Name: visualizing multiciliated cells in the zebrafish through a combined protocol of whole mount fluorescent in situ hybridization and immunofluorescence
Uploaded: 2017-11-18T13:00:00.000+00:00
Duration: 9 min 33 s
Description: Watch this Scientific Journal Video about Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence at JoVE.c

这项工作得到部分支持的赠款 R01DK100237 R.A.W. 和国家科学基金会研究生研究金 No。DGE-1313583 至 A.N.M。我们还感谢科学学院暑期本科生研究奖学金计划, 支持 M.U。我们要感谢在圣母大学的斑马鱼研究中心对我们的斑马鱼的专门护理。我们还要感谢生物科学系以及我们实验室的成员们所提供的所有支持和宝贵的见解。

下面的协议使用斑马鱼在圣母大学的研究中心的维护和照顾。所有与斑马鱼成人和胚胎工作的方法都得到了机构动物保育和使用委员会的批准。
1. 胚胎固定
<ol><li>使用先前描述的方法收集斑马鱼胚胎39,40。在 24 hpf, 添加500µL 的非特异性蛋白酶混合物溶液 (50 毫克/毫升) 到 E3 在一个胚胎盘孵化室温 (RT)。</li>
	<li>一旦胚胎开始脱离绒毛膜, 从胚胎盘中取出所有液体。</li>
	<li>用新鲜的 E3 2-3 倍的 E3, 在盘子里轻轻地旋转 E3, 然后将液体迁成液体废物容器。</li>
	<li>在修复前安乐胚胎, 在 E3 中加入2毫升的 0.2% tricaine。</li>
	<li>将胚胎移植到一个5毫升的玻璃瓶中, 使用塑料的巴斯德吸管。在胚胎已经沉淀到瓶子底部后, 用吸管去除大部分的 tricaine/E3 溶液。</li>
	<li>修复 24 hpf 胚胎与5毫升4% 甲醛 (粉煤灰) 的 2-4 h 在 RT, 没有躁动。 注意: 粉煤灰是有毒的, 粉煤灰解决方案应该在化学罩下处理, 而研究员戴着眼睛保护, 手套, 和实验室大衣。颗粒粉煤灰中的粉煤灰固定剂的制备应格外小心, 因为颗粒状的粉煤灰往往通过静态电荷来分散。 注: 胚胎可以在4° c 的晚上在4% 的粉煤灰中修复。将1x 磷酸盐缓冲盐水 (PBS) 加热至沸腾, 并不断搅拌溶液以完全溶解颗粒状粉煤灰, 制备4% 的粉煤灰。一旦溶液冷却回 RT, 4% 的粉煤灰是过滤消毒通过它通过一个0.45 µm 一次性系统的真空过滤, 然后和 aliquoted 到15毫升或50毫升部分存储在-20 ° c。</li>
	<li>在 rt 中用1X 磷酸缓冲盐和 0.1% Tween-20 (PBST) 去除4% 的粉煤灰和洗涤胚胎两次. 用5毫升的100% 甲醇 (甲醇) 在 rt 冲洗胚胎一次. 在胚胎中加入5毫升新鲜100% 甲醇, 在-20 ° c 下孵育至少20分钟。 注: 胚胎可以储存在100% 甲醇在-20 ° c, 直到准备好胚胎准备。</li>
</ol>2. 胚制备与杂交
<ol><li>水化5毫升的50% 甲醇, 在 1x PBST, 在 rt 的5分钟内洗净胚胎. 继续补液, 洗净的胚胎在5毫升30% 甲醇在 1x PBST 5 分钟, 在 rt. 在5毫升的 1x PBST 洗涤的胚胎每两次5分钟rt.</li>
	<li>Permeabilize 在5毫升的 1:1, 000 蛋白酶 K (pK) (10 毫克/毫升) 的溶液中孵化他们的胚胎在 1x PBST 为2分钟 RT。 注: 在上述 pK 浓度中, 较早的胚胎可以孵育超过2分钟, 但5毫克/毫升的浓度和潜伏期少于2分钟的胚胎建议为小于 24 hpf 的胚。</li>
	<li>删除 pK 解决方案, 并立即在5毫升的 1x PBST 冲洗两次在 rt. 在 rt 中修复5毫升4% 粉煤灰中的胚胎至少20分钟。 注: 胚胎可以在4° c 的晚上在4% 的粉煤灰中修复。</li>
	<li>在 RT 的5毫升的 1x PBST 中去除煤灰和洗涤两次。</li>
