Name: methanol-based whole-mount preparation for the investigation of retinal ganglion cells
Uploaded: 2023-04-07T12:20:00.000+00:00
Duration: 3 min 47 s
Description: Watch this Scientific Journal Video about Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells at JoVE.com

这项工作由湖北省重点实验室开放项目（批准号:2021KFY055）、湖北省自然科学基金（批准号:2020CFB240）和中央高校基本科研业务费（批准号:2042020kf0065）资助。

除非另有说明，否则所有步骤均在室温下进行。所有使用的C57BL / 6J小鼠均从武汉大学实验动物中心获得，所有相关实验均由武汉大学动物实验伦理委员会批准。尽一切努力将小鼠的痛苦降到最低。
1. 眼球摘除和固定
<ol><li>用二氧化碳窒息对小鼠实施安乐死，并使用无牙镊子轻轻地去除眼球。</li>
	<li>用磷酸盐缓冲盐水（PBS）冲洗眼球一次。</li>
	<li>在显微镜下用注射器针刺穿角膜处的眼球（目镜WF:10x/22;连续放大镜:0.67x-4.5x），以加速注视到眼球中并提高固定效率。</li>
	<li>转移到含有4%多聚甲醛（PFA）的24孔板中。</li>
	<li>将眼球固定在4%PFA中45分钟，然后将其转移到1x PBS中以立即去除多余的PFA（洗涤三次，每次5分钟）。</li>
</ol>2. 视网膜隔离和扁平化
<ol><li>将眼球转移到载玻片上，并将其置于解剖显微镜下（目镜WF:10x/22;连续放大镜:0.67x-4.5x）。</li>
	<li>一只手握住镊子，用它来夹住角膜的边缘，以防止眼球运动。用另一只手握住解剖剪刀，穿过角巩膜缘，在角膜周围切开约1毫米的深度。取下晶状体和玻璃体。</li>
	<li>将视盘保持在中心，并使用解剖剪刀沿四个象限放射状切割视网膜，达到视网膜半径的大约 2/3。</li>
	<li>用齿镊钳夹住巩膜边缘之一。用另一只手用无齿镊子握住巩膜的边缘，并从齿镊固定巩膜的位置向巩膜底部移动。小心地剥离视网膜。</li>
	<li>使用移液管 （200 μL） 抽出 PBS 冲洗并润湿视网膜，然后用一小张吸收纸去除任何多余的 PBS。如果需要，用小刷毛刷轻轻压平视网膜。</li>
	<li>将冷甲醇（在-20°C冰箱中预冷）一滴一滴地缓慢移液到视网膜内表面（只要视网膜可以被甲醇完全覆盖，甲醇的体积就不固定）。视网膜变白并发展出更强的韧性。</li>
	<li>轻轻地使视网膜变平，并用小刷毛刷去除残留的色素膜和玻璃体等杂质。使用小刷毛刷倒置视网膜，然后重复上述过程。</li>
	<li>将甲醇处理的视网膜转移到24孔板的孔中，并保持其浸泡在甲醇中。</li>
	<li>将其储存在-20°C下1-2小时以立即免疫染色，或将视网膜留在甲醇中，并将其储存在-20°C以长期储存。</li>
	<li>从24孔板中取出甲醇，并用1x PBS冲洗视网膜。</li>
</ol>3. RGC的免疫荧光染色
<ol><li>小心地将视网膜转移到带有塑料巴斯德移液管的24孔血凝板上，并封闭在5%PBS-TX-BSA（5g牛血清白蛋白粉末溶解在100mL的1x PBST溶液中;通过将10mL的Triton X-100添加到2，000mL的1x PBS）在室温下4小时或在4°C下过夜而形成PBST。 保持神经节面朝上。</li>
	<li>取出 PBS-TX-BSA，并将视网膜在 4 °C 下孵育 48-96 小时，使用 1:400 稀释液加入 100 μL 选定的一抗（豚鼠抗 RBPMS 抗体）。</li>
	<li>去除一抗，用1x PBS冲洗视网膜三次（每次20分钟），在室温下振荡。</li>
	<li>将视网膜在4°C下轻轻摇动过夜，与100μL相应的二抗（参见 材料表），花青Cy3 affini纯驴抗豚鼠IgG（H + L），使用1:400稀释。</li>
	<li>用1x PBS冲洗视网膜三次（每次20分钟）。</li>
	<li>将视网膜平放在载玻片上，神经节细胞层朝上，用移液管在视网膜周围滴适量的抗荧光猝灭剂，并用盖玻片盖住载玻片（注意不要有气泡）。 注意:要确定抗荧光淬灭剂的适当量，请确保它覆盖整个视网膜。之后，盖上载玻片，不要让视网膜漂浮和轻松移动。</li>
	<li>用密封剂密封滑块四周以防止其移动。将样品储存在-20°C，并避光。</li>
	<li>使用荧光/共聚焦显微镜对视网膜进行成像。</li>
</ol>

