Name: in situ hybridization combined with immunohistochemistry in cryosectioned zebrafish embryos
Uploaded: 2022-03-03T13:00:00
Description: Watch this Scientific Journal Video about In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos at JoVE.com

这项工作得到了中国南通市科学技术基金（MS12019011）、中国南通市科学技术基金（JC2021058）和江苏省高等学校自然科学基金（21KJB180009）的支持。

所有动物方案均由南通大学机构动物护理和使用委员会批准（编号S20191210-402）。
1. 斑马鱼胚胎的采集
<ol><li>在收集卵的前一天晚上在繁殖池中设置一对斑马鱼，一条是转基因斑马鱼，另一条是AB野生型斑马鱼（Tg （foxP2：egfp-caax） X AB野生型或 Tg （hb9：egfp） X AB野生型）（见 材料表）。使用对角线塑料隔板将公头和母头?...

<ol>
	<li>Streisinger, G., Walker, C., Dower, N., Knauber, D., Singer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+clones+of+homozygous+diploid+zebra+fish+(Brachydanio+rerio).">Production of clones of homozygous diploid zebra fish (Brachydanio rerio).</a> Nature. 291 (5813), 293-296 (1981).</li><li>Chen, E., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+genomics+research+model.">Zebrafish as a genomics research model.</a> Current Pharmaceutical Biotechnology. 5 (5), 409-413 (2004).</li><li>Howe, K., Clark, M. D., Torroja, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496 (7446), 498-503 (2013).</li><li>Coons, A. H., Creech, H. J., Jones, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+properties+of+an+antibody+containing+a+fluorescent+group.">Immunological properties of an antibody containing a fluorescent group.</a> Proceedings of the Society for Experimental Biology and Medicine. 47 (2), 200-202 (1941).</li><li>The lack of commercial antibodies for model organisms (and how you can deal with it) [Internet]. BenchSci Blog Available from: <a target="_blank" href="https://blog.benchsci.com/the-lack-of-commercial-antibodies-for-model-organisms-and-how-you-can-deal-with-it">https://blog.benchsci.com/the-lack-of-commercial-antibodies-for-model-organisms-and-how-you-can-deal-with-it</a> (2018)</li><li>Akam, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+location+of+ultrabithorax+transcripts+in+Drosophila+tissue+sections.">The location of ultrabithorax transcripts in Drosophila tissue sections.</a> EMBO Journal. 2, 2075-2084 (1983).</li><li>Hafen, E., Levine, M., Garber, R. L., Gehring, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+in+situ+hybridization+method+for+the+detection+of+cellular+RNAs+in+Drosophila+tissue+sections+and+its+application+for+localizing+transcripts+of+the+homeotic+Antennapedia+gene+complex.">An improved in situ hybridization method for the detection of cellular RNAs in Drosophila tissue sections and its application for localizing transcripts of the homeotic Antennapedia gene complex.</a> EMBO Journal. 2, 617-623 (1983).</li><li>Thisse, B., Thisse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+on+whole-mount+zebrafish+embryos+and+young+larvae.">In situ hybridization on whole-mount zebrafish embryos and young larvae.</a> Methods in Molecular Biology. 1211, 53-67 (2014).</li><li>Clay, H., Ramakrishnan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+fluorescent+in+situ+hybridization+in+zebrafish+embryos+using+tyramide+signal+amplification.">Multiplex fluorescent in situ hybridization in zebrafish embryos using tyramide signal amplification.</a> Zebrafish. 2 (2), 105-111 (2005).</li><li>Driever, W., Stemple, D., Schier, A., Solnica-Krezel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+genetic+tools+for+studying+vertebrate+development.">Zebrafish: genetic tools for studying vertebrate development.</a> Trends in Genetics. 10 (5), 152-159 (1994).</li><li>Cunningham, R. L., Monk, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+mount+in+situ+hybridization+and+immunohistochemistry+for+zebrafish+larvae.">Whole mount in situ hybridization and immunohistochemistry for zebrafish larvae.</a> Methods in Molecular Biology. 1739, 371-384 (2018).</li><li>Thisse, C., Thisse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+in+situ+hybridization+to+whole-mount+zebrafish+embryos.">High-resolution in situ hybridization to whole-mount zebrafish embryos.</a> Nature Protocols. 3 (1), 59-69 (2008).</li><li>Heisler, L. K., Zhou, L., Bajwa, P., Hsu, J., Tecott, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+5-HT(2C)+receptors+regulate+anxiety-like+behavior.">Serotonin 5-HT(2C) receptors regulate anxiety-like behavior.</a> Genes, Brain, and Behavior. 6 (5), 491-496 (2007).</li><li>Ferguson, J. L., Shive, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+immunofluorescence+and+immunohistochemistry+on+cryosectioned+zebrafish+embryos.">Sequential immunofluorescence and immunohistochemistry on cryosectioned zebrafish embryos.</a> Journal of Visualized Experiments: JoVE. (147), e59344 (2019).</li><li>Hammond-Weinberger, D. R., ZeRuth, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+mount+immunohistochemistry+in+zebrafish+embryos+and+larvae.">Whole mount immunohistochemistry in zebrafish embryos and larvae.</a> Journal of Visualized Experiments: JoVE. (155), e60575 (2020).</li><li>Santos, D., Monteiro, S. M., Luzio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+whole-mount+immunohistochemistry+of+zebrafish+(Danio+rerio)+embryos+and+larvae+protocol.">General whole-mount immunohistochemistry of zebrafish (Danio rerio) embryos and larvae protocol.</a> Methods in Molecular Biology. 1797, 365-371 (2018).</li><li>O'Hurley, G., Sjostedt, E., Rahman, A., Li, B., Kampf, C., Ponten, F., 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=Garbage+in,+garbage+out:+a+critical+evaluation+of+strategies+used+for+validation+of+immunohistochemical+biomarkers.">Garbage in, garbage out: a critical evaluation of strategies used for validation of immunohistochemical biomarkers.</a> Molecular Oncology. 8, 783-798 (2014).</li></ol>

