Name: in situ hybridization in zebrafish larvae and juveniles during mesonephros development
Uploaded: 2021-08-13T14:45:00
Description: Watch this Scientific Journal Video about In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development at JoVE.com

资金由宾夕法尼亚科学院，宾夕法尼亚生物学家联邦和宾夕法尼亚州印第安纳大学（研究生学习与研究学院，生物学系和辛西娅·苏沙克本科生物学卓越基金）提供。 Tg（lhx1a-EGFP） 转基因系由Neil Hukriede博士（匹兹堡大学）提供。

斑马鱼幼体和幼鱼的使用已获得IUP IACUC的批准（协议#02-1920，#08-1920）。解决方案内容的详细信息列在 材料表中。
1. 培育幼虫
注意：将幼虫和幼体提升到感兴趣的阶段最多需要21天或更长时间。
<ol><li>在最后一顿饭后的傍晚，通过在交配池中加入1条雄性和1条雌性鱼来设置成年斑马鱼进行交配。 注意：并非所有的鱼对都会交配。首先设置2...

<ol>
	<li>Kinth, P., Mahesh, G., Panwar, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+zebrafish+research:+a+global+outlook.">Mapping of zebrafish research: a global outlook.</a> Zebrafish. 10 (4), 510-517 (2013).</li><li>Grunwald, D. J., Eisen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Headwaters+of+the+zebrafish+-+emergence+of+a+new+model+vertebrate.">Headwaters of the zebrafish - emergence of a new model vertebrate.</a> Nature Reviews. Genetics. 3 (9), 717-724 (2002).</li><li>Wingert, R. A., Davidson, A. J. <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+pronephros:+a+model+to+study+nephron+segmentation.">The zebrafish pronephros: a model to study nephron segmentation.</a> Kidney International. 73 (10), 1120-1127 (2008).</li><li>Drummond, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+development+and+disease+in+the+zebrafish.">Kidney development and disease in the zebrafish.</a> Journal of the American Society of Nephrology: JASN. 16 (2), 299-304 (2005).</li><li>Swanhart, L. M., 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=Zebrafish+kidney+development:+basic+science+to+translational+research.">Zebrafish kidney development: basic science to translational research.</a> Birth defects research. Part C, Embryo Today: Reviews. 93 (2), 141-156 (2011).</li><li>Dressler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+early+kidney+specification,+development+and+patterning.">Advances in early kidney specification, development and patterning.</a> Development. 136 (23), 3863-3874 (2009).</li><li>Johnson, C. S., Holzemer, N. F., 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=Laser+ablation+of+the+zebrafish+pronephros+to+study+renal+epithelial+regeneration.">Laser ablation of the zebrafish pronephros to study renal epithelial regeneration.</a> Journal of Visualized Experiments: JoVE. (54), e2845 (2011).</li><li>Marra, A. 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=Visualizing+gene+expression+during+zebrafish+pronephros+development+and+regeneration.">Visualizing gene expression during zebrafish pronephros development and regeneration.</a> Methods in Cell Biology. 154, 183-215 (2019).</li><li>McMenamin, S. K., Chandless, M. N., Parichy, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+with+zebrafish+at+postembryonic+stages.">Working with zebrafish at postembryonic stages.</a> Methods in Cell Biology. 134, 587-607 (2016).</li><li>Diep, C. Q., 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=Development+of+the+zebrafish+mesonephros.">Development of the zebrafish mesonephros.</a> Genesis. 53 (3-4), 257-269 (2015).</li><li>Zhou, W., Boucher, R. C., Bollig, F., Englert, C., Hildebrandt, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mesonephric+development+and+regeneration+using+transgenic+zebrafish.">Characterization of mesonephric development and regeneration using transgenic zebrafish.</a> American Journal of Physiology. Renal Physiology. 299 (5), 1040-1047 (2010).</li><li>Diep, C. Q., 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=Identification+of+adult+nephron+progenitors+capable+of+kidney+regeneration+in+zebrafish.">Identification of adult nephron progenitors capable of kidney regeneration in zebrafish.</a> Nature. 470 (7332), 95-100 (2011).</li><li>Diep, C. Q., Mikeasky, N., Davidson, A. J., Cartner, 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=.">.</a> The Zebrafish in Biomedical Research: Biology, Husbandry, Diseases, and Research Applications. , 145-150 (2020).</li><li>Parichy, D., Elizondo, M., Mills, M., Gordon, T., Engeszer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+table+of+postembryonic+zebrafish+development:+staging+by+externally+visible+anatomy+of+the+living+fish.">Normal table of postembryonic zebrafish development: staging by externally visible anatomy of the living fish.</a> Developmental dynamics: An Official Publication of the American Association of Anatomists. 238 (12), 2975-3015 (2009).</li><li>Guo, R., 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=LIM+Homeobox+4+(lhx4)+regulates+retinal+neural+differentiation+and+visual+function+in+zebrafish.">LIM Homeobox 4 (lhx4) regulates retinal neural differentiation and visual function in zebrafish.</a> Science Reports. 11 (1), 1977 (2021).</li><li>Vauti, F., Stegemann, L. A., Vogele, V., Koster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-age+whole+mount+in+situ+hybridization+to+reveal+larval+and+juvenile+expression+patterns+in+zebrafish.">All-age whole mount in situ hybridization to reveal larval and juvenile expression patterns in zebrafish.</a> PloS One. 15 (8), 0237167 (2020).</li><li>Parichy, D. M., Turner, J. M., Parker, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+for+puma+in+development+of+postembryonic+neural+crest-derived+cell+lineages+in+zebrafish.">Essential role for puma in development of postembryonic neural crest-derived cell lineages in zebrafish.</a> Developmental Biology. 256 (2), 221-241 (2003).</li><li>Yang, H., Wanner, I. B., Roper, S. D., Chaudhari, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+method+for+in+situ+hybridization+with+signal+amplification+that+allows+the+detection+of+rare+mRNAs.">An optimized method for in situ hybridization with signal amplification that allows the detection of rare mRNAs.</a> The Journal of Histochemistry &amp; Cytochemistry. 47 (4), 431-445 (1999).</li><li>McCampbell, K. K., Springer, K. N., 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=Analysis+of+nephron+composition+and+function+in+the+adult+zebrafish+kidney.">Analysis of nephron composition and function in the adult zebrafish kidney.</a> Journal of Visualized Experiments: JoVE. (90), e51644 (2014).</li></ol>

