td>Fixation</td></tr><tr><td>PCR machine</td><td>Bio-Rad</td><td>Model: DNA Engine PTC-200</td><td>96-well plate PCR and probe denature</td></tr><tr><td>Picric acid solution</td><td>Sigma-aldrich</td><td>80456</td><td>For tissue fixation I solution</td></tr><tr><td>Potassium Chloride (KCL)</td><td>Bioshop</td><td>POC308</td><td></td></tr><tr><td>PP lid for 96-well plates, 100/case</td><td>UltiDent Scientific</td><td>24-P-LID-PP</td><td>Lid for storing plates</td></tr><tr><td>Progene disposable Reagent Reservoirs (50 ml, sterile)</td><td>UltiDent Scientific</td><td>825-1-50-5S</td><td>Liquid handling</td></tr><tr><td>Progene Pierceable Aluminum Foil</td><td>UltiDent Scientific</td><td>87-CFILM-AL</td><td>Seal 96-well plate, DNase and RNase free</td></tr><tr><td>Proteinase K</td><td>Sigma-aldrich</td><td>P2308- 5MG</td><td>Embryo permeabilization</td></tr><tr><td>RiboLock Ribonuclease Inhibitor</td><td>Thermo Scientific</td><td>EO0381</td><td>40 Unit/ul</td></tr><tr><td>Ribonucleoside Triphosphate (NTP) set</td><td>Roche</td><td>11277057001</td><td>RNA probe sythesis</td></tr><tr><td>RNase free water</td><td>GIBCO</td><td>10977015-500ml</td><td>RNA probe sythesis</td></tr><tr><td>RNaseZap</td><td>Ambion</td><td>AM9780</td><td>Remove RNase contamination</td></tr><tr><td>Single stranded DNA from salmon testes</td><td>Sigma-aldrich</td><td>D9156</td><td>For hybridization solution</td></tr><tr><td>Skim milk (non fat power)</td><td>Bioshop</td><td>SKI400.500</td><td>Working concentration is 1 % in 1 X PBT or PBTT as a blocking reagent</td></tr><tr><td>Sodium Acetate (NaOAc) 3 M, pH=5.2 (RNase free)</td><td>Bioshop</td><td>SAA333</td><td>Working concentration is 10 % (v/v) for DNA or RNA precipitation</td></tr><tr><td>SP6 RNA Polymerase</td><td>Thermo Scientific</td><td>EP0131</td><td>20 Unit/ul</td></tr><tr><td>Stainless Steel Shot for Sand Bath</td><td>VWR</td><td>13259-274.</td><td>To fill the heating unit</td></tr><tr><td>Stereomicroscope &amp;amp; gooseneck light source</td><td>Leica</td><td>Model: MZ6</td><td>Tissue dissection and mounting</td></tr><tr><td>Streptavidin, horseradish peroxidase (HRP) conjugate</td><td>Molecular Probes</td><td>S911</td><td>Secondary antibody stock: 1 mg/ml, use 1/1000 dilution (1 &amp;mu;g/ml) for in situ.</td></tr><tr><td>T3 RNA Polymerase</td><td>Thermo Scientific</td><td>EP0101</td><td>20 Unit/ul</td></tr><tr><td>T7 RNA Polymerase</td><td>Thermo Scientific</td><td>EP0111</td><td>20 Unit/ul</td></tr><tr><td>Tip station (10 ul)</td><td>Axygen</td><td>T-300-STK-S</td><td>Certified DNase and RNase free</td></tr><tr><td>Tip station (200 ul)</td><td>Sorbio</td><td>27770T</td><td>Certified DNase and RNase free</td></tr><tr><td>Tips ( 1ml)</td><td>Sorbio</td><td>10200</td><td>Certified DNase and RNase free</td></tr><tr><td>TRITON X-100</td><td>Bioshop</td><td>TRX506</td><td>Working concentration is 0.3% in 1 X PBT for tissue in situ</td></tr><tr><td>Tween 20</td><td>Bioshop</td><td>TWN510</td><td>Working concentration is 0.1% in 1 X PBT for embryo in situ</td></tr><tr><td>Whatman qualitative filter paper, Grade 3</td><td>Sigma-aldrich</td><td>WHA1003917</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Solutions&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Preparation&lt;/strong&gt;</td></tr><tr><td>DABCO (1,4-Diazabicyclo[2.2.2]octane) glycerol anti-fade mounting media</td><td></td><td></td><td>DABCO (1.25 g), Glycerol (35 ml or v/v 70 %) and 1 X PBS (15 ml, or v/v 30 %). Keep dark and rock in cold room. store at -20 &amp;deg;C</td></tr><tr><td>1 X PBT (1L)</td><td></td><td></td><td>1 X PBS with 1 ml Tween 20 (0.1 %)</td></tr><tr><td>1 X PBTT (1L)</td><td></td><td></td><td>1 X PBS with 1 ml Tween 20 (0.1 %) and 3 ml of Triton-X-100 (0.3 %)</td></tr><tr><td>1 X PBTB (1L)</td><td></td><td></td><td>1 % Skim milk in 1 X PBT</td></tr><tr><td>1 X PBTTB (1L)</td><td></td><td></td><td>1 % Skim milk in 1 X PBTT</td></tr></tbody></table>

high resolution fluorescent in situ hybridization in drosophila embryos and tissues using tyramide signal amplification