该协议提出了原位杂交和免疫组织化学的组合，这是斑马鱼胚胎共定位实验的重要一步。该方法是同时分析一种mRNA和一种蛋白质的简单有效的方法。对斑马鱼胚胎进行原位杂交和抗体染色。与之前发表的几种方案14,15,16相比，免疫荧光和免疫组织化学用于探索蛋白质表达。很少有斑马鱼特异性抗体经过适当验证，...

斑马鱼是用于发育和遗传学研究的强大脊椎动物模型1,2。斑马鱼和人类具有很高的遗传同源性（70%的基因与人类基因组共享），这使其可以用作人类疾病的模型3。在斑马鱼中，检测两个基因的表达模式及其空间关系是很常见的。免疫组织化学于 1941 年首次用于通过应用 FITC 标记的抗体来检测感染组织中的病原体4.组织切片中的靶蛋白首先用一抗标记，然后用针对一抗宿主物种免疫球蛋白的二抗标记切片。抗体染色是检测蛋白质定位的可靠方法，可在细胞内水平上提供高光学分辨率。然而，斑马鱼中可用的抗体数量非常有限。最近的一项研究表明，大约有112,000种抗体可供小鼠使用;然而，很少有抗体被证明在斑马鱼5中是可靠的。
相反，在斑马鱼中，原位杂交已被广泛用于基因表达模式分析。该方法在1980年代6,7首次用于评估果蝇胚胎中的基因表达，从那时起，该技术得到了不断发展和改进。最初，放射性标记的DNA探针用于检测mRNA转录本;然而，空间分辨率相对较低，并且存在放射性引起的潜在健康风险。随后，原位杂交依赖于用地高辛（DIG）或荧光素（Fluo）标记的RNA探针，这些探针与碱性磷酸酶（AP）偶联或通过荧光酪胺信号放大（TSA）检测8,9。尽管TSA已被用于检测两个或三个基因，但RNA探针的DIG标记和抗DIG AP偶联抗体仍然是高度敏感，稳定且广泛使用的原位杂交方法。因此，商业化抗体与DIG标记的原位探针相结合，有助于深入了解一个基因的蛋白质定位和表达。
由于光学分辨率低，整个胚胎无法揭示基因之间的空间关系，即使斑马鱼胚胎小而透明10。因此，切片对于在细胞内水平上分析基因的表达模式是必要的。冷冻切片已广泛用于斑马鱼，因为它易于操作并且可以有效地保存抗原。因此，斑马鱼冷冻切片中的 原位 杂交结合免疫组织化学为分析两个基因的表达模式提供了一种强有力的方法。 原位 杂交和免疫组织化学的组合已应用于斑马鱼11。然而，蛋白酶K处理用于以牺牲抗原完整性为代价来增强探针穿透。为了克服这一限制，该方案使用加热来诱导抗原修复。该方案不仅适用于不同阶段的胚胎和不同厚度的组织切片（14μm头部切片和20μm脊髓切片），而且还通过使用在两个器官中表达的基因进行了验证，包括头部和脊髓。
本文将介绍如何在冷冻切片中结合斑马鱼胚胎的 原位 杂交和抗体染色。通过使用许多 原位 杂交 - 免疫组织化学组合，包括针对两个不同神经元的 原位 杂交探针，证明了该协议的多功能性。该方法适用于检测不同区域和不同年龄胚胎的mRNA和蛋白质，以及两个基因的表达模式。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 488 secondary antibody</td>
			<td>Invitrogen</td>
			<td>A21202</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin AP Fab fragments</td>
			<td>Roche</td>
			<td>11093274910</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Millipore</td>
			<td>MAB3580</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking solution</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>0.1% Triton X-100, 3% BSA, 10% goat serum in 1x PBS</td>
		</tr>
		<tr>
			<td>BM purple</td>
			<td>Roche</td>
			<td>11442074001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>B2064</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C5670</td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate buffer</td>
			<td>Leagene</td>
			<td>IH0305</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma</td>
			<td>C2404</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryomold for tissue, 15 mm x 15 mm x 5 mm</td>
			<td>Head Biotechnology</td>
			<td>H4566</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC-Treated Water</td>
			<td>Sangon Biotech</td>
			<td>B501005</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera, fluorescence microscope</td>
			<td>Nikon</td>
			<td>NI-SSR 931479</td>
			<td></td>
		</tr>
		<tr>
			<td>E3 embryo medium</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Invitrogen</td>
			<td>AM9342</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>J&#38;K Scientific</td>
			<td>542858</td>
