Name: in situ hybridization techniques for paraffin-embedded adult coral samples
Uploaded: 2018-08-31T13:00:00
Description: Watch this Scientific Journal Video about In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples at JoVE.com

这项工作是由奖项资助的。OCE-1323652 通过国家科学基金会海洋科学博士后奖学金和1012629号奖, 来自宝来康威基金博士后浓缩计划。

1. 石蜡的去除
注意: 在通风罩下执行以下步骤。
<ol><li>脱蜡100% 二甲苯下的薄切片石蜡嵌入的幻灯片在玻璃 Coplin 罐子里10分钟。不要使用塑料 Coplin 罐子, 因为二甲苯熔化塑料。使用前, 在高压釜中消毒 Coplin 罐。</li>
	<li>准备四无菌玻璃 Coplin 罐与以下: 100% 乙醇, 80% 乙醇, 70% 乙醇和60% 乙醇。用无 RNase 的水稀释乙醇。<ol>
<li>将幻灯片转移到无菌玻璃 Coplin 罐中, 用100% 乙醇浸泡1...

<ol>
	<li>Hughes, T. P., 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=Climate+Change,+Human+Impacts,+and+the+Resilience+of+Coral+Reefs.">Climate Change, Human Impacts, and the Resilience of Coral Reefs.</a> Science. 301 (5635), 933 (2003).</li><li>Chen, P. -. Y., Chen, C. -. C., Chu, L., McCarl, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+economic+damage+of+climate+change+on+global+coral+reefs.">Evaluating the economic damage of climate change on global coral reefs.</a> Global Environmental Change. 30 (Supplement C), 12-20 (2015).</li><li>Hoegh-Guldberg, O., 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=Coral+Reefs+Under+Rapid+Climate+Change+and+Ocean+Acidification.">Coral Reefs Under Rapid Climate Change and Ocean Acidification.</a> Science. 318 (5857), 1742 (2007).</li><li>Grasso, L. C., 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=Microarray+analysis+identifies+candidate+genes+for+key+roles+in+coral+development.">Microarray analysis identifies candidate genes for key roles in coral development.</a> BMC Genomics. 9 (1), 540 (2008).</li><li>Miller, D. J., 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=The+innate+immune+repertoire+in+Cnidaria+-+ancestral+complexity+and+stochastic+gene+loss.">The innate immune repertoire in Cnidaria - ancestral complexity and stochastic gene loss.</a> Genome Biology. 8 (4), R59 (2007).</li><li>Shinzato, C., Iguchi, A., Hayward, D. C., Technau, U., Ball, E. E., Miller, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox+genes+in+the+coral+Acropora+millepora:+divergent+expression+patterns+reflect+differences+in+developmental+mechanisms+within+the+Anthozoa.">Sox genes in the coral Acropora millepora: divergent expression patterns reflect differences in developmental mechanisms within the Anthozoa.</a> BMC Evolutionary Biology. 8 (1), 311 (2008).</li><li>Anctil, M., Hayward, D. C., Miller, D. J., Ball, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+and+expression+of+four+coral+G+protein-coupled+receptors+distinct+from+all+classifiable+members+of+the+rhodopsin+family.">Sequence and expression of four coral G protein-coupled receptors distinct from all classifiable members of the rhodopsin family.</a> Gene. 392 (1-2), 14-21 (2007).</li><li>Darby, I. A., Hewitson, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In situ hybridization protocols. , (2006).</li><li>Valentino, K. L., Eberwine, J. H., Barchas, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In situ hybridization. , (1987).</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 (2), 81-85 (1989).</li><li>Traylor-Knowles, N., Rose, N. H., Palumbi, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+specificity+of+gene+expression+in+the+response+to+heat+stress+in+corals.">The cell specificity of gene expression in the response to heat stress in corals.</a> Journal of Experimental Biology. 220 (10), (2017).</li><li>Wolenski, F. S., Layden, M. J., Martindale, M. Q., Gilmore, T. D., Finnerty, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+spatiotemporal+expression+of+RNAs+and+proteins+in+the+starlet+sea+anemone,+Nematostella+vectensis.">Characterizing the spatiotemporal expression of RNAs and proteins in the starlet sea anemone, Nematostella vectensis.</a> Nature Protocols. 8, 900 (2013).</li><li>Schnitzler, C. E., Simmons, D. K., Pang, K., Martindale, M. Q., Baxevanis, A. D. <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+multiple+Sox+genes+through+embryonic+development+in+the+ctenophore+Mnemiopsis+leidyi+is+spatially+restricted+to+zones+of+cell+proliferation.">Expression of multiple Sox genes through embryonic development in the ctenophore Mnemiopsis leidyi is spatially restricted to zones of cell proliferation.</a> EvoDevo. 5 (1), 15 (2014).</li><li>Traylor-Knowles, N. G., Kane, E. G., Sombatsaphay, V., Finnerty, J. R., Reitzel, 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=Sex-specific+and+developmental+expression+of+Dmrt+genes+in+the+starlet+sea+anemone,+Nematostella+vectensis.">Sex-specific and developmental expression of Dmrt genes in the starlet sea anemone, Nematostella vectensis.</a> EvoDevo. 6 (1), (2015).</li><li>Barry, S. N., Crow, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+HoxA11+and+HoxA13+in+the+evolution+of+novel+fin+morphologies+in+a+representative+batoid+(Leucoraja+erinacea).">The role of HoxA11 and HoxA13 in the evolution of novel fin morphologies in a representative batoid (Leucoraja erinacea).</a> EvoDevo. 8 (1), 24 (2017).</li><li>Sharma, P. P., Gupta, T., Schwager, E. E., Wheeler, W. C., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subdivision+of+arthropod+cap-n-collar+expression+domains+is+restricted+to+Mandibulata.">Subdivision of arthropod cap-n-collar expression domains is restricted to Mandibulata.</a> EvoDevo. 5 (1), 3 (2014).</li><li>Piatigorsky, J., Kozmik, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cubozoan+jellyfish:+an+Evo/Devo+model+for+eyes+and+other+sensory+systems.">Cubozoan jellyfish: an Evo/Devo model for eyes and other sensory systems.</a> The International Journal of Developmental Biology. 48 (8-9), 719-729 (2004).</li></ol>

