Name: high resolution fluorescent in situ hybridization in drosophila embryos and tissues using tyramide signal amplification
Uploaded: 2017-10-19T13:00:00
Duration: 14 min 21 s
Description: Watch this Scientific Journal Video about High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification at JoVE.com

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 &#956;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>Ampicilin</td>
			<td>Sigma-aldrich</td>
			<td>A9393-5G</td>
			<td>Stock concentration is 100 g/ml disolved ddH&#8322;O and 0.2&#956;m filtered (use as 1/1000)</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 &#956;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&#956;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>DEPC (Diethyl pyrocarbonate)</td>
			<td>Sigma-aldrich</td>
			<td>D5758</td>
			<td>Working concentration is 0.1 % in ddH&#8322;O. To make RNase free water 0.1 %</td>
		</tr>
		<tr>
			<td>Digoxigenin-11-uridine-5&#8217;-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>Hydrogen peroxide (H&#8322;O&#8322;)</td>
			<td>Sigma-Aldrich</td>
			<td>216763-500 ml</td>
			<td></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>Potassium phosphate monobasic (KH&#8322;PO&#8324;)</td>
			<td>Bioshop</td>
			<td>PPM302.1</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>Sodium citrate (HOC(COONa)(CH&#8322;COONa)&#8322; &#183; 2H&#8322;O</td>
			<td>Sigma-aldrich</td>
			<td>S4641-500G</td>
			<td>Reagent in 20 X SSC</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na&#8322;HPO&#8324;)</td>
			<td>Bioshop</td>
			<td>SPD307.1</td>
			<td>Reagent in 10 X PBS</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 &#38; 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 &#956;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions</td>
			<td></td>
			<td></td>
			<td>Preparation</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 &#176;C</td>
		</tr>
		<tr>
			<td>DEPC (Diethyl pyrocarbonate) treated ddH&#8322;O (1L)</td>
			<td></td>
			<td></td>
			<td>Add 1 ml of DEPC in 999 ml of ddH&#8322;O (0.1 %). Stir O/N in fumehood O/N and then autoclave 45 min liquid cycle</td>
		</tr>
		<tr>
			<td>10 X PBS (1L)</td>
			<td></td>
			<td></td>
			<td>NaCL (80 g), KCL (2 g), Na&#8322;HPO&#8324; (14.4 g), KH&#8322;PO&#8324; (2.4 g) and unautoclaved DECP ddH&#8322;O (800 ml). Adjust pH to 7.4 with 10 N NaOH and then bring the total volume to 1 L. Autoclave 45 min liquid cycle</td>
		</tr>
		<tr>
			<td>20 X SSC (1L)</td>
			<td></td>
			<td></td>
			<td>NaCL (175.3 g), Sodium citrate tribasic dihydrate(88.2 g) and unautoclaved DECP ddH&#8322;O (800 ml). Adjust pH to 7.4 with 10 N NaOH and then bring the total volume to 1 L. Autoclave 45 min liquid cycle</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 ...

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

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