用于研究视网膜神经节细胞的小鼠视网膜分离和甲醇处理

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作者不知道任何可能影响本研究客观性的隶属关系、成员资格、资金或金融控股。

固定是保护视网膜的重要步骤，这可能会影响任何基于形态的后续RGC研究。成功的固定可以快速捕获视网膜在暴露于固定介质时的结构和状态，这对于进一步分析至关重要。尽管甲醛被认为是组织和细胞固定和保存中最常见的固定剂之一，但对于某些研究而言，甲醛本身并不总是作为最佳化学固定剂起作用10，14。不同种类的固定剂具有不同的沉淀氨基酸和核苷酸、稳定蛋白质和维持细胞形态的能力15.通过使用两种固定剂的混合物，可以充分利用每种固定剂的强度16，17。甲醇经常用于许多固定程序，在组织新鲜冷冻或嵌入理想的切割材料16，18之前。对于全卡口实验，使用100%冷（-20°C）甲醇来实现视网膜样品固定，这有助于样品透化，以便随后进行基于荧光的成像19。
甲醇固定的一个关键优点是它易于保存。在该协议中，在视网膜变平后，甲醇在-20°C下逐渐添加到视网膜的前后。很容易观察到，视网膜的颜色迅速从半透明变为乳白色，杂质变得更加清晰。应该注意的是，视网膜应用刷子轻轻压平，以防止在甲醇滴落过程中卷曲。甲醇处理后，视网膜的整体韧性增加，降低了去除杂质和转移视网膜的难度。用甲醇固定后更方便保存已用PFA稍微预先固定的视网膜。但是，当视网膜的韧性提高时，玻璃体和其他杂质的韧性也可能相应增加。可以在使用甲醇之前尝试轻轻去除一些容易去除的杂质，然后在滴注甲醇后去除剩余的杂质，这样更容易执行。早期去除杂质需要足够的实践和经验。建议从视盘向外轻轻刷视网膜上剩余的玻璃体。
在该方案中，在治疗后，将视网膜浸泡在甲醇中并放置在-20°C冰箱中一段时间，然后再进行随后的密封，其他治疗可以进一步增加视网膜的韧性。然而，很难说出一个精确的时间长度，对于浸没固定来说，这是普遍最佳的。如果需要，视网膜可以长时间储存在冷甲醇中。
在这项工作中，使用所提出的方法，研究了甲醇储存对RGC染色的影响 - 这是视网膜定性和定量评估的关键技术。使用甲醇时，RGC的荧光强度（会影响定性和定量测试的结果）在免疫染色过程中不会改变。视网膜可以在染色前无限期地保存在保存溶液冷（-20°C）甲醇中至少12个月。此外，我们之前的研究表明，在甲醇中长期储存后，视神经压碎小鼠模型中的RGC存活率没有显着差异，甲醇保存对氧诱导视网膜病变模型20中视网膜脉管系统和视网膜缺氧的定量分析也影响不大。我们建议依次使用4%PFA和冷（−20°C）甲醇进行样品制备，并在实验室中采用甲醇固定作为储存策略。一个限制是分析不能完全显示树突或轴突水平的细微形态变化，尽管RGC的密度和体细胞的形态似乎没有变化21，22。应该注意的是，在此协议中，我们仅测试了RGC的RBPMS。进一步验证不同的RGC蛋白（例如，Brn3a，神经元核，神经丝-L）将提供更可靠的结果。此外，该方案还有可能应用于视网膜中需要进一步研究的任何其他细胞类型。但长期吸入或直接皮肤接触甲醇可能对人体造成毒性作用。因此，在使用甲醇的过程中要做好预防措施。
我们的方法非常强调使用甲醇固定来储存用于RGC研究的视网膜，并强调了有关RGC定量和荧光强度的重要考虑因素。该协议提供了一种简单实用的方法，以满足保存视网膜样本以供进一步使用的必要性。

