Name: whole mount immunohistochemistry in zebrafish embryos and larvae
Uploaded: 2020-01-29T14:30:00
Description: Watch this Scientific Journal Video about Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae at JoVE.com

国家卫生研究院的资助赠款8P20GM103436 14。

本议定书中描述的斑马鱼繁殖成人和胚胎的手术程序得到了默里州立大学机构动物护理和使用委员会的批准。
1. 胚胎收集和固定
<ol><li>通过将成年斑马鱼混合性对或组放入装有系统水的网状或槽内衬的水箱中，准备产卵池。</li>
	<li>在亮起灯时，将产卵罐水更换为新鲜系统水，以清除粪便。使用 14 小时/10 小时暗循环，上午 9 点亮起灯。</li>
	<li>一旦产卵，让大人回到家里的...

<ol>
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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>Lopez, 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=Combined+in+situ+Hybridization/Immunohistochemistry+(ISH/IH)+on+Free-floating+Vibratome+Tissue+Sections.">Combined in situ Hybridization/Immunohistochemistry (ISH/IH) on Free-floating Vibratome Tissue Sections.</a> Bio-protocol. 4 (18), (2014).</li><li>Morimoto, M., Morita, N., Ozawa, H., Yokoyama, K., Kawata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+glucocorticoid+receptor+immunoreactivity+and+mRNA+in+the+rat+brain:+an+immunohistochemical+and+in+situ+hybridization+study.">Distribution of glucocorticoid receptor immunoreactivity and mRNA in the rat brain: an immunohistochemical and in situ hybridization study.</a> Neuroscience Research. 26 (3), 235-269 (1996).</li><li>Newton, S. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic Molecular Protocols in Neuroscience: Tips, Tricks, and Pitfalls. , 91-103 (2014).</li><li>Vogel, C., Marcotte, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+regulation+of+protein+abundance+from+proteomic+and+transcriptomic+analyses.">Insights into the regulation of protein abundance from proteomic and transcriptomic analyses.</a> Nature Reviews Genetics. 13 (4), 227-232 (2012).</li><li>Semache, M., Ghislain, J., Zarrouki, B., Tremblay, C., Poitout, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+and+duodenal+homeobox-1+nuclear+localization+is+regulated+by+glucose+in+dispersed+rat+islets+but+not+in+insulin-secreting+cell+lines.">Pancreatic and duodenal homeobox-1 nuclear localization is regulated by glucose in dispersed rat islets but not in insulin-secreting cell lines.</a> Islets. 6 (4), e982376 (2014).</li><li>Kang, H. S., Beak, J. Y., Kim, Y. S., Herbert, R., Jetten, 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=Glis3+is+associated+with+primary+cilia+and+Wwtr1/TAZ+and+implicated+in+polycystic+kidney+disease.">Glis3 is associated with primary cilia and Wwtr1/TAZ and implicated in polycystic kidney disease.</a> Molecular and Cellular Biology. 29 (10), 2556-2569 (2009).</li><li>Billova, S., Galanopoulou, A. S., Seidah, N. G., Qiu, X., Kumar, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+expression+and+colocalization+of+somatostatin,+carboxypeptidase-E+and+prohormone+convertases+1+and+2+in+rat+brain.">Immunohistochemical expression and colocalization of somatostatin, carboxypeptidase-E and prohormone convertases 1 and 2 in rat brain.</a> Neuroscience. 147 (2), 403-418 (2007).</li><li>Westerfield, 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> The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio Rerio). , (2000).</li><li>N&#252;sslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish: A Practical Approach. , (2002).</li><li>Cheng, C. N., Li, Y., Marra, A. N., Verdun, V., Wingert, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flat+mount+preparation+for+observation+and+analysis+of+zebrafish+embryo+specimens+stained+by+whole+mount+in+situ+hybridization.">Flat mount preparation for observation and analysis of zebrafish embryo specimens stained by whole mount in situ hybridization.</a> Journal of Visualized Experiments. (89), (2014).</li><li>Brennan, C., Mangoli, M., Dyer, C. E. F., Ashworth, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+and+calcium+signalling+regulates+muscle+fibre+formation+in+the+zebrafish+embryo.">Acetylcholine and calcium signalling regulates muscle fibre formation in the zebrafish embryo.