这项工作得到了美国国家眼科研究所的 R01 EY032056（至 CC）资助。作者感谢斯克里普斯研究核心显微镜设施的Theresa Fassel博士和Kimberly Vanderpool对电子显微镜图像的帮助。

小鼠已根据印第安纳大学布卢明顿分校机构动物护理和使用委员会批准的动物协议进行护理。用于生成代表性数据的小鼠是 C57BL6/J 背景的对照（野生型）动物、雌性和 8-12 周龄。雄性和雌性小鼠都可用于该实验，因为小鼠的性别不太可能影响实验结果。
1.晶状体解剖和开封
<ol><li>按照美国国立卫生研究院的"实验动物护理和使用指南"以及该机构批准的动物使用协议对小鼠实施安乐死。 注:在本研究中，小鼠被 CO2 过量安乐死，然后根据批准的动物方案（印第安纳大学）进行宫颈脱位。</li>
	<li>使用弯曲的镊子将眼睛从小鼠身上移开，方法是用镊子的一侧压住眼睛周围的组织，将眼睛移出眼窝。接下来，关闭眼睛下方的镊子并提起以将眼睛从眼窝中取出。将眼睛转移到解剖托盘中的新鲜1x磷酸盐缓冲盐水（PBS）中。</li>
	<li>用超细剪刀尽可能靠近眼球剪断视神经。小心地将细尖直镊子通过眼后视神经出口插入眼球。</li>
	<li>小心地将剪刀插入与步骤 1.3 中镊子相同的位置，然后开始从后部向角膜-巩膜交界处切开切口。 注意:啮齿动物的晶状体占据了眼睛的 ~30%。如果镊子或剪刀插入眼睛太深，就会发生意外损坏。</li>
	<li>继续沿着角膜-巩膜交界处切割，直到至少一半的交界处分离。</li>
	<li>使用镊子轻轻推动角膜，使晶状体可以通过步骤 1.4 和 1.5 的切口退出。</li>
	<li>使用细尖直镊子小心地从镜片上去除任何大块组织。检查镜头以找到赤道区域。</li>
	<li>使用细尖直镊子浅刺穿镜片，然后取出镜片囊。晶状体纤维细胞的质量将保持完整，晶状体上皮单层将保持附着在晶状体囊上。丢弃镜片胶囊。</li>
</ol>2.晶状体单纤维细胞染色
<ol><li>将晶状体纤维细胞团转移到装有500μL新制备的1%多聚甲醛（PFA;在1x PBS溶液中）的孔板中，并将样品在4°C下孵育过夜，轻轻振动（图2A）。 注意:给定溶液的体积针对 48 孔板进行了优化。如果使用其他尺寸的孔板或试管，请相应地调整溶液的体积。用10x PBS和双蒸水（ddH2O）制成固定，封闭和洗涤（1x PTX，0.1%Triton X-100在1x PBS中）溶液至终浓度为1x PBS。</li>
	<li>将晶状体纤维细胞转移到含有 1% PFA 的 60 mm 培养皿中。使用锋利的手术刀沿其前后轴将纤维细胞球分成两半（图2B）。沿同一轴再次将两半切成两半，以产生四分之一。 注意:前后轴很容易通过组织团块中纤维细胞的方向来识别。</li>
	<li>使用直镊子从晶状体纤维细胞区去除细胞核区域（图2C）。 注意:啮齿动物晶状体的晶状体核区域是刚性的，晶状体的中心很容易与较软的皮质纤维分离。</li>
	<li>在室温（RT）下轻轻摇动（在板振荡器上300rpm），将晶状体皮层区域的四分之一固定在200μL的1%PFA中15分钟。</li>
	<li>在750μL的1x PBS中洗涤组织四分之一两次，每次5分钟，在室温下轻轻摇动。</li>
	<li>使用200μL封闭溶液（5%血清，0.3%Triton X-100,1x PBS）在室温下轻轻摇动封闭样品1小时。 注:对于本协议中的代表性数据，样品未用一抗或二抗染色。封闭步骤后，将样品与小麦胚芽凝集素（WGA;1:100）和鬼笔环肽（1:100）（见材料表）在室温下孵育3小时，轻轻摇晃并避光。一抗染色已在先前的出版物中得到证实22,23。</li>