视网膜神经节细胞（RGC）是视网膜中唯一的投射神经元，它们整合并将外部视觉信息传递给大脑1。许多神经退行性疾病，如青光眼和创伤性视神经病变，其特征是RGCs2，3的不可逆转的损伤和丧失。分析RGCs的形态和数量变化是确定神经退行性疾病如何发展和进展的关键步骤4，5。
间接免疫荧光测定是一种广泛接受的监测蛋白质分布和细胞计数的方法。在实验室中，全卡口视网膜免疫染色通常用于RGCs的实验研究，以评估视网膜的生理和病理状况6。用于整个视网膜中RGC定量的最常见标志物包括Brn3a，具有多次剪接的RNA结合蛋白（RBPMS）等7，8。表征RGC的数量和分布需要高质量的全安装视网膜免疫染色。通常，在免疫染色方案中，视网膜在抗体中孵育之前浸入化学固定剂中。理想的固定剂不应改变细胞的形状、抗体表位的可及性或亲和力或组织的线性尺寸9，10。
由于视网膜结构复杂，在制作全贴片视网膜贴片时容易出现视网膜脆性和折叠等问题，以及细胞收缩和细胞核不清晰等一些常见困难，这对实验研究产生了负面影响。此外，并非所有视网膜都会立即进行免疫染色，特别是当涉及到价格昂贵且来源珍贵的转基因小鼠的视网膜时，需要保存额外的视网膜样本以供进一步使用。
适当的固定液可以快速固定组织，避免组织自溶，保持组织细胞的正常形态和结构，并保持蛋白质和其他物质的抗原性10。目前，基于甲醛的固定已广泛应用于各种组织，包括分离的视网膜，半切的眼罩和整个眼球10。组织收缩和细胞形态改变是浸泡在甲醛11中后遇到的两个关键挑战。此外，改良的固定配方正在越来越多地出现，以最大限度地保留视网膜和靶细胞的原始特性9，10。不同的视网膜固定治疗对视网膜结构、蛋白质免疫原性、荧光激发和衰减猝灭周期的影响可能不同12，13。与用福尔马林固定的视网膜相比，用戴维森溶液固定的视网膜在形态上更完整，但戴维森溶液与某些抗体的相容性较差，例如小胶质细胞标记电离钙结合接头分子112。考虑到视网膜的脆弱性质，研究人员自然会想知道视网膜完整性以及靶细胞的性质和形态在长期储存后是否会发生变化。然而，固定液在储存数月后对视网膜和RGC细胞形态的可能影响很少有报道。视网膜固定的优化对于RGC的评估和保存至关重要。
我们详细介绍了一种可靠且技术上简单的方法，该方法用于全安装鼠视网膜染色。我们的方法强调为RGC研究正确准备和储存视网膜，同时考虑到视网膜组织长期储存的需要以及荧光团形成或降解的特定方面。

<table><tbody><tr><td>24-well cell culture cluster</td><td>Costar</td><td></td><td>Eyeball fixation</td></tr><tr><td>24-well hemagglutination plate</td><td>Labedit Company</td><td></td><td>Incubation antibody</td></tr><tr><td>Adhesion microscope slides</td><td>Citotest</td><td></td><td>Or similar</td></tr><tr><td>Anti-fluorescent quenching mountant</td><td>Servicebio</td><td>G1401</td><td>Slow down fluorescence quenching</td></tr><tr><td>BSA (bovine serum albumin)</td><td>Servicebio</td><td>GC305010</td><td>Blocking reagent</td></tr><tr><td>Confocal microscope</td><td>OLYMPUS</td><td></td><td>Apply 40x objective lens</td></tr><tr><td>Curved scissors</td><td>Jiangsu Kanghua Medical Equipment Co., Ltd.</td><td></td><td>Dissecting tools</td></tr><tr><td>Dissecting microscope</td><td>RWD Life science Co.,LTD&amp;nbsp;</td><td>77001S</td><td>Dissecting tools</td></tr><tr><td>Forceps</td><td>Jiangsu Kanghua Medical Equipment Co., Ltd.</td><td></td><td>Dissecting tools</td></tr><tr><td>Methanol</td><td>Sinopharm Chemical Reagent Co., Ltd.</td><td>20210624</td><td>GC&amp;ge;99.5%</td></tr><tr><td>Nail polish</td><td>SecheVite</td><td></td><td>Sealing agent</td></tr><tr><td>Needles&amp;nbsp;</td><td>Shanghai Kindly Enterprises Development Group Co., Ltd.</td><td></td><td>Accelerate the fixation</td></tr><tr><td>Paraformaldehyde solution</td><td>Servicebio</td><td>G1101</td><td>Eyeball fixation</td></tr><tr><td>PBS (phosphate buffered saline pH 7.4)</td><td>Servicebio</td><td>G0002</td><td>Rinse the eyeball&amp;nbsp;</td></tr><tr><td>Primary antibody: guinea pig anti-RNA-binding protein with multiple splicing (RBPMS)</td><td>PhosphoSolutions</td><td>Cat. #1832-RBPMS</td><td>For immunofluorescence. Used at 1:400</td></tr><tr><td>Secondary antibody: Cy3 affiniPure donkey anti-guinea pig IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>706-165-148</td><td>For immunofluorescence. Used at 1:400</td></tr><tr><td>Straight scissors</td><td>Jiangsu Kanghua Medical Equipment Co., Ltd.</td><td></td><td>Dissecting tools</td></tr></tbody></table>