这里描述 的原位 杂交方法旨在研究中胚层发育。然而，它可以应用于研究蜕变过程中其他组织和器官的发育，例如肠道，神经系统，鳞片和色素沉着14。可以为转基因系中的内源性基因或荧光标记物生成探针。
幼虫保持完整至关重要，以便观察其原生环境中的器官和组织。为了保持组织的完整性，重要的是要尽量减少蛋白酶K治疗的时间。重要的是要...

斑马鱼胚胎是研究组织发育的重要模型，因为它的尺寸小，透明度，可用的工具，并且在不进食的情况下存活长达五天1，2。它极大地促进了对肾脏发育的理解以及斑马鱼和哺乳动物之间的保护3，4，5。肾脏在维持液体稳态、过滤血液和排泄废物方面起着至关重要的作用6。肾单位是肾脏的功能单位，包括连接到长小管的血液过滤器。在斑马鱼中，两个肾脏结构在其一生中形成。前发肾（临时胚胎肾）在早期发育期间首先形成。它由两个沿前后轴运行的肾单位组成，并在受精后约2天（dpf）变得功能性。前静脉的效用在于其简单性，只有两个肾单位，它们大多是线性的，易于观察（尽管近端卷曲的小管开始以三 dpf的速度盘绕）3。这不仅促进了从中间中胚层对其早期发展的研究，还促进了分割模式和小管修复的研究7，8。
斑马鱼的用处在五分后变得有限，此时蛋黄减少，幼虫依靠水生系统中的觅食9。在大约2周龄时，幼虫蜕变为幼体，形成新组织，旧组织丢失和/或重组9。这也是中胚层（永久成人肾脏）形成10，11，12的时候。第一个成年肾单位在第六个体参体附近形成，并与前发性肾病的远端早期小管融合。额外的肾单位最初向后添加到该位置，但稍后也向前部添加。第一波中的主肾单位融合到前发管的小管中，并共享一个共同的腔来沉积它们的废物。次级肾单位在下一波中形成并融合到初级肾单位。这种重复过程会产生一个分支的中胚层，有点类似于哺乳动物的肾脏。据推测，前驱肾小管最终会失去其小管特性，并成为两个主要的收集管，所有肾单位都排出废物13。
在第一个成体肾单位形成之前，单个祖细胞开始出现在〜4毫米（总长度）幼虫（〜10 dpf）中。这些细胞在 Tg（lhx1a-EGFP） 转基因系中标记，粘附在前发细胞上，似乎迁移到未来的肾病发生部位。单个细胞合并成簇，分化成新的肾单位12。目前尚不清楚这些细胞位于何处，也不清楚它们在介子发生发生之前表达的基因。
了解中胚层发育可以以前肾无法实现的方式深入了解哺乳动物肾脏。这些包括从单个祖细胞形成肾源聚集体，新肾单位与现有肾单位的功能整合以及分支形态发生。然而，研究胚胎后发育存在局限性。幼虫由于其体型较大且具有色素沉着而不太透明。个体动物之间的发育时间表不同步，并且高度依赖于环境因素，例如进食和拥挤9，14。虽然存在敲低试剂，但与胚胎相比，它们在幼虫中的效果较差15。在该协议中，描述了一种 原位 杂交方法，以确定斑马鱼中胚层发育过程中的基因表达。优化了几个步骤，以提高中胚层的可视化以及探针和抗体的穿透和洗涤。 Tg（lhx1a-EGFP） 转基因系与EGFP探针一起使用，以标记单个祖细胞，肾源聚集体和新生肾单位的远端小管。这种方法将提供对中胚层发育的更深入理解和对再生疗法的见解。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-fluorescein antibody</td>
			<td>Roche/Sigma-Aldrich</td>
			<td>11426338910</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleaching solution</td>
			<td></td>
			<td></td>
			<td>0.8% KOH, 0.9% H2O2 in PBST</td>
		</tr>
		<tr>
			<td>Blocking reagent</td>
			<td>Roche/Sigma-Aldrich</td>
			<td>11096176001</td>
			<td>Use for blocking solution, prepare according to manufacture&#39;s instruction</td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>Fisher Scientific</td>
			<td>&#160;22-363-549</td>
			<td>100 &#956;m</td>
		</tr>
		<tr>
			<td>E3 medium</td>
			<td></td>
			<td></td>
			<td>5 mM NaCl, 0.33 mM CaCl2, 0.33 mM MgSO4, 0.17 mM KCl, 0.0001% methylene blue</td>
		</tr>
		<tr>
			<td>Eyelash manipulator</td>
			<td>Fisher Scientific</td>
			<td>NC1083208</td>
			<td>Use to manipulate larvae</td>
		</tr>
		<tr>
			<td>Fixing solution</td>
			<td></td>
			<td></td>
			<td>4% paraformaldehyde, 1% DMSO in PBS; heat at 65&#176;C while shaking until the powder dissolves, then add DMSO after it cools down</td>
		</tr>
		<tr>
			<td>Fluorescein probe synthesis</td>
			<td>Roche/Sigma-Aldrich</td>
			<td>11685619910</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vial</td>
			<td>Fisher Scientific</td>
			<td>03-338B</td>
			<td></td>
		</tr>
		<tr>
			<td>Hatchfry encapsulation</td>