	<li>用100μL一抗溶液在4°C下孵育过夜，轻轻摇动。 注:抗体在封闭液中稀释。与带有组织切片的载玻片相比，这种类型的制剂中有更多的细胞。应增加一抗浓度，以提供充足的抗体来染色更多细胞。建议将组织切片上使用的抗体浓度增加一倍。这同样适用于二抗。</li>
	<li>在室温下用1x PTX（0.1%Triton X-100,1x PBS）洗涤组织区三次，每次5分钟，轻轻摇动。</li>
	<li>将纤维细胞与 100 μL 二抗/染料溶液在室温下轻轻摇动孵育 3 小时。在此步骤和后续步骤中保护样品免受光照。 注:WGA、鬼笔环肽和其他荧光染料可以添加到二抗溶液中，用于同时标记细胞膜、细胞骨架或其他细胞器，同时标记一抗。</li>
	<li>在室温下用1x PTX洗涤纤维细胞四次，每次5分钟，轻轻摇动。</li>
	<li>在将组织区转移到载玻片上之前，将一滴或 50 μL 的封片介质添加到带正电荷的显微镜载玻片上。</li>
	<li>使用镊子轻轻地将纤维细胞彼此分开，并尝试限制细胞束的重叠。</li>
	<li>在封固剂中轻轻地将 #1.5 盖玻片涂在样品顶部。封固介质应扩散到盖玻片的边缘;如果没有发生这种情况，请在盖玻片的边缘添加一些额外的封固剂。吸走盖玻片边缘周围多余的封片介质，并使用指甲油将盖玻片的边缘密封在载玻片上。 注:为共聚焦显微镜配制的任何类型的封固剂都可用于这些实验。</li>
</ol>3.晶状体核单纤维细胞染色
<ol><li>完成第 1 节中概述的剖析。</li>
	<li>通过轻轻地将纤维细胞团转移到湿的戴手套的指尖并轻轻滚动组织团块，从晶状体纤维细胞球中机械地去除皮质纤维，离开晶状体核。 注:在小鼠晶状体中，在戴手套的指尖之间滚动晶状体纤维细胞球是从硬晶状体核中去除皮质纤维细胞的有效方法28,30。对于这种机械方法无效的晶状体，可以使用仔细解剖或涡旋方法29去除皮质纤维细胞。</li>
	<li>将晶状体核转移到孔板中新鲜制备的1%PFA溶液中，并在4°C下轻轻摇动孵育过夜。</li>
	<li>将样品转移到含有1%PFA的60mm培养皿中，并使用锋利的手术刀沿前后轴分裂晶状体核。再次将组织样本减半以产生细胞核四分之一。</li>
	<li>按照步骤 2.4-2.13 中概述的过程进行免疫染色和样品封片。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65638/65638fig02v2.jpg"> 图 2:详细介绍晶状体纤维细胞制备和 免疫染色的图形摘要。 （A） 该 48 孔板已按色谱柱进行颜色编码，以演示所述方法的样品板设置，通过使用镊子轻柔处理，允许在各种免疫染色步骤之间轻松转移样品。虽然该方案的代表性数据未与一抗一起孵育，但该图包括用于一抗孵育的柱，并且在通过抽吸除用过的洗涤缓冲液后，可以重复使用用于洗涤的孔。（B）固定晶状体纤维细胞团后，组织沿前后轴（红色虚线）分裂，以保留细胞的原始结构。一旦组织团块减半，将样品旋转，并将两半沿前后轴（红色虚线）分成四分之一。（C） 使用镊子从皮质纤维细胞（蓝色）中挖出致密的中央组织，很容易去除晶状体核区域（粉红色）。卡通图部分是使用 BioRender.com 创建的，而不是按比例绘制的。 <a href="https://www.jove.com/files/ftp_upload/65638/65638fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