</a> Journal of Cell Science. 118 (22), 5181 (2005).</li><li>Liu, D. W., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clustering+of+muscle+acetylcholine+receptors+requires+motoneurons+in+live+embryos,+but+not+in+cell+culture.">Clustering of muscle acetylcholine receptors requires motoneurons in live embryos, but not in cell culture.</a> The Journal of Neuroscience. 12 (5), 1859 (1992).</li><li>Borodinsky, L. N., Spitzer, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+neurotransmitter-receptor+matching+at+the+neuromuscular+junction.">Activity-dependent neurotransmitter-receptor matching at the neuromuscular junction.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (1), 335-340 (2007).</li><li>Brunelli, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamatergic+reinnervation+through+peripheral+nerve+graft+dictates+assembly+of+glutamatergic+synapses+at+rat+skeletal+muscle.">Glutamatergic reinnervation through peripheral nerve graft dictates assembly of glutamatergic synapses at rat skeletal muscle.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (24), 8752-8757 (2005).</li><li>Hans, F., Dimitrov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H3+phosphorylation+and+cell+division.">Histone H3 phosphorylation and cell division.</a> Oncogene. 20 (24), 3021-3027 (2001).</li><li>Yee, N. S., Pack, M. <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+model+for+pancreatic+cancer+research.">Zebrafish as a model for pancreatic cancer research.</a> Methods in Molecular Medicine. 103, 273-298 (2005).</li><li>Seth, A., Stemple, D. L., Barroso, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+use+of+zebrafish+to+model+metabolic+disease.">The emerging use of zebrafish to model metabolic disease.</a> Disease Models &amp; Mechanisms. 6 (5), 1080-1088 (2013).</li><li>Fontana, B. D., Mezzomo, N. J., Kalueff, A. V., Rosemberg, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developing+utility+of+zebrafish+models+of+neurological+and+neuropsychiatric+disorders:+A+critical+review.">The developing utility of zebrafish models of neurological and neuropsychiatric disorders: A critical review.</a> Experimental Neurology. 299 (Pt A), 157-171 (2018).</li><li>Richter, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glyoxal+as+an+alternative+fixative+to+formaldehyde+in+immunostaining+and+super-resolution+microscopy.">Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy.</a> The EMBO journal. 37 (1), 139-159 (2018).</li><li>Elsalini, O. A., Rohr, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenylthiourea+disrupts+thyroid+function+in+developing+zebrafish.">Phenylthiourea disrupts thyroid function in developing zebrafish.</a> Development genes and evolution. 212 (12), 593-598 (2003).</li><li>Wang, W. D., Wang, Y., Wen, H. J., Buhler, D. R., Hu, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenylthiourea+as+a+weak+activator+of+aryl+hydrocarbon+receptor+inhibiting+2,3,7,8-tetrachlorodibenzo-p-dioxin-induced+CYP1A1+transcription+in+zebrafish+embryo.">Phenylthiourea as a weak activator of aryl hydrocarbon receptor inhibiting 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1A1 transcription in zebrafish embryo.</a> Biochemical pharmacology. 68 (1), 63-71 (2004).</li><li>Bohnsack, B. L., Gallina, D., Kahana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenothiourea+sensitizes+zebrafish+cranial+neural+crest+and+extraocular+muscle+development+to+changes+in+retinoic+acid+and+IGF+signaling.">Phenothiourea sensitizes zebrafish cranial neural crest and extraocular muscle development to changes in retinoic acid and IGF signaling.</a> PloS one. 6 (8), e22991 (2011).</li><li>Inoue, D., Wittbrodt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+for+all--a+highly+efficient+and+versatile+method+for+fluorescent+immunostaining+in+fish+embryos.">One for all--a highly efficient and versatile method for fluorescent immunostaining in fish embryos.</a> PLoS One. 6 (5), e19713 (2011).</li></ol>