在我们的努力, 以确定的表达模式和亚细胞定位的果蝇rna 在全基因组的基础上, 并在各种组织, 我们已经开发了许多修改和改进, 我们原来的荧光原位杂交 (鱼) 协议。为了方便吞吐量和成本效益, 所有步骤, 从探头生成到信号检测, 都是使用外显子 96-孔板。Digoxygenin (挖掘)-标签反义 RNA 探针的生产使用的是 cDNA 克隆或基因组 DNA 作为模板。在组织固定和性后, 探针杂交到感兴趣的抄本, 然后用一系列抗性的抗体结合生物素, 亲和共轭辣根过氧化物酶 (HRP) 和荧光共轭tyramide, 在 HRP 的存在下, 产生一个高度反应性的中间体, 结合到紧邻的蛋白质的电子密集区域。这些放大和定位步骤产生了一个强大的和高度本地化的信号, 有利于细胞和亚单元转录定位。所提供的协议经过了优化, 可以在各种组织和发育阶段产生高度特异的信号。此外, 还提供了额外的变异, 同时检测多个成绩单, 或抄本和蛋白质。

图 6显示了使用此过程获得的结果的示例。前3面板显示了早期的果蝇胚胎 (图 6, 面板 A-C) 的示例, 接下来的3面板显示了在不同组织 (amnioserosa、肌肉和中枢神经系统中有信号的晚期果蝇胚胎的示例。分别 (图 6, 面板 D-f)。接下来是第三龄幼虫组织的例子 (图 6, J 型面板)。K 和 L 组显示成人性腺 (卵巢和睾丸分别) 的代表性图像。在我们可搜索的 "荧光原位果蝇数据库" (FlyFISH (http://fly-fish.ccbr.utoronto.ca)) 上上载并标注了数以百计的图像。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56281/56281fig1.jpg"> 
图1。荧光原位杂交 (fish) 协议和关键设备概述.(A) cDNA 的 pcr 扩增。(b) 体外转录在一个96井板中生成基因特异的反义挖掘标记 RNA 探针 (如 B 所示的板的照片)。(C)和(D):成人 (女性: 男性比率 2:1. 300-400 苍蝇/瓶) 苍蝇可以交配3-4 天。在25° c 的标准玉米面食品上过夜收集的胚胎被转移到一个通风良好的塑料盒 (1 升的玉米面食品在一个容器大小: 8 "x 8" x 3 "高) 或新瓶保持适当的幼体密度。对于组织收集, 活性酵母粉应洒在食物的顶部 24, 48 小时后产卵 (快)。(E 到 H):荧光原位杂交试验连续3天进行。对于不漂浮的胚胎或组织, 使用96井 PCR 板, 如图 b 所示, 8 通道器 (相片 c)。对于漂浮的组织, 像大多数幼体组织一样, 使用一个滤底板 (在 d 中的照片), 并使用低压下的流形器从底部去除液体 (e 中的照片)。<a href="//ecsource.jove.com/files/ftp_upload/56281/56281fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56281/56281fig2.jpg"> 
图2。RNA 探针产量的测定。在1% 琼脂糖凝胶 (5 µL/车道) 上观察到的新合成反义 RNA 探针。产量可以被分类 (根据带的强度) 作为 "低收益" 在车道3和 10, ' 典型的出产量 ' 在车道 1, 4, 5, 6 和 8, 或 "高收益" 在车道 2, 7, 9, 11 和12。对于具有 "典型产量" 的样品, 使用10-15 µL 探针稀释100µL 的杂交溶液为每个原位实验。根据波段强度相对于 "典型探针" 强度, 将探针的 "高收率" 稀释样品与附加杂交溶液。目的是在每个井中有大致相等的最终探针浓度。箭头指向一个 ' RNA 探针 ', 在每条车道上的更高的波段是原始的 DNA 模板。<a href="//ecsource.jove.com/files/ftp_upload/56281/56281fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56281/56281fig3.jpg"> 
图3。large-scale 胚胎采集和固定的主要步骤.<a href="//ecsource.jove.com/files/ftp_upload/56281/56281fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56281/56281fig4.jpg"> 
图4。自制的管子, 用于从幼虫或漂浮的组织中除去液体.一些幼虫和成人组织在固定期间漂浮在溶液中。这个简单的工具由一个尼龙网格由一个200µL 削减尖端, 后跟一个1毫升小费作为适配器连接到一个器。液体可以安全地去除, 而不会丢失任何组织。<a href="//ecsource.jove.com/files/ftp_upload/56281/56281fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56281/56281fig5.jpg"> 
图5。Tyramide 信号放大.(1) 用内源感 rna hybridizes 的反义 rna 探针。(2) 生物素-共轭主抗体与挖双绞线之间的免疫亲和结合。(3) HRP 共轭亲和结合生物素。(4) HRP 催化菁 3-tyramide 和过氧化氢反应形成短命的菁 3-tyramide 基。(5) 菁 3-tyramide 基与邻近蛋白的酪氨酸残留物结合。<a href="//ecsource.jove.com/files/ftp_upload/56281/56281fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56281/56281fig6.jpg"> 
图6。代表原位杂交图像.每个面板显示一个不同的 RNA (显示在青色) 和细胞核染色的 DAPI (以红色显示) 的例子。(A-C)早期的果蝇胚胎的例子。(D、F)晚期果蝇胚胎的示例。(G-J)例第三龄幼虫果蝇幼体组织 (malphigian 管、中肠、涎腺和肌肉分别)。(K) 成人卵巢。(L) 成人睾丸。每个面板都显示探针与之杂交的基因的名称。缩放栏 = 10 µm.<a href="//ecsource.jove.com/files/ftp_upload/56281/56281fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification at JoVE.com