晶状体纤维细胞中膜叉指的免疫荧光染色

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lens+actin+filament+cytoskeleton:+Diverse+structures+for+complex+functions.">The lens actin filament cytoskeleton: Diverse structures for complex functions.</a> Experimental Eye Research. 156, 58-71 (2017).</li><li>Goldberg, M. W., Fiserova, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogold+labeling+for+scanning+electron+microscopy.">Immunogold labeling for scanning electron microscopy.</a> Methods in Molecular Biology. 1474, 309-325 (2016).</li><li>Goldberg, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+scanning+electron+microscopy+and+immuno-gold+labeling+of+the+nuclear+lamina+and+nuclear+pore+complex.">High-resolution scanning electron microscopy and immuno-gold labeling of the nuclear lamina and nuclear pore complex.</a> Methods in Molecular Biology. 1411, 441-459 (2016).</li><li>Hermann, R., Walther, P., Muller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogold+labeling+in+scanning+electron+microscopy.">Immunogold labeling in scanning electron microscopy.</a> Histochemistry and Cell Biology. 106 (1), 31-39 (1996).</li><li>Gokhin, D. S., 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=Tmod1+and+CP49+synergize+to+control+the+fiber+cell+geometry,+transparency,+and+mechanical+stiffness+of+the+mouse+lens.">Tmod1 and CP49 synergize to control the fiber cell geometry, transparency, and mechanical stiffness of the mouse lens.</a> PLoS One. 7 (11), e48734 (2012).</li></ol>

作者没有什么可透露的。

该方案已经证明了固定，保存和免疫染色方法，这些方法忠实地保留了晶状体中不同深度的束或单个晶状体纤维细胞的3D膜形态。将染色的晶状体纤维与长期用于研究晶状体纤维细胞形态的SEM制剂进行比较。结果表明，两种制剂之间的膜结构相当。EM 仍然是研究细胞形态的金标准，但在 SEM 样品中定位蛋白质33、34、35 的免疫标?...

晶状体是眼睛前房中的透明卵形组织，由两种细胞类型组成，即上皮细胞和纤维细胞1（图1）。有一层单层上皮细胞覆盖晶状体的前半球。纤维细胞从上皮细胞分化而来，构成晶状体的大部分。高度特化的纤维细胞经历伸长、分化和成熟编程，其特征是从晶状体外围到晶状体中心的细胞膜形态发生明显变化 2,3,4,5,6,7,8,9,10,11,12 ，也称为晶状体核。晶状体对从不同距离射入视网膜的光线进行精细聚焦的功能取决于其生物力学特性，包括刚度和弹性 13、14、15、16、17、18、19。晶状体纤维的复杂交叉已被假设 20,21，最近被证明对晶状体刚度很重要 22,23。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65638/65638fig01.jpg"> 图 1:晶状体纤维的晶状体解剖图和代表性扫描电子显微镜 （SEM） 图像。这幅漫画显示了上皮细胞前单层（浅蓝色阴影）和大量晶状体纤维细胞（白色）的纵向（从上到下从前到后）视图。晶状体的中心（粉红色阴影）被称为细胞核，由高度致密的纤维细胞组成。在右边，一幅横截面的卡通图显示了排列成蜂窝状的透镜纤维的细长六边形细胞形状。纤维细胞有两个宽边和四个短边。底部的代表性SEM图像显示了晶状体不同深度的晶状体纤维细胞之间的复杂膜状交叉。从右边看，晶状体周边新形成的透镜纤维沿短边有小突起，沿宽边有球窝（红框）。在成熟过程中，晶状体纤维会形成大的桨状结构域，这些桨状结构域由沿短边的小突起（蓝色框）装饰。成熟的纤维细胞具有大的桨状结构域，由小突起表示。这些互锁域对晶状体的生物力学特性很重要。晶状体核中的纤维细胞沿其短边具有较少的小突起，并具有复杂的榫槽状指（紫色框）。细胞的宽边显示球状膜形态。这幅漫画是从22,32 修改而来的，没有按比例绘制。比例尺 = 3 μm。 <a href="https://www.jove.com/files/ftp_upload/65638/65638fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
晶状体通过添加覆盖在前几代纤维24,25 上的新纤维细胞壳来生长。纤维细胞具有细长的六边形横截面形状，具有两个宽边和四个短边。这些细胞从晶状体的前极延伸到后极，根据物种的不同，晶状体纤维的长度可以达到几毫米。为了支持这些细长和瘦小细胞的结构，沿着宽边和短边的专门的指状结构创造了互锁结构，以保持晶状体形状和生物力学特性。电子显微镜 （EM） 研究广泛记录了纤维细胞分化和成熟过程中细胞膜形状的变化 2,3,4,5,6,7,8,9,10,20,26,27,28,29 .新形成的纤维细胞在其宽边上有球窝，短边有非常小的突起，而成熟纤维的短边有互锁的突起和桨。核纤维呈榫槽状交叉和球状膜形态。对于这些复杂的互锁膜所需的蛋白质知之甚少。以前关于纤维细胞中蛋白质定位的研究依赖于晶状体组织切片，这无法清晰地可视化复杂的细胞结构。
这项工作创造并完善了一种新方法，可以固定单个和成束的晶状体纤维细胞，以保持复杂的形态，并允许对细胞膜和细胞质内的蛋白质进行免疫染色。该方法忠实地保留了细胞膜结构，可与EM研究的数据相媲美，并允许使用特定蛋白质的一抗进行染色。我们之前对正在经历分化和成熟的皮质晶状体纤维进行了免疫染色22,23。在该协议中，还有一种从晶状体细胞核染色纤维细胞的新方法。该协议为了解纤维细胞成熟和晶状体细胞核压实过程中膜指的形成和变化机制打开了大门。