	<li>将5毫升 1x PBST 的胚胎移植到平底离心管中, 放置在 RT 离心机架上, 以促进每个胚胎与随后的杂交溶液之间的相互作用。</li>
	<li>在 RT 中, 在1.5 毫升杂交溶液 (Hyb +) 中两次洗净胚胎. 加入1.5 毫升的新鲜 Hyb + 和孵化胚胎在70° c 的杂交烤箱4-6 小时。</li>
	<li>用一卷探针溶液 (10 µL RNA probe/500 µL Hyb +) 取代1.5 毫升的 Hyb + 充分覆盖胚胎。 注: 使用先前描述的方法合成反义 RNA 探针39。</li>
	<li>杂交在杂交烤箱过夜, 在70° c 与单独的管定位直立在离心架, 没有晃动。</li>
</ol>3. 热洗和堵塞
注: 通过将适当的溶液放在胚胎上, 然后在70° c 的杂交烤箱中孵化, 进行热洗。为了保持70° c 的洗涤解决方案, 当探针/Hyb + 混合物加入时, 将每个溶液的50毫升管放在杂交烤箱中。
<ol><li>卸下探头。在70° c 的1.5 毫升的 50% formamide/2x 生理盐水 (SSC) 的缓冲液中清洗两次胚胎, 每次20-30 分钟。 注: 探头可储存在 Hyb +-20 ° c, 并在以后再用。</li>
	<li>在70° c 的1.5 毫升的 2x ssc 中清洗一次胚胎15分钟, 在70° c 的1.5 毫升 0.2X ssc 中洗涤胚胎两次, 每20-30 分钟。用1.5 毫升的阻断试剂取代 0.2x SSC, 在4° c 夜间孵育。 注: 在 RT 中孵育阻塞试剂的可能性为4小时, 而不是隔夜。</li>
</ol>4. 抗体孵化和马来酸缓冲洗涤
注意: 保持胚胎免受环境光线的保护。
<ol><li>用0.2 毫升的防挖荚剂, 以1:1、000的比例, 用铝箔或另一遮光盖在 RT 下孵育 3 h, 以取代堵塞试剂。 注: 或者, 抗体可以在4° c 的夜间孵育。</li>
	<li>3小时后, 在1.5 毫升的顺丁烯二酸缓冲液中清洗2-4 次, 每次在 RT 时为10-15 分钟. 在4° c 的新鲜马来酸缓冲液中, 将胚胎一夜孵化。 注: 不需要在一夜之间在马来酸缓冲液中孵育, 但这样做会降低探头的背景。</li>
</ol>5. 过氧化物酶的探针检测与去除
注意: 保持胚胎免受环境光线的保护。
<ol><li>去除马来酸缓冲液, 更换1.5 毫升 1x PBS。在1.5 毫升的 1x PBS 中冲洗两次胚胎, 每次5分钟在 rt. 0.2 毫升的 Cy3 荧光染色溶液中孵育胚胎60分钟。 注意: 不同探头的孵化时间可能有所不同。</li>
	<li>用1.5 毫升的甲醇在 1X PBS 10 分钟的 RT: 30% 甲醇, 50% 甲醇, 75% 甲醇和100% 甲醇。</li>
	<li>在甲醇中孵育1.5 毫升的 1% H2O2的胚胎, 在 rt 中为 30 min. 在 1x PBS 中, 用1.5 毫升的下列甲醇溶液冲洗胚胎一次:10 分钟甲醇、75% 甲醇和50% 甲醇。在1.5 毫升的 1x PBS 中, 在 RT 中每两次清洗一次胚胎5分钟。 注: 胚胎可储存在 PBS 夜间在4° c。</li>
</ol>6. 免疫荧光
注意: 保持胚胎免受环境光线的保护。
<ol><li>在 RT 中, 用1.5 毫升的 ddH2O 清洗胚胎5分钟. 将1.5 毫升的 pre-chilled 丙酮 (保持在-20 ° c) 中的胚胎洗净, 7 分钟. 在 rt 中用1.5 毫升的 ddH2O 清洗胚胎一次, 5 分钟. 在1.5 中清洗胚胎一次毫升的 1x PBST 与1% 的亚砜 (PBDT) 为5分钟的 RT。</li>
	<li>孵育1.5 毫升的 PBDT + 10% 胎牛血清 (FBS) 在 RT 的一个摇杆2小时的胚胎。</li>
	<li>去除 PBDT + FBS 和替换与1.5 毫升的主要抗体, anti-acetylated 蛋白 (生产的老鼠) 和反γ蛋白 (生产的兔), 稀释一起在1:400 在 PBDT + 1% FBS。在4° c 的夜间孵育胚胎。 注意: 潜伏期可能会因主抗体而异。测试不同的时间间隔可能是必要的优化标签。</li>
</ol>7. 二级抗体
注意: 保持胚胎免受环境光线的保护。