			<td></td>
		</tr>
		<tr>
			<td>HYB</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>preHYB plus 50 &#181;g/mL heparin sodium salt, 100 &#181;g/mL ribonucleic acid diethylaminoethanol salt</td>
		</tr>
		<tr>
			<td>Immunohistochemical wet box</td>
			<td>Mkbio</td>
			<td>MH10002</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>Low profile leica blades</td>
			<td>Leica</td>
			<td>819</td>
			<td></td>
		</tr>
		<tr>
			<td>MABT (1x)</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>0.1 M maleic acid, 0.15 M NaCl, 0.02% Tween-20, pH 7.5</td>
		</tr>
		<tr>
			<td>Maleic acid</td>
			<td>Sigma</td>
			<td>M0375</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>J&#38;K Scientific</td>
			<td>116481</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Macklin</td>
			<td>M859248</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>NTMT</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>0.1M Tris-HCl, 0.1M NaCl, 1% Tween-20</td>
		</tr>
		<tr>
			<td>OCT medium</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>Enzo Life Sciences</td>
			<td>ADI-950-233</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 4%</td>
			<td>Abbexa</td>
			<td>abx082483</td>
			<td>made in lab in 1x PBS</td>
		</tr>
		<tr>
			<td>PBST (1x)</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>1x PBS plus 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>Phenylthiourea</td>
			<td>Merck</td>
			<td>103-85-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (10x)</td>
			<td>Invitrogen</td>
			<td>AM9624</td>
			<td></td>
		</tr>
		<tr>
			<td>preHYB</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>50% formamide, 5x SSC, 9.2 mM citric acid (pH 6.0), 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Roche</td>
			<td>1092766</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribonucleic acid diethylaminoethanol salt</td>
			<td>Sigma</td>
			<td>R3629</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free 1.5 mL tubes</td>
			<td>Ambion</td>
			<td>AM12400</td>
			<td></td>
		</tr>
		<tr>
			<td>SSC (20x)</td>
			<td>Invitrogen</td>
			<td>AM9770</td>
			<td></td>
		</tr>
		<tr>
			<td>SSCT (0.2x)</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>0.2x SSC plus 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>SSCT (1x)</td>
			<td>made in lab</td>
			<td>N/A</td>
			<td>1x SSC plus 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Invitrogen</td>
			<td>15503022</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Zebrafish</td>
			<td>Laboratory Animal Center of Nantong University</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ hybridization combined with immunohistochemistry in cryosectioned zebrafish embryos

