Name: Advancing Lens Biomechanics Research Through a Novel Protocol for Imaging Complex Interdigitations and Protein Staining (Video) | JoVE
Uploaded: 2023-06-09T14:50:00
Description: Watch this Scientific Journal Video about Advancing Lens Biomechanics Research Through a Novel Protocol for Imaging Complex Interdigitations and Protein Staining at JoVE.com

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?...

Immunofluorescence Staining of Membrane Interdigitations in Lens Fiber Cells

<ol>
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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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >100% Triton X-100</td>
			<td >FisherScientific</td>
			<td >BP151-500</td>
			<td ></td>
		</tr>
		<tr >
			<td >60mm plate</td>
			<td >FisherScientific</td>
			<td >FB0875713A</td>
			<td></td>
		</tr>
		<tr >
			<td >16% paraformaldehyde</td>
			<td >Electron Microscopy Sciences</td>
			<td >15710</td>
			<td></td>
		</tr>
		<tr >
			<td >10X phosphate buffered saline</td>
			<td >ThermoFisher</td>
			<td >70011-044</td>
			<td></td>
		</tr>
		<tr >
			<td >1X phosphate buffered saline</td>
			<td >ThermoFisher</td>
			<td >14190136</td>
			<td></td>
		</tr>
		<tr >
			<td >48-well plate</td>
			<td >CytoOne</td>
			<td >CC7672-7548</td>
			<td></td>
		</tr>
		<tr >
			<td >Cover slips (22 x 40 mm)</td>
			<td >FisherScientific</td>
			<td >12-553-467</td>
			<td></td>
		</tr>
		<tr >
			<td >Curved tweezers</td>
			<td >World Precision Instruments</td>
			<td >501981</td>
			<td></td>
		</tr>
		<tr >
			<td >Dissection microscope</td>
			<td >Carl Zeiss</td>
			<td >Stereo Discovery V8</td>
			<td></td>
		</tr>
		<tr >
			<td >Fine tip straight tweezers</td>
			<td >Electron Microscopy Sciences</td>
			<td >72707-01</td>
			<td></td>
		</tr>
		<tr >
			<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>
		</tr>
		<tr >
			<td >Nail polish</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Normal donkey serum</td>
			<td >Jackson ImmunoResearch</td>
			<td >017-000-121</td>
			<td></td>
		</tr>
		<tr >
			<td >Phalloidin (rhodamine)</td>
			<td >ThermoFisher</td>
			<td >R415</td>
			<td></td>
		</tr>
		<tr >
			<td >Primary antibody</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Scalpel Feather Disposable, steril, No. 11</td>
			<td >VWR</td>
			<td >76241-186</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr >
			<td >Ultra-fine scissors</td>
			<td >World Precision Instruments</td>
			<td >501778</td>
			<td></td>
		</tr>
		<tr >
			<td >VECTASHIELD Antifade Mounting Medium with DAPI</td>
			<td >Vector Laboratories</td>
			<td >H-1200</td>
			<td></td>
		</tr>
		<tr >
			<td >Wheat germ agglutinin (fluorescein)</td>
			<td >Vector Laboratories</td>
			<td >FL-1021-5</td>
			<td></td>
		</tr>
	</tbody>
</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。

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

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

Advancing Lens Biomechanics Research Through a Novel Protocol for Imaging Complex Interdigitations and Protein Staining (Video) | JoVE

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。