<ol><li>为了去除胚胎中多余的未绑定抗体, 在1.5 毫升的 PBDT + 1% FBS + 0.1 米的氯化钠中, 在摇杆上进行1分钟的冲洗。</li>
	<li>在1.5 毫升的 PBDT + 1% FBS + 0.1 M 氯化钠清洗胚胎5次, 每在一个摇杆上的30分钟. 用1.5 毫升的 PBDT + 1% FBS 在摇杆上的30分钟清洗胚胎。</li>
	<li>在4° c 夜间孵育200µL 的二次抗体, alexa 488 山羊 anti-mouse igg 和 alexa 647 山羊兔 igg, 稀释在 PBDT 在1:500。</li>
</ol>8. DAPI 染色
注意: 保持胚胎免受环境光线的保护。
<ol><li>在1.5 毫升的 PBDT 中快速冲洗胚胎两次。用1.5 毫升的 DAPI 取代 PBDT, 稀释1:15000 在 1X PBST, 在 RT 上孵育15分钟的摇杆。</li>
	<li>在 RT 中洗涤1.5 毫升的 PDBT + 1% FBS + 0.1 米氯化钠三次, 每次15-20 分钟。将胚胎储存在1.5 毫升的 PBDT 在4° c, 直到准备好安装和图像。</li>
</ol>9. 斑马鱼 Pronephros 的安装和成像
<ol><li>将胚侧放在玻璃上, 小体积, 大约10µL, PBDT。</li>
	<li>用一副精致的镊子把眼睛后面的胚胎挤压, 去掉头部和一些蛋黄球。</li>
	<li>用细钳将剩余的蛋黄球从胚体中轻轻刮去。用薄的纸巾从滑梯上取下游离的蛋黄和多余的液体。</li>
	<li>在胚胎和位置的剩余尾部添加一滴安装介质, 这样尾巴就会横向放置。将片放在尾部的顶部, 以使样品扁平化, 然后放置在覆盖的滑动框中。</li>
	<li>图像肾脏纤毛在60X 放大的共聚焦显微镜下使用以下渠道: DAPI 在蓝色 (laser 设置在408.0 毫微米), FITC 在绿色 (laser 设置在488.0 毫微米), Cy3 染料标记在红色 (laser 设置在561.0 毫微米) 和 Alexa 680 在白色 (laser 设置在637.0 毫微米)。</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tbx2a/b+transcription+factors+direct+pronephros+segmentation+and+corpuscle+of+Stannius+formation+in+zebrafish.">The tbx2a/b transcription factors direct pronephros segmentation and corpuscle of Stannius formation in zebrafish.</a> Dev Biol. 421 (1), 52-66 (2017).</li><li>Jaffe, K. M., Thiberge, S. Y., Bisher, M. E., Burdine, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cilia+in+zebrafish.">Imaging cilia in zebrafish.</a> Methods Cell Biol. 97, 415-435 (2010).</li><li>Lauter, G., Soll, I., Hauptmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+fluorescent+in+situ+hybridization+to+define+abutting+and+overlapping+gene+expression+in+the+embryonic+zebrafish+brain.">Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain.</a> Neural Dev. 6, (2011).</li><li>Sorrells, S., Toruno, C., Stewart, R. 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作者没有什么可透露的。