作为一种脊椎动物，斑马鱼已被广泛用于生物学研究。斑马鱼和人类具有很高的遗传同源性，这使其可以用作人类疾病的模型。基因功能研究基于基因表达模式的检测。尽管免疫组织化学提供了一种测定蛋白表达的有效方法，但斑马鱼中市售抗体的数量有限，限制了成本分配的应用。 原位 杂交广泛用于斑马鱼胚胎中以检测mRNA表达。该协议描述了如何通过结合斑马鱼胚胎切片的 原位 杂交和免疫组织化学来获取图像。在冷冻切片之前进行 原位 杂交，然后进行抗体染色。原 位 杂交后进行免疫组织化学和单次冷冻切片成像。该方案有助于揭示两个基因的表达模式，首先通过 原位 转录本检测，然后通过针对同一部分中蛋白质的免疫组织化学。

该协议可用于同时检查一种mRNA和一种蛋白质的表达模式。图 1 显示了实验性工作流程。5-HT2C受体是由神经递质血清素（5-羟色胺，5-HT）结合的5-HT受体的亚型。它广泛分布在中枢神经系统（CNS）中，可以显着调节多种大脑功能，包括食欲，情绪，焦虑和生殖行为13。5-HT2C在CNS中的表达由转基因系Tg（foxp2：egfp-caax）检测，其中foxP2神经元由荧光素...

Watch this Scientific Journal Video about In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos at JoVE.com

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

该协议描述了如何通过结合斑马鱼胚胎切片的 原位 杂交和免疫组织化学来获取图像。在冷冻切片之前进行 原位 杂交，然后进行抗体染色。如果抗体缺乏，检测斑马鱼中两个基因的表达模式是有用的。

原位 冷冻切片斑马鱼胚胎中的杂交结合免疫组织化学

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a ...