免疫组织化学是一种多功能的工具，可用于描述生物体中几乎所有感兴趣的蛋白质的时空表达。免疫组织化学用于各种组织和模型生物体。该协议已针对斑马鱼的使用进行了优化。不同物种的免疫组织化学可能需要不同的固定和处理技术，根据物种和内源性过氧化物酶的存在阻塞溶液，以及由于组织的厚度和组成而孵育的时间。斑马鱼中的IHC在增进我们对癌症24、代谢性疾病<sup ...

斑马鱼（达尼奥再鱼）已成为研究生物过程的有力模型，原因包括发电时间短、发展迅速、遗传技术的可及性等。因此，斑马鱼通常用于高通量小分子屏幕的毒理学研究和药物发现。Zebrafish 也是研究发育过程的一个有吸引力的模型，因为单个雌性可以一次正常生产 50-300 个卵子，而光学清晰的胚胎在外部发育，从而能够有效地可视化发育过程。然而，由于在建立反向遗传技术方面的挑战，早期研究主要依靠使用N-乙基-N-硝基苏雷亚（ENU）或其他诱变基因的正向遗传屏幕。大约20年前，斑马鱼首次使用变形金刚来敲除靶向基因1。形态诺是一种小的反义寡核苷酸，在早期发育阶段将微注射到胚胎后抑制目标mRNA的转化。形态软糖的一个主要弱点是，当细胞分裂时，它们被稀释，通常在受精后72小时（hpf）时失去效力。虽然形态动物仍然是斑马鱼基因破坏的有力工具，但转录活化样核酸酶（TALEN）、锌指核酸酶（ZFNs）和定期聚因间隔短回溯重复（CRISPRs）最近被用于直接靶向斑马鱼基因组2，3。这些反向遗传策略，结合正向遗传学和高通量屏幕，建立了斑马鱼作为研究基因表达和功能的强大模型。
研究基因功能的能力通常需要评估基因或基因产物表达的时空分布。在发育早期可视化这种表达模式的两种最常用的技术是原位杂交（ISH）和全装载免疫组织化学（IHC）。原位杂交于1969年首次开发，依靠使用标记的反义RNA探针来检测生物体4中的mRNA表达。相反，标记抗体用于免疫组织化学，以可视化蛋白质表达。标记蛋白质进行检测的想法可追溯到20世纪30年代的5世纪，1941年，第一个IHC实验发表于1941年，当时FITC标记的抗体被用来检测受感染组织中的致病细菌。ISH和IHC在随后几十年中已经发展并显著改善，现在都常规地用于分子和诊断研究实验室7，8，9，10，11。虽然这两种技术都有优点和缺点，但 IHC 比 ISH 具有若干优点。实际上，IHC 比 ISH 耗费少得多，并且根据原抗体的成本通常较低。此外，mRNA表达并不总是蛋白质表达的可靠指标，正如在小鼠和人类中已经证明的，只有大约三分之一的蛋白质丰度变异可以通过mRNA丰度12来解释。因此，IHC 是确认 ISH 数据的重要补充，如果可能的话。最后，IHC可以提供亚细胞和共同本地化数据，这些数据不能由ISH13、14、15确定。在这里，我们描述了一个分步法，通过免疫性化学在整座斑马鱼胚胎和幼虫中可靠地检测蛋白质。这项技术的目的是确定整个胚胎中感兴趣的蛋白质的时空表达。该技术利用抗原特异性原抗体和荧光标记的二级抗体。该协议易于适应，适用于滑动安装的组织部分，并与致变色基质一起用于荧光。使用这个协议，我们证明，除了乙酰胆碱受体外，开发斑马鱼骨骼肌表达的有益性谷氨酸受体。NMDA型谷氨酸受体亚单位在23 hpf的纵向肌肉上可检测到。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP160-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil, heavy duty</td>
			<td>Kirkland</td>
			<td></td>
			<td>Any brand may be substituted</td>
		</tr>
		<tr>
			<td>Anti-NMDA antibody</td>
			<td>Millipore Sigma</td>
			<td>MAB363</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-phospho-Histone H3 (Ser10), clone RR002</td>
			<td>Millipore Sigma</td>
			<td>05-598</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-pan-AMPA receptor (GluR1-4)</td>
			<td>Millipore Sigma</td>
			<td>MABN832</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Fisher Scientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Nitrate [Ca(NO3)2]</td>
			<td>Sigma Aldrich</td>
			<td>C4955</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 1.5 mL</td>
			<td>Axygen</td>
			<td>MCT150C</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear nail polish</td>
			<td>Sally Hanson</td>
			<td></td>
			<td>Any nail polish or hardener may be subsituted</td>
		</tr>
		<tr>
			<td>Depression (concavity) slide</td>
			<td>Electron Miscroscopy Sciences</td>
			<td>71878-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Diaminobenzidine</td>
			<td>Thermo Scientific</td>
			<td>1855920</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryo medium, Danieau, 30%</td>
			<td></td>
			<td></td>
			<td>17.4 mM NaCl, 0.21 mM KCl, 0.12 mM MgS04, 0.18 mM Ca(NO3)2, 1.5 mM HEPES in ultrapure water.</td>
		</tr>
		<tr>
			<td>Embryo medium, E2</td>
			<td></td>
			<td></td>
			<td>7.5 mM NaCl, 0.25 mM KCl, 0.5 mM MgSO4, 75 uM KH2PO4, 25 uM Na2HPO4, 0.5 mM CaCl2, 0.35 mM NaHCO3, 0.5 mg/L methylene blue</td>
		</tr>
		<tr>
			<td>Floating tube holder</td>
			<td>Thermo Scientific</td>
			<td>59744015</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence compound microscope</td>
			<td>Leica Biosystems</td>
			<td>DMi8</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence stereomicroscope</td>
			<td>Leica Biosystems</td>
			<td>M165-FC</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips 18 x 18</td>
			<td>Corning</td>
			<td>284518</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips 22 x 60</td>
			<td>Thermo Scientific</td>
			<td>22-050-222</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG Alexa 488</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES solution</td>