上面详细的协议是优化的标签在 pronephros 与odf3b转录和α-蛋白在 24 hpf 斑马鱼胚胎。为了取得最佳效果, 建议使用新鲜固定和准备好的胚胎。胚胎被固定和储存在甲醇或 Hyb + 在-20 ° c 超过一周可以使用, 但不必要的背景染色的概率增加的时间, 胚胎保存在储存。
这项技术的修改和故障排除是必要的, 以便为其他特定标记 (例如其他反义 riboprobes 和其他抗体) 量身定做这种方法。在整个装入原位杂交过程中, 许多调整步骤参数的方法已经被我们的组和其他人记录下来了.31,3436,38, 39。同样, 每种蛋白质抗体都需要在染色时间方面进行一些故障诊断, 而每个主要抗体的稀释和潜伏期的近似范围最好通过对记录结果的评估28,35. 对于小于 24 hpf 的胚胎, pk 治疗应短于2分钟, 其中超过 24 hpf 的胚胎应以 pk 治疗超过2分钟。此外, 早于 24 hpf 的胚胎应在 pK 治疗前漂白色素。
染色后的荧光染色溶液, 这是至关重要的, 以消除任何残留的污渍, 抗体和过氧化物, 执行一系列的甲醇和过氧化氢洗涤。紧接着的 PBS 冲洗是从胚胎中去除过量甲醇的关键。重要的是, 在进行 IF 抗体, 我们发现, 丙酮和去离子水冲洗使胚胎不太可能坚持彼此和/或离心管。在我们的经验中, 特定转录因子和其他基因的抗体, 虽然它们可能有效地在西方的印迹, 如果在斑马鱼的效果不好。然而, 高度保守和丰富的蛋白质, 如α-蛋白和β-蛋白, 在斑马鱼的工作很好, 如果16,37。
有了鱼, 就有可能想象出在斑马鱼身上还没有特定抗体的 RNA 转录基因。通过结合鱼类与 IF, 如本议定书所示, 共存的 RNA 转录和蛋白质可以看到在体内(图 2)。该协议灵活的性质, 使快速故障排除的各种 RNA 转录和蛋白质在许多组织和时间点的可视化。当与已有的斑马鱼胚胎的特征相结合时, 本议定书的鱼类 + 如果提供了另一种工具来探索基因和蛋白质的表达对发育途径的重要调节。