该协议使用原位杂交和免疫组织化学的组合，它是同时分析一种mRNA和一种蛋白质的简单有效的方法。该方案适用于不同阶段的胚胎，不同厚度的组织切片和不同的器官。首先，将大约 10 个胚胎与 0.5 至 1 毫升新鲜的 4% 多聚甲醛固定在 1.5 毫升微量离心管中，在 4 摄氏度下过夜 12 至 14 小时。通过在室温下依次用PBST中的25,50和75%甲醇洗涤5分钟，逐渐脱水胚胎。然后，在室温下用100%甲醇洗涤胚胎五分钟。将胚胎在零下20摄氏度的100%甲醇中孵育至少过夜，持续12至14小时。通过在室温下依次用75%，50和25%甲醇在PBST中洗涤5分钟，逐渐使胚胎再水化。再次，在室温下用PBST洗涤胚胎三次，每次五分钟。在室温下用PBST中每毫升蛋白酶K10微克消化胚胎，并用PBST洗涤胚胎三次五分钟。然后，在室温下将洗涤的胚胎重新固定在4%多聚甲醛中15分钟。同样，用PBST洗涤胚胎三次，同时在每次洗涤期间孵育五分钟。对于胚胎的预杂交，将它们在65摄氏度的预杂交溶液中孵育五分钟。然后，用杂交溶液替换预杂交溶液，并预杂交至少四个小时。在加入胚胎之前，在杂交溶液中加热探针在95摄氏度下加热五分钟。尽可能多地去除预杂交溶液，不要让胚胎与空气接触。向装有胚胎的管中，在杂交溶液中加入预热的探针，并使探针在50至70摄氏度下杂交12至14小时。第二天，用移液管吸出探针溶液并将其储存在零下20摄氏度的管中，以便可以多次重复使用。用100%杂交溶液洗涤胚胎，然后在65摄氏度下用75%，50和25%杂交溶液在两倍浓度的SSCT中连续洗涤15分钟。然后，在65摄氏度下以SSCT浓度的0.2倍洗涤胚胎两次15分钟。在室温下在MABT中洗涤胚胎两次10分钟。在室温下封闭杂交和洗涤的胚胎至少两个小时，用2%封闭溶液-1。在新鲜的2%封闭溶液-1中用抗地高辛碱性磷酸酶替换封闭溶液-1，并在4摄氏度下摇动12至14小时。然后，在室温下在MABT中洗涤胚胎四次30分钟。同样，在NTMT中洗涤胚胎两次，并使用移液管从胚胎中取出尽可能多的NTMT。用BM紫色碱性磷酸酶底物替换，并在室温下在黑暗中染色胚胎。一旦污渍形成，用NTMT短暂冲洗两次来停止反应。原位杂交后，用PBST冲洗胚胎三次20分钟。将胚胎浸入PBS中的5%蔗糖中过夜，在4摄氏度下过夜12至14小时。然后，将覆盖胚胎的溶液在PBS中更改为15%蔗糖，并在4摄氏度下孵育12至16小时。同样，将覆盖胚胎的溶液更换为PBS中的30%蔗糖，并在4摄氏度下孵育一到两天。将胚胎转移到新的冷冻体中，并用最佳切割温度介质轻轻填充，同时避免形成气泡。接下来，使用低温恒温器将样品切成 12 至 20 微米厚的切片，并将切片快速转移到载玻片上。让样品达到室温，并将切片储存在零下80摄氏度的密封载玻片盒中，以备后续使用。用PBS清洗包含切片的载玻片五分钟。然后，将载玻片放入缓冲液中，将溶液加热约20分钟，使其接近沸腾温度。沥干多余的溶液，并用纸巾小心地擦干每个部分周围的区域。然后，用防水笔在该部分周围画一个圆圈以形成疏水屏障，并在室温下将该部分在封闭溶液-2中封闭两个小时。将每张载玻片移取一抗溶液，并将载玻片在4摄氏度的免疫组织化学湿盒中孵育过夜。用PBS洗涤载玻片三次，每次洗涤期间孵育10分钟，并排出多余的PBS。然后，将载玻片与适当的二抗在室温下在PBS中孵育一小时。同样，用PBS洗涤载玻片三次，同时在每次洗涤期间孵育10分钟，并排出多余的PBS。随后，将安装介质移液到载玻片上，并用载玻片盖玻片安装。在转基因foxP2 egfp-caax系中检测到中枢神经系统中5-HT2C受体的表达。在转基因系中观察到5-HT2C与foxP2神经元同时表达，狐狸P2神经元被荧光素绿色荧光蛋白标记。在转基因系中检测到斑马鱼脊髓和运动神经元中的锌指转录因子insm1a的表达。在转基因系中观察到insm1a与hb9神经元同时表达，hb9神经元由荧光素绿色荧光蛋白标记。斑马鱼必须用新鲜制备的多聚甲醛固定。

Research

JoVE Journal

Neuroscience

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Double Fluorescence in situ Hybridization in Fresh Brain Sections

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

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

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 ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

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

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

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

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

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

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

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

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

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

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

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

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

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

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

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

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

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

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

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

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

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

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

原位 冷冻切片斑马鱼胚胎中的杂交结合免疫组织化学

原位冷冻切片斑马鱼胚胎中的杂交结合免疫组织化学