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			<td >100% Triton X-100</td>
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			<td >BP151-500</td>
			<td ></td>
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			<td >60mm plate</td>
			<td >FisherScientific</td>
			<td >FB0875713A</td>
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			<td >16% paraformaldehyde</td>
			<td >Electron Microscopy Sciences</td>
			<td >15710</td>
			<td></td>
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			<td >10X phosphate buffered saline</td>
			<td >ThermoFisher</td>
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			<td >1X phosphate buffered saline</td>
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			<td >48-well plate</td>
			<td >CytoOne</td>
			<td >CC7672-7548</td>
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			<td >Cover slips (22 x 40 mm)</td>
			<td >FisherScientific</td>
			<td >12-553-467</td>
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			<td >Curved tweezers</td>
			<td >World Precision Instruments</td>
			<td >501981</td>
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			<td >Dissection microscope</td>
			<td >Carl Zeiss</td>
			<td >Stereo Discovery V8</td>
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			<td >Fine tip straight tweezers</td>
			<td >Electron Microscopy Sciences</td>
			<td >72707-01</td>
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			<td >Fisherbrand Superfrost Plus Microscope Slides</td>
			<td >FisherScientific</td>
			<td >12-550-15</td>
			<td></td>
		</tr>
		<tr >
			<td >LSM 800 confocal microscope with Airyscan (63X) and Zen 3.5 Software</td>
			<td >Carl Zeiss</td>
			<td ></td>
			<td></td>
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		<tr >
			<td >Nail polish</td>
			<td ></td>
			<td ></td>
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			<td >Normal donkey serum</td>
			<td >Jackson ImmunoResearch</td>
			<td >017-000-121</td>
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			<td >Phalloidin (rhodamine)</td>
			<td >ThermoFisher</td>
			<td >R415</td>
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			<td >Primary antibody</td>
			<td ></td>
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			<td >Scalpel Feather Disposable, steril, No. 11</td>
			<td >VWR</td>
			<td >76241-186</td>
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		<tr >
			<td >Secondary antibody</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Straight forceps</td>
			<td >World Precision Instruments</td>
			<td >11252-40</td>
			<td></td>
		</tr>
		<tr >
			<td >Thermo Scientific&#160;Nunc MicroWell MiniTrays (dissection tray)</td>
			<td >FisherScientific</td>
			<td >12-565-154</td>
			<td></td>
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		<tr >
			<td >Ultra-fine scissors</td>
			<td >World Precision Instruments</td>
			<td >501778</td>
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			<td >VECTASHIELD Antifade Mounting Medium with DAPI</td>
			<td >Vector Laboratories</td>
			<td >H-1200</td>
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		<tr >
			<td >Wheat germ agglutinin (fluorescein)</td>
			<td >Vector Laboratories</td>
			<td >FL-1021-5</td>
			<td></td>
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</table>

preparation and immunofluorescence staining of bundles and single fiber cells from the cortex and nucleus of the eye lens

晶状体是眼睛前房中的透明椭圆形器官，它改变形状以将光线精细地聚焦到视网膜上以形成清晰的图像。这种组织的大部分由特化的分化纤维细胞组成，这些细胞具有六边形横截面，从晶状体的前极延伸到后极。这些又长又细的细胞与相邻的细胞紧密相对，并且沿细胞的长度具有复杂的指间。晶状体的正常生物力学特性需要特殊的互锁结构，并且已使用电子显微镜技术进行了广泛的描述。该协议展示了第一种方法来保存和免疫染色单一以及小鼠晶状体纤维细胞束，以允许在这些复杂形状的细胞内详细定位蛋白质。代表性数据显示晶状体所有区域的外周细胞、分化细胞、成熟细胞和核纤维细胞的染色。该方法可用于从其他物种的晶状体中分离的纤维细胞。

晶状体纤维细胞由晶状体皮层（分化纤维和成熟纤维）和细胞核制备，细胞用鬼笔环肽染色用于F-肌动蛋白，用WGA染色用于细胞膜。观察细胞束或单晶状体纤维的混合物（图3）并成像。从晶状体皮层中，发现了两种类型的细胞（图3A）。晶状体周边的分化纤维细胞是直的，沿其短边有非常小的突起。随着细胞的分化，纤维细胞变成波浪状，并带有小的?...