本协议中描述的方法已从以前在医学和进化发展研究8、9、10、12、17中的工作中进行了修改。该协议的重点是在原位杂交的细微差别与挖掘标记 RNA 反意识探针的成年珊瑚, 已保存和嵌入石蜡。这种方法可以很容易地转移到生物样本以外的珊瑚, 也已石蜡嵌入。最后, 这?...

珊瑚是重要的生态系统建设者和重要的生物多样性的海洋和人类健康1,2,3。由于气候变化和其他人为的压力, 它们受到威胁, 许多珊瑚物种被认为是严重濒危的。因此, 需要细胞和分子工具来诊断珊瑚在压力下。此外, 对于在成人珊瑚组织中表达基因的地方, 几乎没有什么了解, 因此对这些基因的功能了解甚少。为了解决这个问题, 我们已经修改了原位杂交协议, 通常用于人类医学和进化发育生物学, 用于在成人珊瑚的石蜡嵌入组织样本。这项技术是最强大的, 当用于成年珊瑚已经经历了紧张的事件, 如接触热应力。然而, 这种技术可用于珊瑚的广泛的组织和生命阶段, 并不仅限于热应激珊瑚4,6,7。此外, 这种技术可以用于任何后生的组织或细胞只要有 cDNA 序列信息可用。
这种方法的目的是在成年珊瑚组织中可视化 RNA 探针, 这些细胞已经保存并嵌入石蜡切片并切片到幻灯片上。这种方法是一个强大的诊断工具, 允许在成人珊瑚组织中的核酸可视化。最初这种方法是为医疗诊断而开发的, 此后它已成为发展生物学和进化发育生物学8,9,10等领域的流行工具。当基因组和 transcriptomic 序列数据可用, 但空间基因表达模式未知时, 它也是一种关键方法, 特别是在非模型系统中。对于非模型系统的诊断工作, 这种技术是强大的, 因为它可以表明哪些细胞和组织表达感兴趣的基因, 并可以导致更有针对性的治疗方法8,9,10, 11,12。最后, 该技术在与定量基因表达数据配对时, 质量和功能更强11。
本文概述的方法将感兴趣的研究人员已经设计了辛 (挖) 标记的 RNA 探针 (这两种感觉和反义探针), 现在准备进行原位杂交探针到一个样本。为了执行这种方法, 每一个正在测试的探针都需要两个连续切片的石蜡珊瑚组织。一节将用于感官探针, 另一个用于反义探针。感官探测器将是指示非特定绑定的控件。如果在感官探针中观察到染色, 则反义探针并不特定于感兴趣的 RNA。探针可以为任何表达的基因设计。在本议定书中, 以前发现在珊瑚热应激期间表达的几个例子: FBJ 小鼠骨肉瘤病毒癌基因同源 b (Fos b), 激活蛋白 (AP1), 肿瘤坏死因子受体 41 (TNFR 41)11。使用挖掘标记的 RNA 探针比使用放射性探针更可取, 因为它们的处理更安全10。此外, 这项技术是高度敏感的, 可以执行广泛的组织和胚胎以外的热应力成人珊瑚13,14,15,16。