			<td>Argent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hyb- solution</td>
			<td></td>
			<td></td>
			<td>50% formamide, 5X SSC, 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>Hyb+ solution</td>
			<td></td>
			<td></td>
			<td>HYB-, 5 mg/mL torula RNA, 50 ug/mL heparin</td>
		</tr>
		<tr>
			<td>MAB (10X)</td>
			<td></td>
			<td></td>
			<td>1 M maleic acid, 1.5 M NaCl, pH 7.5</td>
		</tr>
		<tr>
			<td>MABT</td>
			<td></td>
			<td></td>
			<td>1X MAB, 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>Maleic acid</td>
			<td>Sigma-Aldrich</td>
			<td>M0375</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X)</td>
			<td></td>
			<td></td>
			<td>8% NaCl, 0.2% KCl, 1.44% Na2HPO4, 0.24% KH2PO4</td>
		</tr>
		<tr>
			<td>PBST</td>
			<td></td>
			<td></td>
			<td>1X PBS, 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>PBST2</td>
			<td></td>
			<td></td>
			<td>1X PBS, 0.2% Tween-20</td>
		</tr>
		<tr>
			<td>Powder food</td>
			<td></td>
			<td></td>
			<td>Mix 2 g of each of spirulina and hatchfry encapsulon in 50 mL of fish system water and shake well</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-Aldrich</td>
			<td>P5568</td>
			<td>Use to permeabilize larvae</td>
		</tr>
		<tr>
			<td>Proteinase K solution</td>
			<td></td>
			<td></td>
			<td>20&#160; &#956;g/mL, 1% DMSO final concentration in PBST</td>
		</tr>
		<tr>
			<td>Spirulina microfine powder</td>
			<td>Argent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SSC (20X)</td>
			<td></td>
			<td></td>
			<td>3 M NaCl, 0.3 M sodium acetate anhydrous, pH 7, autoclave</td>
		</tr>
		<tr>
			<td>SSCT (0.2X)</td>
			<td></td>
			<td></td>
			<td>Dilute from 20X SSC, 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>SSCT (2X)</td>
			<td></td>
			<td></td>
			<td>Dilute from 20X SSC, 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>Staining buffer</td>
			<td></td>
			<td></td>
			<td>100 mM Tris pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween-20</td>
		</tr>
		<tr>
			<td>Staining solution</td>
			<td></td>
			<td></td>
			<td>200 &#956;g/mL iodonitrotetrazolium chloride, 200 &#956;g/mL 5-Bromo-4-chloro-3-indolyl phosphate disodium salt, in staining buffer</td>
		</tr>
		<tr>
			<td>Stopping solution</td>
			<td></td>
			<td></td>
			<td>1 mM EDTA, pH 5.5, in PBST</td>
		</tr>
		<tr>
			<td>Torula (yeast) RNA</td>
			<td>Sigma-Aldrich</td>
			<td>R6625</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma Aldrich</td>
			<td>E10521</td>
			<td>2%, pH 7</td>
		</tr>
		<tr>
			<td>Wash buffer</td>
			<td></td>
			<td></td>
			<td>50% formamide, 2X SSC, 0.1% Tween-20</td>
		</tr>
	</tbody>
</table>