			<td>Sigma Aldrich</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>Humid chamber with lid</td>
			<td>Simport</td>
			<td>M920-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide, 30%</td>
			<td>Fisher Scientific</td>
			<td>H325-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunedge pap pen</td>
			<td>Vector labs</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Insect pins, size 00</td>
			<td>Stoelting</td>
			<td>5213323</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (MgSO4 &#183; 7H2O)</td>
			<td>Sigma Aldrich</td>
			<td>63138</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh strainer</td>
			<td>Oneida</td>
			<td></td>
			<td>Any brand may be substituted</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Sigma Aldrich</td>
			<td>M9140</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-tube cap lock</td>
			<td>Research Products International</td>
			<td>145062</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave oven</td>
			<td>Toastmaster</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG</td>
			<td>Sigma Aldrich</td>
			<td>I8765</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Millipore Sigma</td>
			<td>S02L1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutating mixer</td>
			<td>Fisher Scientific</td>
			<td>88-861-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>04042-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr>
			<td>PBTriton</td>
			<td></td>
			<td></td>
			<td>1% TritonX-100 in 1x PBS</td>
		</tr>
		<tr>
			<td>Permount mounting medium</td>
			<td>Fisher Chemical</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (glass)</td>
			<td>Pyrex</td>
			<td>3160100</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (plastic)</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>1-phenyl 2-thiourea</td>
			<td>Acros Organics</td>
			<td>207250250</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS), 10x, pH 7.4</td>
			<td>Gibco</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS), 1x</td>
			<td></td>
			<td></td>
			<td>1x made from 10x stock diluted in dH2O</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Hydroxide (KOH)</td>
			<td>Fisher</td>
			<td>P250-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic (KH2PO4)</td>
			<td>Sigma Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Sigma Aldrich</td>
			<td>10165921001</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Invitrogen</td>
			<td>AM2544</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic (Na2HPO4)</td>
			<td>Sigma Aldrich</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr>
			<td>Spawning tank with lid and insert</td>
			<td>Aquaneering</td>
			<td>ZHCT100</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperBlock PBS</td>
			<td>Thermo Scientific</td>
			<td>37515</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost + slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Superglue gel</td>
			<td>3M Scotch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TNT</td>
			<td></td>
			<td></td>
			<td>100 mM Tris, pH 8.0; 150 mM NaCl; 0.1% Tween20; made in dH2O</td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Fisher</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloracetic Acid (Cl3CCOOH)</td>
			<td>Sigma Aldrich</td>
			<td>T6399</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Sigma Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrafine forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, ultrapure/double distilled</td>
			<td>Fisher Scientific</td>
			<td>W2-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