<table>
	<tbody>
		<tr>
			<td>Denhardt&#39;s solution</td>
			<td>Affymetrix</td>
			<td>70468 50 ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioworld Alkaline phosphatase buffer</td>
			<td>Fisher</td>
			<td>50-198-724</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide mailers</td>
			<td>Fisher</td>
			<td>12-587-17B</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioworld Alkaline phosphatase buffer</td>
			<td>Fisher</td>
			<td>50-198-724</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon tubes</td>
			<td>Fisher</td>
			<td>14-959-49A</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Salmon Sperm DNA solution</td>
			<td>Invitrogen</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS - Phosphate-Buffered Saline (10X) pH 7.4</td>
			<td>Invitrogen</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure DNase/RNase-Free Distilled Water, 10 x 500 mL</td>
			<td>Invitrogen</td>
			<td>10977-023</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure DNase/RNase-Free Distilled Water, 10 x 500 mL</td>
			<td>Invitrogen</td>
			<td>10977-023</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Salmon Sperm DNA solution</td>
			<td>Invitrogen</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide white apex superior adhesive</td>
			<td>Leica Biosystems</td>
			<td>3800080</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS solution, pH 7.4</td>
			<td>Life Technologies</td>
			<td>10010072</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K, Molecular Grade, 2 mL</td>
			<td>New England Biolabs</td>
			<td>P8107S</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Pap Pen Liquid Blocker</td>
			<td>Promega</td>
			<td>22309</td>
			<td></td>
		</tr>
		<tr>
			<td>DIG Anti-Digoxigenin-AP Fab fragments</td>
			<td>Roche</td>
			<td>11093274910</td>
			<td></td>
		</tr>
		<tr>
			<td>BM Purple, 100 mL</td>
			<td>Roche</td>
			<td>11442074001</td>
			<td></td>
		</tr>
		<tr>
			<td>DIG Wash and Block Buffer Set</td>
			<td>Roche</td>
			<td>11585762001</td>
			<td></td>
		</tr>
		<tr>
			<td>NBT/BCIP</td>
			<td>Roche</td>
			<td>11681451001</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution, 500 mL size</td>
			<td>Sigma-Aldrich</td>
			<td>252549-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>SSC Buffer 20X concentration</td>
			<td>Sigma-Aldrich</td>
			<td>S6639-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Anhydride</td>
			<td>Sigma-Aldrich</td>
			<td>320102-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma-Aldrich</td>
			<td>47670-250ML-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>90279-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine intestinal mucosa</td>
			<td>Sigma-Aldrich</td>
			<td>H3149-10KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes, AR (ACS), For Histological Use</td>
			<td>VWR</td>
			<td>MK866806</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>EM-EX0276-4S</td>
			<td></td>
		</tr>
		<tr>
			<td>TE buffer</td>
			<td>VWR</td>
			<td>PAV6232</td>
			<td></td>
		</tr>
		<tr>
			<td>hybridization oven</td>
			<td>VWR</td>
			<td>97005-252, 97005-254</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>VWR</td>
			<td>89032-088</td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ hybridization techniques for paraffin-embedded adult coral samples

珊瑚是重要的海洋无脊椎动物, 对整个海洋健康和人类健康至关重要。然而, 由于海洋温度上升和海洋酸化等人类影响, 珊瑚越来越受到威胁。为了应对这些挑战, 细胞和分子生物学的进步已经证明对诊断珊瑚的健康至关重要。修改人类医学常用的一些技术可以大大提高研究人员治疗和拯救珊瑚的能力。为了解决这一问题, 现已将一种主要用于人类医学和进化发育生物学的原位杂交协议用于在压力下的成年珊瑚中使用。
该方法的目的是可视化的 RNA 探针在成人珊瑚组织, 已嵌入石蜡和切片到玻璃幻灯片的空间表达。该方法的重点是去除样品的石蜡和补液, 对样品进行预处理, 以保证样品的渗透性, 预杂交孵化, rna 探针的杂交, rna 探针的可视化。这是一个强有力的方法, 当使用非模型有机体发现特定基因的表达, 并且协议可以很容易地适应其他非模型有机体。然而, 该方法是有限的, 因为它主要是定性的, 因为表达强度可能会因在可视化步骤中使用的时间量和探针的浓度而异。此外, 需要有耐心, 因为这个协议可能需要5天 (而且在许多情况下, 更长的时间) 取决于所使用的探头。最后, 非特异背景染色是常见的, 但这一限制可以克服。

完成该协议后, 将会对表达 RNA 探针的细胞和组织进行识别。该协议的代表性结果为 AP-1、FosB 和 TNFR41。这些结果, 以前发表的特雷勒-诺里斯等。11、在暴露于热应激的成人珊瑚上显示 RNA 探针的空间表达。图 1给出了两种不同染色类型的例子。图 1A是一种弥漫性组织染色的例子。染色是在整个组织中发现?...

Watch this Scientific Journal Video about In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples at JoVE.com

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

本议定书的目的是对已嵌入石蜡切片的成人珊瑚样品进行原位杂交, 并将其切片到玻璃滑梯上。这是一种定性的方法, 用于可视化的 RNA 反感探针在石蜡嵌入组织的空间表达。

就地石蜡镶嵌成人珊瑚样品的杂交技术

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In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

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Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

用原位曝光法测定淡水珍珠贻贝 Bioindication 的流环境适宜性

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

用于表征复杂天然有机物混合物的单吞吐量互补高分辨率分析技术

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

在西图化学分析中，以检验水生生态系统中的微生物行为

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread ...

Determination of Total Lipid and Lipid Classes in Marine Samples

海洋样品中总脂质和脂质类别的测定

Lipids are largely composed of carbon and hydrogen and, therefore, provide a greater specific energy than other organic macromolecules in the sea. Being ...