Watch this Scientific Journal Video about Advancing Lens Biomechanics Research Through a Novel Protocol for Imaging Complex Interdigitations and Protein Staining at JoVE.com

作者聚焦:通过对复杂叉指和蛋白质染色进行成像的新型方案推进晶状体生物力学研究 

该协议描述了制备用于免疫荧光染色的外周，成熟和核眼晶状体纤维细胞的方法，以研究复杂的细胞间指和膜结构。

晶状体皮层和晶状体核束和单纤维细胞的制备和免疫荧光染色

The lens is a transparent and ellipsoid organ in the anterior chamber of the eye that changes shape to finely focus light onto the retina to form a clear ...

晶状体是眼睛前房中的透明椭球体器官，负责将光线精细聚焦到视网膜上以产生清晰的图像。晶状体的功能依赖于其生物力学特性，晶状体纤维细胞之间的复杂交叉最近已被证明对晶状体刚度很重要。虽然晶状体纤维细胞的特殊形态之前已经通过电子显微镜描述过，但对这些互锁膜所需的蛋白质知之甚少。以前的研究依赖于晶状体组织切片，这不允许复杂细胞结构的清晰可视化。我们创造并完善了一种新技术，可以忠实地保留晶状体纤维细胞之间复杂的膜交叉，用于特定蛋白质的染色和共聚焦显微镜成像。在该协议中，还有一种从晶状体中心对纤维细胞进行染色的新方法，也称为晶状体核。该方法通过研究膜指间的形成机制和变化，为理解纤维细胞分化和成熟打开了大门。

preparation-immunofluorescence-staining-bundles-single-fiber-cells

Research

JoVE Journal

Developmental Biology

Preparation and Immunofluorescence Staining of Bundles and Single Fiber Cells from the Cortex and Nucleus of the Eye Lens

1.3K 次观看.  Indiana University. 晶状体是眼睛前房中的透明椭球体器官，负责将光线精细聚焦到视网膜上以产生清晰的图像。晶状体的功能依赖于其生物力学特性，晶状体纤维细胞之间的复杂交叉最近已被证明对晶状体刚度很重要。虽然晶状体纤维细胞的特殊形态之前已经通过电子显微镜描述过，但对这些互锁膜所需的蛋白质知之甚少。以前的研究依赖于晶状体组织切片，这不允许复杂细胞结构的清?...

视频: 作者聚焦:通过对复杂叉指和蛋白质染色进行成像的新型方案推进晶状体生物力学研究 

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

视网膜色素上皮细胞的干细胞高效的推导

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

科研

发育生物学

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

器官培养和鼠标沃尔弗导管的全山免疫染色

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient&#39;s blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.

Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.

患者外周血单核细胞中人心肌细胞的生成和扩增

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. ...

Rapid Isolation of Single Cells from Mouse and Human Teeth

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental pulp tissue, rich in the extracellular matrix, requires a long and tedious dissociation process that is typically beyond the reasonable time for single-cell transcriptomics. For avoiding artificial changes in gene expression, the time elapsed from euthanizing an animal until the analysis of single cells needs to be minimized. This work presents a fast protocol enabling to obtain single-cell suspension from mouse and human teeth in an excellent quality suitable for scRNA-seq (single-cell RNA-sequencing). This protocol is based on accelerated tissue isolation steps, enzymatic digestion, and subsequent preparation of final single-cell suspension. This enables fast and gentle processing of tissues and allows using more animal or human samples for obtaining cell suspensions with high viability and minimal transcriptional changes. It is anticipated that this protocol might guide researchers interested in performing the scRNA-seq not only on the mouse or human teeth but also on other extracellular matrix-rich tissues, including cartilage, dense connective tissue, and dermis.

The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human teeth.

从小鼠和人类牙齿中快速分离单个细胞

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...