whole mount immunohistochemistry in zebrafish embryos and larvae

免疫组织化学是一种广泛使用的技术，用于在正常发育和疾病状态下探索蛋白质表达和定位。尽管许多免疫组织化学方案已针对哺乳动物组织和组织部分进行了优化，但这些协议通常需要对非哺乳动物模型生物体进行修改和优化。斑马鱼越来越多地作为基础、生物医学和转化研究的模型系统，用于研究发育过程的分子、遗传和细胞生物学机制。斑马鱼作为模型系统具有许多优点，但也需要经过修改的技术来优化蛋白质检测。在这里，我们提供斑马鱼胚胎和幼虫全装荧光免疫基化学方案。该协议还描述了可以使用的几种不同的安装策略，并概述了每种策略提供的优缺点。我们还描述了对该协议的修改，以允许检测整个安装组织中的染色体基质和切片幼虫组织的荧光检测。该议定书广泛适用于许多发育阶段和胚胎结构的研究。

全装免疫组织化学利用抗体检测完整动物中蛋白质表达的空间模式。免疫组织化学的基本工作流程（如图1所示）包括繁殖斑马鱼、培育和制备胚胎、阻断非特异性抗原、使用抗原特异性原抗体靶向感兴趣的蛋白质、检测带有标记的次级抗体的初级抗体、安装标本和记录表达。
全山免疫性化学是研究斑马?...

Watch this Scientific Journal Video about Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae at JoVE.com