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

所描述的 rna原位杂交协议允许检测整个果蝇胚胎或解剖组织中的 rna。使用96井孔板和 tyramide 信号放大, 记录可以检测高分辨率, 灵敏度和吞吐量, 并以较低的成本。

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

high-resolution-fluorescent-situ-hybridization-drosophila-embryos

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

12.8K 次观看.  University of Toronto. 所描述的 rna原位杂交协议允许检测整个果蝇胚胎或解剖组织中的 rna。使用96井孔板和 tyramide 信号放大, 记录可以检测高分辨率, 灵敏度和吞吐量, 并以较低的成本。

视频: High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

科研

遗传学

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

斑马鱼整装高分辨率双荧光原位杂交

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

生物学

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

为介观荧光体层摄影术在体内成像的发展果蝇</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

可视化 果蝇 胚胎中含脂质细胞器的细胞骨架依赖性运输

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

Using Expansion Microscopy to Physically Enlarge Whole-Mount Drosophila Embryos for Super-Resolution Imaging

The workhorse of developmental biology is the confocal microscope, which allows researchers to determine the three-dimensional localization of tagged molecules within complex biological samples. While traditional confocal microscopes allow one to resolve two adjacent fluorescent point sources located a few hundred nanometers apart, observing the finer details of subcellular biology requires the ability to resolve signals in the order of tens of nanometers. Numerous hardware-based methods for super-resolution microscopy have been developed to allow researchers to sidestep such resolution limits, although these methods require specialized microscopes that are not available to all researchers. An alternative method for increasing resolving power is to isotropically enlarge the sample itself through a process known as expansion microscopy (ExM), which was first described by the Boyden group in 2015. ExM is not a type of microscopy per se but is rather a method for swelling a sample while preserving the relative spatial organization of its constituent molecules. The expanded sample can then be observed at an effectively increased resolution using a traditional confocal microscope. Here, we describe a protocol for implementing ExM in whole-mount Drosophila embryos, which is used to examine the localization of Par-3, myosin II, and mitochondria within the surface epithelial cells. This protocol yields an approximately four-fold increase in sample size, allowing for the detection of subcellular details that are not visible with conventional confocal microscopy. As proof of principle, an anti-GFP antibody is used to distinguish distinct pools of myosin-GFP between adjacent cell cortices, and fluorescently labeled streptavidin is used to detect endogenous biotinylated molecules to reveal the fine details of the mitochondrial network architecture. This protocol utilizes common antibodies and reagents for fluorescence labeling, and it should be compatible with many existing immunofluorescence protocols.

Using Expansion Microscopy to Physically Enlarge Whole-Mount Drosophila Embryos for Super-Resolution Imaging

Here, a protocol for the implementation of expansion microscopy in early Drosophila embryos to achieve super-resolution imaging using a conventional laser-scanning confocal microscope is presented.

使用扩增显微镜物理放大 整个果蝇 胚胎以进行超分辨率成像

The workhorse of developmental biology is the confocal microscope, which allows researchers to determine the three-dimensional localization of tagged ...

通过抗体介导的实时成像增强蛋白质可视化

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

作者聚焦：揭示动物形态发生研究中机械和生化信号的动力学 

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

摘要

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此视频中的章节

在果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

多目标显色全安装

斑马鱼整装高分辨率双荧光原位杂交

为介观荧光体层摄影术在体内成像的发展果蝇

可视化果蝇胚胎中含脂质细胞器的细胞骨架依赖性运输

使用扩增显微镜物理放大整个果蝇胚胎以进行超分辨率成像

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

全山RNA荧光

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

摘要

探索更多视频

此视频中的章节

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

多目标显色全安装

斑马鱼整装高分辨率双荧光原位杂交

为介观荧光体层摄影术在体内成像的发展果蝇

可视化 果蝇 胚胎中含脂质细胞器的细胞骨架依赖性运输

使用扩增显微镜物理放大 整个果蝇 胚胎以进行超分辨率成像

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

全山RNA荧光

在果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

可视化果蝇胚胎中含脂质细胞器的细胞骨架依赖性运输

使用扩增显微镜物理放大整个果蝇胚胎以进行超分辨率成像