在过去的几十年中, 斑马鱼 (斑马鱼) 已经成为研究发育生物学的主要模型生物体。胚胎在母亲的外面开发并且是光学上透明的。此外, 眼部、肾脏和前脑等重要器官的形成也迅速发生, 其结构由仅 24 h 后受精 (hpf) 组成。重要的是, 斑马鱼基因组是高度保守的哺乳动物1,2,3。此外, 斑马鱼和哺乳动物的器官也有相似的解剖和生理学。斑马鱼的胚胎肾脏, 或 pronephros, 证明了模型系统的价值检测基因功能在早期 nephrogenesis 和命运确定的保存上皮细胞种群的脊椎动物肾单位4,5,6,7,8,9,10. 同样, 斑马鱼胚胎在检查监控化学品的个体发育中变得越来越重要11、12、13、14、15、16,17。
正如他们的名字所表明的, 监控化学品是上皮细胞的特点是一束运动纤毛位于顶端表面17。在斑马鱼中, 监控化学品在流体流动中的作用, 并在 pronephros 的每一个肾单位的中间分布着一个 "salt-and 胡椒", 24 hpf11,12,13,14,15,16,17. 虽然它们只在少数人类肾脏疾病病例中被注意18、19、20、21, 但在其他哺乳动物组织 (如大脑和气管22,23,24, 这给实验设计带来了许多挑战。优雅的研究, 包括斑马鱼在内的各种脊椎动物模型显示了 mcc 命运的保守通路, 以缺口信号通路作为 mcc 的抑制剂开发12,17,25 ,26,27。因此, 斑马鱼 pronephros 提供了一个易于访问的模型来研究 MCC 发展的遗传机制在体内11,12,13,14,15,16,17。
在研究控制细胞命运、组织生长和早期胚胎发育的基因和分子通路时, 斑马鱼胚胎的透明和容易的基因操作已经证明是非常宝贵的特性1,2 ,3。因此, 传统的技术来可视化蛋白质和基因转录, 如原位杂交和整个装载如果, 已被应用到和优化斑马鱼16,28,29, 30,31,32,3334353637通过结合流行的鱼的协议和如果, 它是可能的标签和分析监控化学品在体内16,28,37,38。

<table><tbody><tr><td>non-specific protease mixture solution</td><td>Roche</td><td>11459643001, pronase from Streptomyces griseus</td><td>Dilute in E3 &lt;strong&gt;without&lt;/strong&gt; methylene blue to make 50mg/mL stock solution; store at -20&amp;deg;C</td></tr><tr><td>E3 solution</td><td></td><td></td><td>Dilute 50X E3 (250 mM NaCl, 8.5 mM KCl, 16.5 mM CaCl2, 16.5 mM MgSO4 in distilled water) in distilled water; add 1 x 10-5 M methylene blue (sigma M9140) to inhibit contamination</td></tr><tr><td>tricaine (ethyl 3-aminobenzoate methanesulfonate)</td><td>Fluka</td><td>A5040-250G</td><td>To make a 0.2% stock solution, dissolve 1 g of tricaine powder in 10 mL Tris, pH 9.5 and distilled water up to 500 mL</td></tr><tr><td>embryo dishes</td><td>VWR</td><td>351029</td><td></td></tr><tr><td>5 mL glass vials</td><td>Wheaton</td><td>225012</td><td></td></tr><tr><td>disposable plastic Pasteur pippettes</td><td>VWR</td><td>414004-004</td><td></td></tr><tr><td>4% paraformaldehyde (PFA) solution</td><td>Electron Microscopy Services</td><td>19210</td><td>Dissolve 4% PFA in 1X PBS and bring to a boil under a fume hood. Cool and aliquot, then store at -20&amp;deg;C. Do not refreeze once thawed for use.</td></tr><tr><td>10X PBS</td><td>American Bioanalytical</td><td>AB11072</td><td>Dilute in distilled water to make a 1X stock</td></tr><tr><td>Tween-20 stock</td><td>American Bioanalytical</td><td>AB02038</td><td>Make a 0.1% Tween-20 stock by diluting in distilled water.</td></tr><tr><td>1X phosphate buffered saline with 0.1% Tween 20 (PBST)</td><td>Sigma</td><td>P9416</td><td>0.1% Tween-20 in 1X PBS</td></tr><tr><td>methanol (MeOH)</td><td>Sigma</td><td>34860-4L</td><td></td></tr><tr><td>proteinase K</td><td>Roche</td><td>3115879001</td><td>Dissolve in distilled water to make a 10 mg/mL stock; aliquot and store at -20&amp;deg;C</td></tr><tr><td>flat bottom microcentrifuge tubes</td><td>VWR</td><td>87003-300; 87003-298</td><td></td></tr><tr><td>formamide</td><td>American Bioanalytical</td><td>AB00600</td><td>store at -20&amp;deg;C</td></tr><tr><td>hybridization solution (HYB+)</td><td></td><td></td><td>50% formamide, 5X SSC, 0.1% Tween-20, 5 mg/mL yeast torula RNA, 50 &amp;mu;g/&amp;mu;L heparin; store at -20&amp;deg;C</td></tr><tr><td>hybridization oven</td><td>Fisher Scientific</td><td>13-247-10Q</td><td></td></tr><tr><td>20X saline-sodium citrate (SSC) buffer solution</td><td>American Bioanalytical</td><td>AB13156</td><td>dilute in distilled water to make 2X and 0.2X stocks</td></tr><tr><td>blocking reagent solution</td><td>Roche</td><td>11096176001</td><td>dilute in maleic acid buffer to make a 10% stock solution; store at 4&amp;deg;C</td></tr><tr><td>maleic acid buffer solution</td><td>Sigma</td><td>M0375 and S7653</td><td>150mM maleic acid, 100mM NaCl (pH 7.5)</td></tr><tr><td>anti-Digoxigenin-POD, Fab fragments</td><td>Roche</td><td>11207739910</td><td>store at 4&amp;deg;C</td></tr><tr><td>Cy3 fluorescent staining solution</td><td>PerkinElmer, Inc.</td><td>NEL744001KT, TSA Plus Cyanin3 system</td><td>store at 4&amp;deg;C; prepare staining solution fresh by making a 1:50 dilution of TSA reagent (dissolved in 60 uL of DMSO) in the kit buffer</td></tr><tr><td>Hydrogen peroxide (H2O2)</td><td>Sigma</td><td>H1009-500mL</td><td>store at 4&amp;deg;C</td></tr><tr><td>molecular grade distilled water (ddH2O)</td><td>Mediatech</td><td>25-055-CM</td><td></td></tr><tr><td>acetone</td><td>American Bioanalytical</td><td>AB00636-01000</td><td>store an aliquot at -20&amp;deg;C</td></tr><tr><td>DMSO</td><td>American Bioanalytical</td><td>AB00435-01000</td><td></td></tr><tr><td>fetal bovine serum (FBS)</td><td>Gibco</td><td>10438-034</td><td>aliquot and store at -20&amp;deg;C</td></tr><tr><td>monoclonal anti-acetylated tubulin clone 6-11B-1</td><td>Sigma-Aldrich</td><td>T6793</td><td>aliquot and store at -20&amp;deg;C</td></tr><tr><td>anti-&amp;gamma;-tubulin anitbody produced in rabbit</td><td>Sigma-Aldrich</td><td>T5192</td><td>aliquot and store at -20&amp;deg;C</td></tr><tr><td>odf3b cDNA clone MGC:63985</td><td>OpenBiosystems</td><td>IMAGE:6792478</td><td>store bacterial glycerol stock at -80&amp;deg;C</td></tr><tr><td>NaCL</td><td>American Bioanalytical</td><td>AB01915-05000</td><td></td></tr><tr><td>Alexa flour 647 goat anti-rabbit IgG</td><td>Life Technologies</td><td>A21245</td><td>store at 4&amp;deg;C; protect from light</td></tr><tr><td>Alexa fluor 488 goat anti-mouse IgG</td><td>Life Technologies</td><td>A11029</td><td>store at 4&amp;deg;C; protect from light</td></tr><tr><td>DAPI</td><td>Life Technologies</td><td>D1306</td><td>aliquot and store at -20&amp;deg;C</td></tr><tr><td>glass slide</td><td>Thermo-Fisher</td><td>4445</td><td></td></tr><tr><td>glass coverslip</td><td>Thermo-Fisher</td><td>12-540A</td><td>18 x 18 mm</td></tr><tr><td>fine forceps</td><td>Roboz</td><td>RS-1050</td><td>Dumont Tweezers Pattern #55</td></tr><tr><td>mounting media</td><td>Polysciences, Inc.</td><td>18606, Aqua-Poly/Mount</td><td>store at 4&amp;deg;C</td></tr><tr><td>confocal microscope and associated software</td><td></td><td></td><td>We use a Nikon C2plus Confocal Microscope with NIS-elements AR software</td></tr><tr><td>rocker</td><td>Bio-Rad</td><td>1660710EDU</td><td></td></tr></tbody></table>

visualizing multiciliated cells in the zebrafish through a combined protocol of whole mount fluorescent in situ hybridization and immunofluorescence

近年来, 斑马鱼胚胎已成为一个流行的模式, 研究发展生物学, 由于特点, 如前子宫胚胎发育和光学透明度。特别是斑马鱼胚胎已成为研究脊椎动物肾脏器官和 multiciliated 细胞 (MCC) 发育的重要机体。为了可视化监控器在胚胎斑马鱼肾脏, 我们已经开发了一个联合协议的全贴装荧光原位杂交 (鱼) 和全贴装免疫荧光 (如果), 使高分辨率成像。这份手稿描述了我们的技术 co-localizing RNA 转录和蛋白质作为一个工具, 以更好地理解对发展计划的调节, 通过表达各种血统因素。

野生型斑马鱼的胚胎是固定在 24 hpf, 并立即准备如上所述。图 1描述了一个实验工作流以及所选的插图阶段。步骤1-8 中概述的工作流程包含了样品的采购、固定和操纵的过程, 固定组织用反义 riboprobes 标记内源性转录, 其次是免疫荧光标记感兴趣的蛋白质。工作流中的步骤9是指为优化斑马鱼胚胎躯干的可视化而专门设计的安装和成像技术。在伴随步骤9的绘图中, 我们演示了将组织放置在玻璃幻灯片上的操作方法。在这一步, 胚胎头和蛋黄球被删除, 留下的尾巴是横向定位之间的玻璃幻灯片和片。蛋黄球和头部的去除建议的最佳成像的居民 MCC 人口在 pronephros, 作为蛋黄球 auto-fluoresces 和阻碍安装过程。
使用共聚焦显微镜获得的代表性图像在图 2中提供, 它在60X 放大倍数和同一放大倍数下显示相同的野生类型胚。顶部的面板提供每个单独通道的图像, 而底部的两个面板是这些成像数据的复合叠加。白色框 (在左列图像中) 概述了数字缩放 (右列图像) 的焦点所在的区域。在数字变焦的最后一个面板中突出显示, 细胞核被标记在 DAPI 中, 并且根据反义 riboprobe 检测了监控化学品, 其目的是识别odf3b, γ-蛋白的抗体表示基底体, α-蛋白指纤毛。在复合叠加的数字变焦中, 黄色虚线圆圈表示 MCC 的周长, 这是由于拥有odf3b抄本、多个基底体和纤毛而被识别的。相邻的单纤毛细胞, 标有一个黄色的圆点, 是基于表型的拥有一个单一的基础体和单一的纤毛。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56261/56261fig1.jpg"> 图 1: 实验流程图示意图这个流程图显示了一个实验工作流程, 伴随着雪花表示冷孵化的关键阶段的插图, 三条曲线代表热量, 而时钟代表长时间的潜伏期。工作流可以至少执行6天 (请参见过程中每个阶段的右上角的日数), 尽管有些步骤可以在较长的时间段内执行, 如协议中所述。对安装过程进行了详细的描述, 展示了最终安装在玻璃滑梯和片之间的胚胎尾部。黑色虚线框概述了在60X 放大倍数下成像的区域。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56261/56261fig1large.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/56261/56261fig2.jpg"> 图 2: 对斑马鱼 pronephros 的可视化监控化学品的代表性结果24 hpf 野生型斑马鱼胚胎在60X 倍放大时的最大图像投影以及在同一胚的60X 放大倍率上的数字变焦。白色方框表示聚焦的区域为变焦。DAPI (细胞核)、 odf3b (监控化学品)、γ-蛋白 (基底体) 和α-蛋白 (纤毛) 的单个污点被标记, 然后在底部两个面板中合并在一起。在数字变焦中, 我们提供了以下单元格位置的近似: 一个 MCC 由虚线黄色圆圈勾勒, 虚线黄色圆圈勾勒出一个单纤毛细胞。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56261/56261fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence at JoVE.c

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

纤毛发育对正常器官的形成至关重要。该协议描述了一种优化的方法来标记和可视化纤毛细胞的斑马鱼。

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 ...

visualizing-multiciliated-cells-zebrafish-through-combined-protocol

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

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

8.1K 次观看.  University of Notre Dame. 纤毛发育对正常器官的形成至关重要。该协议描述了一种优化的方法来标记和可视化纤毛细胞的斑马鱼。

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

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

在斑马鱼后脑细胞标记和成像

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

科研

神经科学

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

发育生物学

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

免疫染色磷酸化表位整装斑马鱼胚胎纤毛器官

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

化学

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-&#946;-Hydroxysteroid dehydrogenase /&#916;5-4 isomerase (3&#946;-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3&#946;-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3&#946;-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.

The interrenal gland in zebrafish is the teleostean counterpart of the mammalian adrenal gland. This protocol introduces how to perform the 3-&#946;-hydroxysteroid dehydrogenase (&#916;5-4 isomerase; 3&#946;-Hsd) enzymatic activity assay, which detects differentiated steroidogenic cells in the developing zebrafish.

可视化Interrenal类固醇合成组织及其血管微环境斑马鱼

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

生物学

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

斑马鱼整装高分辨率双荧光原位杂交

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of 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. ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

平山准备观察斑马鱼胚胎标本染色，并通过分析全山<em&gt;原位</em&gt;杂交

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

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 model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry of zebrafish embryonic sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. It is useful to detect the expression patterns of two genes in zebrafish if there is a paucity of antibodies.

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 ...

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

摘要

探索更多视频

此视频中的章节

在斑马鱼后脑细胞标记和成像

在活斑马鱼胚胎成像的亚细胞结构

免疫染色磷酸化表位整装斑马鱼胚胎纤毛器官

可视化Interrenal类固醇合成组织及其血管微环境斑马鱼

现场共聚焦显微镜对斑马鱼的胚胎脑成像

斑马鱼整装高分辨率双荧光原位杂交

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

斑马鱼胚胎和幼虫的整个山免疫性化学

平山准备观察斑马鱼胚胎标本染色，并通过分析全山

原位冷冻切片斑马鱼胚胎中的杂交结合免疫组织化学

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

摘要

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此视频中的章节

在斑马鱼后脑细胞标记和成像

在活斑马鱼胚胎成像的亚细胞结构

免疫染色磷酸化表位整装斑马鱼胚胎纤毛器官

可视化Interrenal类固醇合成组织及其血管微环境斑马鱼

现场共聚焦显微镜对斑马鱼的胚胎脑成像

斑马鱼整装高分辨率双荧光原位杂交

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

斑马鱼胚胎和幼虫的整个山免疫性化学

平山准备观察斑马鱼胚胎标本染色，并通过分析全山

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