in situ hybridization in zebrafish larvae and juveniles during mesonephros development

斑马鱼在其一生中形成两个肾脏结构。前发肾（胚胎肾）在胚胎发育过程中形成，并在受精后2天开始发挥作用。前发肾上腺仅由两个肾单位组成，在幼虫存活期间充当唯一的肾脏，直到由于体重增加而需要更多的肾功能。为了应对这种更高的需求，中胚层（成人肾脏）在蜕变过程中开始形成。新的初级肾单位融合到前静脉并形成连接的管腔。然后，次级肾单位融合到初级肾单位（依此类推）以在中胚层中形成分支网络。绝大多数研究都集中在前驱虫上，因为使用胚胎很容易。因此，有必要开发技术来研究较老和较大的幼虫和幼鱼，以更好地了解中胚层的发育。在这里，用于基因表达分析 的原位 杂交方案针对探针穿透，探针和抗体的洗涤以及色素的漂白进行了优化，以更好地可视化中胚层。 Tg（lhx1a-EGFP） 转基因系用于标记新生肾单位的祖细胞和远端肾小管。该协议填补了中胚层研究的空白。它是了解新肾组织如何形成并与现有肾单位整合并提供再生疗法见解的关键模型。

使用Tg（lhx1a-EGFP）转基因系，证明该原位杂交方案在中胚层发育过程中有效标记肾脏祖细胞和各种肾单位结构。正如预期的那样，中枢神经系统也被标记在这个转基因系中（未显示）。初始的中胚层肾单位在大约5.2毫米处形成，背侧到前几发（图2A），并且该肾单位的远端肾小管由EGFP10，12标记。祖细胞簇存在于这个?...

Watch this Scientific Journal Video about In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development at JoVE.com

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

斑马鱼是了解肾脏发育的重要模型。在这里， 对原位 杂交方案进行了优化，以检测斑马鱼幼虫和幼鱼在中胚层发育过程中的基因表达。

原位 斑马鱼幼体和幼鱼在雌雄同体发育过程中的杂交

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at ...