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

在这里，我们提出了一个方案，荧光抗体介导检测蛋白质在斑马鱼胚胎和幼虫的整个制剂。

斑马鱼胚胎和幼虫的整个山免疫性化学

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

全安装免疫组织化学是一种相对快速和简单的技术，允许使用标记抗体直接在组织或生物体中对蛋白质进行空间和时间观察。这项技术的实用性是，它允许染色一个完整的组织或生物体，而不必第一节它和共物显微镜可以用来产生蛋白质表达模式的三维表示。免疫史托化学在生物医学研究中被广泛用于了解疾病的病因和分子机制。它也是一个有价值的诊断工具，用于识别病理样本中的肿瘤。首先，准备装满系统水的产卵池，并准备好网状或槽槽衬垫。将成年斑马鱼混合性对成组在水箱过夜。使用 14 小时/10 小时的光/暗循环，9 点 9 .m。第二天早上，随着灯的亮起，用新鲜的系统水改变产卵罐水以清除粪便。一旦产卵，将成人返回家中。使用转移移液器绘制鸡蛋来收集它们。将50个卵子转移到每个装满胚胎培养的培养皿中。取出任何不透明和多云的卵子，这些鸡蛋已经死亡或无法分割。在28.5摄氏度下孵育鸡蛋，直到受精后23小时。如果胚胎在受精后超过24小时，用移液器将胚胎移植到胚胎介质中的200微摩尔PTU，以防止黑色素生成。在立体显微镜下，使用超细的尖端钳子对未带帽子的胚胎进行脱光。使用火抛光的巴斯特移液器，以尽量减少损害，将它们转移到玻璃或塑料培养皿涂有1-2%的阿加罗斯溶解在胚胎介质中。然后使用塑料或火抛光移液器将胚胎移植到1.5毫升离心管。用微管取出胚胎介质。只留下足够的液体来覆盖胚胎。在室温下轻轻摇动，将胚胎在4%PFA中固定一到两个小时。接下来，在1XPBS和1%PBTriton中清洗胚胎三次，洗涤5分钟。立即使用胚胎或在4摄氏度下储存长达一周。对于长期储存，脱水和储存胚胎在100%甲醇在零下20摄氏度。要补充胚胎水分，在室温下进行连续孵育五分钟，同时减少甲醇、PBS和PBTriton的量，进行最后的洗涤。根据手稿继续胚胎制备。选择与二级抗体宿主物种匹配的阻断溶液，例如 PBTriton 中每毫升 BSA 有或没有两毫克的 10% 山羊血清。在含有胚胎的管子中加入0.5毫升的阻块溶液，并在室温下将管子放在摇杆上一至三个小时。然后在震撼时在四摄氏度下孵育原抗体稀释的阻断溶液和PBTriton。早晨，在PBTriton中清洗胚胎五次，每次在室温下摇动10分钟。然后根据初级抗体的宿主种类和488纳米所需的波长选择二级抗体。将稀释的二级抗体放入含有胚胎的管子中。用铝箔盖住管子，在室温下孵育两小时，同时摇动。之后，在PBTriton中清洗胚胎三次，在室温下清洗10分钟，同时用铝箔和摇动覆盖。使用移液器，将胚胎转移到PBS中50%甘油溶液的床上，在胚胎介质中，在2%的阿加罗斯的床上。要去除蛋黄，请将 200 升 1X PBS 微升转移到凹陷滑梯或普通玻璃滑梯上。使用塑料转移移液器将一个或多个胚胎移动到 PBS 液滴。使用超细钳和双零昆虫针来分离蛋黄，非常轻轻地从胚胎的腹体表面刮黄粒。取出蛋黄颗粒，并根据需要补充 PBS。安装后，将样品放在显微镜舞台上。通过选择相对明亮的示例找到感兴趣的区域。调整摄像机曝光和增益，使信号足够明亮而不饱和。在比较实验抗体标记胚胎和控制抗体胚胎时，使用相同的暴露设置。全山免疫基础化学的这一协议是利用斑马鱼发育研究时空蛋白表达的宝贵工具。NMDA型谷氨酸受体的GluN1亚单位在受精后23小时显示谷氨酸受体亚基在发育斑马鱼肌肉过程中的表现。受精后72小时胚胎的冷冻部分被安装在幻灯片上，并免疫，用于磷-H3，这是增殖细胞的标志。几个细胞表示磷-H3，在心室区域最显著。小鼠IgG控制抗体和排除原抗体揭示了非特异性表达水平低。重要的是要选择适当的阻断解决方案和二级抗体基于主要抗体宿主物种，以确保检测和尽量减少非特异性染色。必须验证免疫历史化学，以确保所观测到的信号实际上是感兴趣的蛋白质。后续实验通常涉及使用基因或环境改造的生物体。免疫组织化学使研究人员能够就地观察蛋白质，大大加快了我们对基础和应用生物学中许多系统的理解，特别是在胚胎发育过程中。甲醇和甲醛是有毒的，应谨慎处理。废物需要按照体制程序收集和处理。

Research

JoVE Journal

Developmental Biology

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

科研

发育生物学

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

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

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 ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

全斑马鱼胚胎的延长时移共聚焦显微镜的分层安装方法

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

使用全载免疫作用化学和三维重建对咽弓动脉的可视化与分析

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart ...