我们要感谢病理学服务在 Investigaciones Cardiovasculares (CNIC) 中心的技术援助。我们还感谢 Motoharu 博士精机为 Mt4-mmpLacZ小鼠和阿罗约博士提供支持我们的项目和她对手稿的批判性阅读。我们要感谢彼得邦尼对这篇文章的校对。这项工作得到马德里大学欧洲大学的支持, 2017UEM01 授予 C.S.C。

所有实验程序都得到了 CNIC 动物实验伦理学委员会 (Investigaciones Cardiovasculares) 和马德里 Comunidad 国立的批准, 以确保最小的动物痛苦。
1. 从怀孕小鼠中采集胚胎 (从 E8.5 到 E12.5)
<ol><li>通过宫颈脱位或一氧化碳2吸入来牺牲怀孕的老鼠。第一个被观察的阴道插头的天被考虑了胚胎天 0.5 (E0.5)。</li>
	<li>将动物放在吸水垫的仰卧位上, 用70% 乙醇清洁老鼠的腹部皮肤。 注意: 以下步骤不要求不育。</li>
	<li>用小鼠牙钳捏起腹部皮肤。</li>
	<li>切口通过皮肤和腹壁, 2 到4厘米垂直横跨中线的腹部使用手术剪刀。</li>
	<li>用手术剪刀将子宫内弯曲的血管切开, 从母亲身上取出子宫角。</li>
	<li>去掉胚胎的字符串, 将其转移到含有10毫升冰冷0.01 米磷酸盐缓冲盐水 pH 值 7.4 (PBS) 的冰上的培养皿中。</li>
	<li>仔细解剖胚胎从子宫下的显微镜在一个培养皿与10毫升新鲜冰冷 PBS。用制表钳抓住肌壁以暴露羊膜囊, 然后用镊子的提示撕去羊膜以除去封闭的胚胎和蛋黄囊。</li>
	<li>从怀孕小鼠早期 (从 E8.5 到 E12.5) 收集 littermate 胚胎 (图 2)。</li>
	<li>用弯曲钳将胚胎移植到10毫升冷 1x PBS 的新鲜菜肴上, 或者, 对于非常小的胚胎 (E8.5-E9.5), 使用塑料转移吸管收集没有胚胎损伤的样品。</li>
	<li>在开始固定之前, 在离心管中取一个胚胎样本进行基因分型, 方法是切割一小部分胚胎, 或将最小胚胎中的羊膜 (从 E8.5 到 E9.5) 与制表钳结合。 注: LacZ 基因分型由聚合酶链反应 (PCR) 引物确定: 向前, 5´-ATCGTGCGGTGGTTGAACTG 3´;反转, 5´-CGAGGCGTTAACCGTCAGCA-3´。</li>
</ol>2. 小鼠胚胎的固定
<ol><li>将每个胚胎移植到一个2毫升的离心管中, 其中包含1毫升 1x PBS 的圆形底座。 注意: 使用滚动振动筛在这些管道中执行所有步骤。</li>
	<li>在室温下将 1x PBS 中的胚胎洗净1分钟, 以消除剩余的血液。使用塑料转移吸管改变管中的液体。</li>
	<li>在冰上固定1毫升0.125% 戊二醛的胚胎。 注: 固定时间取决于胚的大小: E8.5 E9.5 胚20分钟, E10.5 胚30分钟, E12.5 胚1小时。</li>
	<li>在处理 X-加仑染色样品前, 取出固定剂并在 1x PBS 中清洗两次, 室温为10分钟。</li>
</ol>3. 小鼠胚胎全芒乳糖酶组织化学研究
<ol><li>在开始操作之前, 准备好 x-加仑冲洗缓冲液和 x-gal 染色溶液 (见表 1和材料表)。使用野生型 (littermate) 胚胎作为对酶反应的阴性对照。</li>
	<li>在室温下以10分钟的温度 (图 2) 在 X-加仑中孵化胚胎。</li>
	<li>在 X-加仑染色溶液中孵化胚胎, 从2小时到隔夜, 在37摄氏度保护免受光照。通过解剖显微镜定期监测颜色反应, 直到在没有胚胎背景的情况下观察到足够的蓝色染色强度。保持溶液 pH 值介于8和9之间, 以减少整个染色过程中的背景。如果染色溶液开始变亮, 用新鲜的基质溶液取代, 以延长酶的反应。</li>
	<li>当所需信号通过在室温下每两次1毫升 1x PBS 的离心管中的10分钟内洗涤时, 停止反应。</li>
	<li>将染色的胚胎转移到1毫升4% 多聚甲醛 (粉煤灰) 重新修复, 1 小时至一夜4摄氏度。 注: 胚胎可贮存在4% 粉煤灰/PBS 中, 较长时间为4摄氏度, 或可立即加工切片。这一最后的固定步骤是长期保存染色模式的关键。</li>
	<li>在室温下, 将1毫升 1x PBS 中的胚胎清洗两次, 10 分钟。</li>
	<li>备选方案 1: 通过浸泡在一系列解决方案中, 清除胚胎, 增加甘油浓度 (20%、40%、60% 和80% 甘油 1x PBS), 每种溶液室温下为1小时。 注: 在80% 甘油中洗涤胚胎, 时间长, 甚至数天, 直到清除, 然后在80% 甘油4摄氏度储存在这个步骤 (图 2)。建议在甘油或 1x PBS 中添加极少量的叠氮化钠麝香晶体, 以防止霉菌生长。清除后, 全芒胚可用于拍照 (步骤 4)。</li>
	<li>选项 2: 使用切片进行石蜡嵌入和切片的加工胚 (图 2)。在切片前记录整个染色胚的表达模式 (步骤4和 5)。</li>
</ol>4. 全芒胚摄影
<ol><li>要在切片前拍摄全芒染色的胚胎, 将其转移到一个培养皿中, 并在 1x PBS 中固化1-2% 琼脂糖。</li>
	<li>完全覆盖胚胎与10毫升 1x PBS 在琼脂糖涂层的菜肴, 以防止脱水和反射, 而在拍摄的液体。</li>
	<li>用镊子将胚胎浸入 1x PBS 中。对于年长的胚胎 (E10.5-E12.5), 用镊子在琼脂糖中做一个小孔, 以不同的角度放置和放置标本, 避免摄影过程中的自由漂浮。</li>
	<li>将盘子放在显微镜台上, 使用透射 (下) 光。改变 "黑暗场" 和 "光明场" 的调整, 以设定在摄影过程中所需的照明和优化样品的半透明性。 注: 对后期胚胎使用光纤照明, 以避免胚胎表面的反射。当拍摄被清除的胚胎, 遵循相同的程序, 但转移到一个含有100% 甘油的培养皿, 并充分浸泡他们。</li>
</ol>5. X-加仑染色胚的石蜡嵌入和切片
<ol><li>石蜡嵌入<ol>
<li>从4°c (步骤 3.5) 中取出 X-加仑染色的胚胎, 并用1毫升 1x PBS 清洗它们, 以消除多余的固定剂。</li>
		<li>用镊子将每个胚胎移植到组织学盒中。</li>
		<li>将含有胚胎的卡带放入烧杯中, 然后在室温下通过分级乙醇系列将其脱水: 70% 乙醇, 一次为30分钟;96% 乙醇, 每两次30分钟;100% 乙醇, 每两次30分钟。 注: 胚胎可以储存在70% 乙醇的不确定时间在4摄氏度, 直到开始脱水过程。</li>
		<li>在室温下, 在异丙醇上孵化整盒30分钟。</li>
		<li>使用镊子, 将盒式磁带转移到含有预热异丙醇的玻璃染色槽中, 并在烤箱中留出60摄氏度, 30 分钟。</li>
		<li>将卡带放在一个新的玻璃染色槽中, 其中含有50% 异丙醇/50% 熔融石蜡, 并在60摄氏度烤箱中离开4小时。</li>
		<li>用两个熔融石蜡变化的胚胎孵化盒, 每30分钟60摄氏度。</li>
		<li>最后石蜡冲洗结束后, 打开卡带, 将每个胚转移到一个单独的组织嵌入模具中, 以查看显微镜下的样品。</li>
		<li>用60摄氏度的熔融石蜡填充井。使用加热钳保持蜡熔化, 并仔细定位在模具的胚胎。 注意: 样品的正确方向是在所需平面切片胚胎的关键步骤。</li>
		<li>在霉菌凝固之前, 放置一个没有盖子的盒式卡带, 用熔融石蜡填充。让剩余的蜡冷却, 直到一夜之间室温凝固。</li>
		<li>石蜡完全固化后, 从模具中取出实心块, 在室温或4摄氏度前将石蜡块贮存至切片14。</li>
	</ol>
</li>
	<li>石蜡切片<ol>
<li>将刀片定向到切片上, 并设置5-7 µm 的厚度。5.2.2. 冷却冰上的石蜡块, 修剪它以产生立方体或金字塔。</li>
		<li>使用少量的熔融蜡将块连接到切片的支架上。</li>
		<li>将块定向到最佳切割。用标准切片在室温下将石蜡块分成5-7 µm 厚片。</li>
		<li>使用精美的画笔, 将部分放置在室温下的水浴中, 用于操作和选择切片。</li>
		<li>从水浴中拿起显微镜滑动的部分, 并将它们转移到42摄氏度的水浴中伸展。</li>
		<li>从水浴中移除部分, 然后轻轻地将它们放到粘附显微镜幻灯片上。</li>
		<li>干燥的幻灯片在一个烤箱在37°c 过夜, 使各部分完全坚持, 否则, 他们可能会在染色过程中丢失。 注: 将幻灯片保存在4摄氏度, 直到开始染色程序。</li>
	</ol>
</li>
	<li>Deparaffinization 和 Counterstaining 染色石蜡切片的研究<ol>
<li>将幻灯片放在显微镜下, 在60摄氏度的烤箱中, 至少45分钟, 使石蜡蜡更流畅。</li>
		<li>将幻灯片转移到机架上, 并使用玻璃染色碟执行以下步骤: 将机架浸入含有减少浓度醇的染色罐中, 以完成石蜡去除和组织水化: 5 分钟的异丙醇60°c;异丙醇3分钟;100% 乙醇2分钟;96% 乙醇1分钟;70% 乙醇在室温下1分钟。</li>
		<li>将蒸馏水中的部分冲洗两次, 以确保没有酒精残留物。</li>
		<li>Counterstain 部分与污点 (例如核快速的红色解答) 为5分钟 (通常在3-8 分钟之间) 在室温 (图 2)。 注意: 这种染色有助于揭示切片的整体组织结构。</li>
		<li>染色后, 将蒸馏水中的滑梯清洗1分钟, 并在显微镜下检查切片染色。 注: 如果所需染色太弱, counterstain 节再长一段时间。</li>
		<li>脱水部分在以下系列以增加的乙醇的浓度: 二洗涤在95% 乙醇为2分钟每, 二洗涤在100% 乙醇为2分钟每, 并且, 为了避免溶化蓝色沉淀, 仅一个快速洗涤在二甲苯。</li>
		<li>将幻灯片从机架中一个一个地取出, 然后放在一张纸上。然后安装和盖玻片每张幻灯片使用合成, 永久安装介质。 注: 二甲苯是一种易燃产品, 其蒸气对人体健康和水生生物有害。它需要在引擎盖内操作, 并需要使用适当的保护设备。</li>
	</ol>
</li>
</ol>

提交人宣布, 他们没有竞争的经济利益。

大肠杆菌LacZ 基因因其灵敏度高、检测方便, 在基因表达模式研究中得到了广泛的应用。本议定书描述了一种基于酶反应的检测β-gal 的经典方法, 它既简单又快速, 而且价格低廉。这种方法也可以应用在整个芒胚, 完整的器官, 恒温器组织切片或培养细胞没有重大的修改。
这种方法的准确应用导致了解释染色的鲁棒性。但是, 强烈建议同时控制和随后的验证步骤。在这方?...

为了描述特定的基因表达模式, 使用报告基因作为标记是最重要的从果蝇到哺乳动物。在涉及转基因和击倒动物的实验中, 大肠杆菌 (大肠杆菌) 的细菌β -乳糖酶基因(LacZ) 是其中最广泛使用的1、2、3、4. β-乳糖酶 (β-加仑) 催化β-galactosides (如乳糖) 的水解成其单糖 (葡萄糖和半乳糖)5。它最常用的基质是 X-加仑 (5-溴-4-氯-3-哚β D-galactopyranoside), 一种由β-乳糖酶水解而成的糖苷, 它能产生 5-溴-4-氯-3-羟基吲哚和半乳糖。第一种被氧化成二聚体, 当与费里钾和氰化物结合使用时, 产生一种特征不溶的蓝色沉淀 (图 1)6。
在三十年前的7,8, LacZ基因开始被用作一个记者基因。通常, LacZ是插入在一个内源启动子下游的开放阅读框架, 所以它可以用于细菌和细胞培养可视化的细胞含有特定的插入物, 以及在转基因动物作为内源性的示踪剂基因表达模式在发展期间9。在这方面, β-乳糖酶活性的可视化在果蝇中得到了广泛的应用, 以了解从单细胞到整个组织的发育和细胞过程。果蝇遗传学倾向于稳定线的生成 , 其中一个含有报告基因LacZ的修饰 P 元素结构入到基因组的随机位置。因此, 当置于增强因子的影响下, 它可能会以组织特定的方式驱动其表达, 这使得在过去的两年中, 对许多基因的表达模式进行系统分析10。此外, 利用转基因小鼠监测LacZ基因的表达也可以通过 loxP 介导的重组来检测基因重组事件, 并在嵌合体分析中定位突变胚胎干细胞衍生物11, 促进LacZ表达的控制在特定组织并且世俗地。此外, 在整个胚胎中, β-乳糖酶活性的检测可能产生不同强度的差异染色模式, 可以在不同的发育阶段方便地观察, 以分析基因表达的时间变化。8,12。
在这篇文章中, 我们提出了一个协议, 通过 X-gal 染色在小鼠胚胎发育阶段的整个组织中对基因表达进行可视化。我们提出这种组织化学的方法是一种高度敏感和廉价的技术, 有利于准确检测的标记细胞, 无论是在整个装载标本或在细胞水平后石蜡嵌入组织或胚胎。该方法可使小鼠组织中染色的直接可视化与其他方法13相比具有最小背景。

<table>
	<tbody>
		<tr>
			<td>REAGENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>SIGMA-ALDRICH</td>
			<td>24137-1L-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>SCHARLAU</td>
			<td>50004/ LE3Q2014</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqueous mounting medium</td>
			<td>VECTOR LABS</td>
			<td>H-5501</td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic mounting media</td>
			<td>MERCK</td>
			<td>100579</td>
			<td></td>
		</tr>
		<tr>
			<td>96% Ethanol</td>
			<td>PROLABO</td>
			<td>20824365</td>
			<td></td>
		</tr>
		<tr>
			<td>99.9% Ethanol absolute</td>
			<td>SCHARLAU</td>
			<td>ET00021000</td>
			<td></td>
		</tr>
		<tr>
			<td>50% Glutaraldehyde solution</td>
			<td>SIGMA-ALDRICH</td>
			<td>G6403-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>85% Glycerol</td>
			<td>MERCK</td>
			<td>104094</td>
			<td></td>
		</tr>
		<tr>
			<td>99.9% Glycerol</td>
			<td>SIGMA-ALDRICH</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>SIGMA-ALDRICH</td>
			<td>63064</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonionic surfactant (Nonidet P-40)</td>
			<td>SIGMA-ALDRICH</td>
			<td>542334</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear Fast Red counterstain</td>
			<td>SIGMA-ALDRICH</td>
			<td>N3020</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin pastilles</td>
			<td>MERCK</td>
			<td>111609</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>SIGMA-ALDRICH</td>
			<td>158127-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (tablets)</td>
			<td>SIGMA-ALDRICH</td>
			<td>P4417-50TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium ferrocyanate</td>
			<td>MERCK</td>
			<td>1049840500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium ferrocyanide</td>
			<td>MERCK</td>
			<td>1049731000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>SIGMA-ALDRICH</td>
			<td>S8032</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>SIGMA-ALDRICH</td>
			<td>30970</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate monohydrate</td>
			<td>SIGMA-ALDRICH</td>
			<td>106346</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic dihydrate</td>
			<td>SIGMA-ALDRICH</td>
			<td>71638</td>
			<td></td>
		</tr>
		<tr>
			<td>Thymol</td>
			<td>SIGMA-ALDRICH</td>
			<td>T0501</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris hydrochloride (Tris HCl)</td>
			<td>SIGMA-ALDRICH</td>
			<td>10812846001 (Roche)</td>
			<td></td>
		</tr>
		<tr>
			<td>X-GAL</td>
			<td>VENN NOVA</td>
			<td>R-0004-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR CHEMICALS</td>
			<td>VWRC28973.363</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable plastic cryomolds 15x15x5 mm</td>
			<td>SAKURA</td>
			<td>4566</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotatory Microtome</td>
			<td>Leica</td>
			<td>RM2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Cassettes</td>
			<td>Oxford Trade</td>
			<td>OT-10-9046</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glasses 24x60 mm</td>
			<td>VWR</td>
			<td>ECN631-1575</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Thermo Scientific, MENZEL-GL&#196;SER</td>
			<td>AGAA000001#12E</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesion microscope slides</td>
			<td>Thermo Scientific, MENZEL-GL&#196;SER</td>
			<td>J1820AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Flotation Water bath</td>
			<td>Leica</td>
			<td>HI1210</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Low Profile Microtome Blades</td>
			<td>Feather</td>
			<td>UDM-R35</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin oven</td>
			<td>J.R. SELECTA</td>
			<td>2000205</td>
			<td></td>
		</tr>
		<tr>
			<td>Wax Paraffin dispenser</td>
			<td>J.R. SELECTA</td>
			<td>4000490</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>DM500</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene microcentrifuge tubes 2.0 mL</td>
			<td>SIGMA-ALDRICH</td>
			<td>T2795</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene microcentrifuge tubes 1.5 mL</td>
			<td>SIGMA-ALDRICH</td>
			<td>T9661</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>IKA Labortechnik</td>
			<td>HS250 BASIC</td>
			<td></td>
		</tr>
		<tr>
			<td>Stirring Hot Plate</td>
			<td>Bibby</td>
			<td>HB502</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Shaker</td>
			<td>IKA Labortechnik</td>
			<td>MS1</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory scale</td>
			<td>GRAM</td>
			<td>FH-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision scale</td>
			<td>Sartorius</td>
			<td>ISO9001</td>
			<td></td>
		</tr>
		<tr>
			<td>pHmeter</td>
			<td>Crison</td>
			<td>Basic 20</td>
			<td></td>
		</tr>
		<tr>
			<td>Optic fiber</td>
			<td>Optech</td>
			<td>PL2000</td>
			<td></td>
		</tr>
	</tbody>
</table>

tracing gene expression through detection of &#946;-galactosidase activity in whole mouse embryos

大肠杆菌 LacZ基因, 编码β-乳糖酶, 主要是作为一个记者的基因表达和作为示踪剂在细胞谱系研究。经典的组织化学反应是基于基片 X-gal 与铁和铁离子的水解, 产生了一种易于可视化的不溶性蓝色沉淀。因此, β-乳糖酶活动作为发展过程中感兴趣基因表达模式的标志。在这里, 我们描述了检测早期全小鼠胚胎β-乳糖酶活性的标准协议, 以及随后的石蜡切片和 counterstaining 方法。此外, 还提供了一个澄清整个胚胎的程序, 以更好地可视化 X-gal 染色在更深的区域的胚胎。通过执行此过程获得一致的结果, 尽管需要优化反应条件以最小化背景活动。还应考虑试验中的局限性, 特别是关于整个芒染色中胚胎的大小。我们的协议为在小鼠发育过程中的β乳糖酶检测提供了一种灵敏和可靠的方法, 可进一步应用于恒温器切片以及整个器官。因此, 在整个胚胎中使用该协议可以很容易地分析整个发育过程中的动态基因表达模式, 而且在石蜡切片后可以评估细胞水平的详细表达。

在这里, 我们显示了应用标准协议的β-乳糖酶组织化学反应使用 X-gal 作为基质在整个小鼠胚胎 (图 1和图 2)。利用该协议, 在不同胚胎发育阶段 (E9.5、E11.5 和 E12.5) 上, 利用 Mt4-mmp 突变小鼠, 在内源性控制下, 对膜型 4-基质金属蛋白酶 (Mt4-mmp) 的表达进行了研究。Mt4-mmp 启动子 (图 3,图 4,<s...

Watch this Scientific Journal Video about Tracing Gene Expression Through Detection of Î²-galactosidase Activity in Whole Mouse Embryos at JoVE.com

Tracing Gene Expression Through Detection of &amp;#946;-galactosidase Activity in Whole Mouse Embryos

在这里, 我们描述了检测早期全小鼠胚胎β乳糖酶活性的标准协议, 以及石蜡切片和 counterstaining 的方法。这是一个简单和快速的过程, 以监测基因表达在发展期间, 也可以应用于组织切片, 器官或培养细胞。

通过检测全小鼠胚胎β乳糖酶活性追踪基因表达

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

This method can have answer key questions in the developmental biology field such as the study of gene expression patterns during embryonic development.The main advantages of this technique are that it is highly sensitive, easy and quick to perform and allows direct visualization of the staining in the whole mount tissue or at higher resolution after paraffin sectioning of the sample.Demonstrating the procedure will be Dr Maria Jose Blanco, Dr Anabel Learte, Dr Emma Munoz and Dr Miguel Marchena.All of them are senior post-doc from my lab.Begin by incubating glutaraldehyde-fixed transgenic embryos expressing galactosidase in freshly prepared X-gal rinse buffer for 10 minutes at room temperature.Use wild type litter-made embryos as negative controls for the enzymatic reaction.Next, replace the rinse buffer with X-gal staining solution at pH 8-9.Protect from light and incubate for 2 hours to overnight at 37

tracing-gene-expression-through-detection-galactosidase-activity

Research

JoVE Journal

Developmental Biology

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

25.8K 次观看.  Universidad Europea de Madrid. This method can have answer key questions in the developmental biology field such as the study of gene expression patterns during embryonic development.The main advantages of this technique are that it is highly sensitive, easy and quick to perform and allows direct visualization of the staining in the whole mount tissue or at higher resolution after paraffin sectioning of the sample.Demonstrating the procedure will be Dr Maria Jose Blanco, Dr Anabel Learte, Dr Emma Munoz and Dr Miguel Marche...

视频: Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

科研

发育生物学

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

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

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

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

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 ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

在嵌合斑马鱼胚胎的个别视网膜祖细胞转基因表达的体内成像来研究细胞非自治的影响

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

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

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

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 ...

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 ...

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

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

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 ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. The high transfection efficiency permits at least 4 distinct mRNAs to be co-transfected with ~87% efficiency. Most of the electroporated mRNAs are degraded during the first 2 h post-electroporation, permitting time-sensitive experiments to be carried out in the developing embryo. Finally, we describe how to dynamically image live embryos electroporated with mRNAs that encode various subcellular targeted fluorescent proteins.

We report messenger RNA (mRNA) electroporation as a method that permits fast and efficient expression of multiple proteins in the quail embryo model system. This method can be used to fluorescently label cells and record their in vivo movements by time-lapse microscopy shortly after electroporation.

使用mRNA电穿孔快速有效地表达Avian胚胎中的多种蛋白质

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its longevity can be a powerful way to reveal mechanisms of tissue development and maintenance. One of the most important tools currently available to monitor cells in vivo is the Confetti mouse model. The Confetti model can be used to genetically label individual cells in living mice with various fluorescent proteins in a cell type-specific manner and monitor their fate, as well as the fate of their progeny over time, in a process called clonal genetic tracing or clonal lineage tracing. This model was generated almost a decade ago and has contributed to an improved understanding of many biological processes, particularly related to stem cell biology, development, and renewal of adult tissues. However, preserving the fluorescent signal until image collection and simultaneous capturing of various fluorescent signals is technically challenging, particularly for mineralized tissue. This publication describes a step-by-step protocol for using the Confetti model to analyze growth plate cartilage that can be applied to any mineralized or nonmineralized tissue.

This method describes the use of the R26R-Confetti (Confetti) mouse model to study mineralized tissues, covering all steps from the breeding strategy to the image acquirements. Included is a general protocol that can be applied to all soft tissues and a modified protocol that can be applied to mineralized tissues.

使用纸屑小鼠研究矿化组织的克隆遗传追踪

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

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. ...