该协议具有重要意义，因为它允许在变质过程中测定老年斑马鱼幼体和幼鱼的基因表达。这种技术的优点是已经针对探针穿透和肾脏可视化优化了几个步骤。首先，在最后一顿饭后的傍晚，通过在交配池中添加一条雄性和一条雌性鱼来设置成年斑马鱼进行交配。第二天，将胚胎收集在含有E3培养基的培养皿中。施肥后五天，将400微米的筛网放入2.8升的水箱中，并用2厘米的系统水填充。然后从一个培养皿中加入幼虫。要固定幼虫，请在所需的时间点取出一罐幼虫，然后使用网和转移移液器，其尖端被切断，将幼虫转移到培养皿中。接下来，加入两毫升2%三卡因以固定幼虫。固定后，用20毫升固定溶液代替三卡因。30分钟后，将幼虫转移到含有25毫升新鲜固定溶液的50毫升管中。然后，确保盖子是紧的，慢慢地在四摄氏度下摇晃管子两天。在第三天，用20毫升PBST替换固定溶液，然后将幼虫转移到培养皿中。要测量幼虫，将培养皿放在解剖显微镜下的平面尺子的顶部，然后使用睫毛操纵器将每个幼虫移动到尺子上以测量其总长度。测量所有幼虫后，将几个长度相似的幼虫与PBST混合到一个5.5毫升的玻璃小瓶上。对于脱水，用四毫升100%甲醇代替玻璃瓶中的PBST，然后将小瓶在零下20摄氏度下储存两天。对于补液，用四毫升75%甲醇和25%PBST溶液代替100%甲醇，并摇动小瓶五分钟。然后，用四毫升新鲜PBST替换甲醇和PBST溶液，并再次摇动小瓶10分钟。对于蛋白酶K消化，用两毫升蛋白酶K溶液代替PBST，并在室温下摇动小瓶30分钟。接下来，为了漂白，将幼虫转移到六孔板中，并用三毫升新鲜漂白溶液代替PBST。在沿中胚层色素沉着完全消失后，将幼虫移回玻璃小瓶中，用四毫升PBST代替漂白溶液，然后摇动小瓶10分钟。对于预杂交，用4毫升PBST替换固定溶液，并将小瓶摇动10分钟，然后，用4毫升Hyb溶液代替PBST并岩石10分钟。在Hyb溶液中另外孵育两次后，用四毫升Hyb +溶液代替溶液。同时将EGFP荧光素探针稀释一至100在500微升杂交加溶液中，并将小瓶和探针在70摄氏度下孵育过夜。第二天，为了杂交探针，用预热的探针替换小瓶中的Hyb +溶液，并在70摄氏度下孵育过夜。第二天，将50毫升预热的0.2倍SSCT加入50毫升管中。然后，将100微米的细胞过滤器插入管的顶部，将幼虫从玻璃小瓶转移到细胞过滤器中，并确保幼虫浸没在缓冲液中，将小瓶在70摄氏度下孵育两个小时。孵育后，将细胞过滤器转移到含有预热0.2倍SSCT的新管中，并在70摄氏度下再次孵育两小时。接下来，为了阻断，将幼虫转移到新的玻璃瓶中，并使其冷却到室温。然后，用4毫升67%0.2倍SSCT和33%MABT溶液代替0.2倍SSCT溶液，并在室温下摇动小瓶10分钟。接下来，用四毫升新鲜MABT替换SSCTT MABT溶液，并将小瓶在摇臂上孵育10分钟。然后，用四毫升封闭溶液代替MABT，并在四摄氏度下孵育过夜。第二天，用抗体溶液代替封闭溶液，并在四摄氏度下孵育两天。对于抗体清洗，将幼虫转移到50毫升的管中，然后加入40毫升PBST2，并将管放在其侧面，以便在4摄氏度下孵育过夜。在第12天，将熔岩转移到六孔板后，用三毫升染色缓冲液代替PBST2。在摇臂上孵育五分钟后，用三毫升染色溶液代替染色缓冲液。当达到所需的染色强度时，用三毫升停止溶液和岩石代替染色溶液30分钟。然后，将幼虫转移到新的玻璃瓶中，用四毫升新鲜固定溶液代替停止溶液，并在室温下孵育一小时。对于成像，用四毫升新鲜PBST代替固定溶液。在摇杆上孵育10分钟后，将幼虫转移到六孔板中，然后用四毫升新鲜PBST替换PBST，并再次摇动板10分钟。接下来，在PBST中加入三毫升50%甘油并岩石10分钟，然后使用解剖显微镜直接在六孔板中对幼虫进行成像。这种原位杂交方案使用中胚层发育有效地标记肾脏祖细胞和各种肾单位结构。初始的中胚层肾单位在前静脉背侧约 5.2 毫米处形成。除了单个祖细胞外，在中胚层发育过程中还存在祖细胞簇。在较大的幼体中，背景染色可能发生在体细胞中。总体而言，除了解剖的成人器官外，这种方法还可用于研究在过程中形成的其他组织。

Research

JoVE Journal

Developmental Biology

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

了解早期器官使用简体<em&gt;原位</em&gt;杂交协议<em&gt;爪</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

科研

发育生物学

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

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 ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

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 ...

Wholemount In Situ Hybridization for Astyanax Embryos

Wholemount In Situ Hybridization for Astyanax Embryos

阿斯蒂亚纳克斯胚胎的全原位杂交

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

斑马鱼原位杂交染色的基因分型与定量

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

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 ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

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. ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

荧光钙成像及其后续原位杂交，用于异种性前体特征

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

原位 斑马鱼幼体和幼鱼在雌雄同体发育过程中的杂交

原位斑马鱼幼体和幼鱼在雌雄同体发育过程中的杂交