methanol-based whole-mount preparation for the investigation of retinal ganglion cells

视网膜神经节细胞（RGC）是视网膜的投射神经元，将外部视觉信息传播到大脑。RGCs的病理变化与许多视网膜退行性疾病密切相关。全卡口视网膜免疫染色经常用于RGC的实验研究，以评估视网膜的发育和病理状况。在某些情况下，一些有价值的视网膜样本，例如来自转基因小鼠的样本，可能需要长期保留，而不会影响RGC的形态或数量。为了获得可信和可重复的实验结果，使用有效的保存介质至关重要。在这里，我们描述了甲醇作为辅助固定培养基的作用，用于视网膜整体安装制备和长期储存。简而言之，在分离过程中，将冷甲醇（-20°C）移液到视网膜表面以帮助固定组织并促进其通透性，然后将视网膜储存在冷甲醇（-20°C）中，然后进行免疫染色。该协议描述了视网膜分离工作流程和组织样本储存协议，这对于RGC的研究是有用和实用的。

解剖后，视网膜应该看起来像一个扁平的四叶草。在这项研究中，通过使用上述方案，添加甲醇后视网膜变白（图1）。同时，视网膜从柔软变为柔韧平坦。接下来，RGC被标记为抗RBPMS8。使用共聚焦显微镜（目镜:10x;物镜:40x）在整个安装视网膜（n = 3）中拍摄四个图像场。 图2显示了甲醇加工前和甲醇长期保存12个月后视网膜可视化RGC的代表性视图。荧光强度无明显变化。甲醇保存不影响RGC免疫染色。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65222/65222fig01.jpg"> 图1:甲醇治疗后视网膜变化 。 （A）甲醇处理前。（二）甲醇处理后。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65222/65222fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65222/65222fig02.jpg"> 图2:甲醇对视网膜神经节细胞（RGC）免疫染色的影响 。 （A）未经甲醇处理的视网膜。（B）视网膜在甲醇中储存12个月。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65222/65222fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells at JoVE.com

作者聚焦:用于研究视网膜神经节细胞的基于甲醇的整装制剂  

甲醇可作为视网膜全膜制剂和长期储存的辅助固定培养基，可用于视网膜神经节细胞的研究。

基于甲醇的全支架制备用于视网膜神经节细胞的研究

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in ...

该研究旨在提供一种改进的视网膜分离工作流程和组织样本储存方案，这对于研究RGCs是有用和实用的。 视网膜是脆弱和折叠的，这限制了制作整个视网膜贴片时的实验研究。此外，所有视网膜都不需要立即免疫染色。有时，额外的视网膜样本需要保存以备进一步使用。我们详细介绍了一种可靠且技术上简单的整体视网膜染色方法，该方法强调了适当的准备和长期储存视网膜以进行RGC研究的必要性。我们将继续致力于退行性视神经病变的基础研究。进一步探讨视神经损伤和事故再生的分子机制以及诊断和治疗神经退行性疾病的潜在临床靶点。

methanol-based-whole-mount-preparation-for-investigation-retinal

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells

3.2K 次观看.  Renmin Hospital of Wuhan University. 该研究旨在提供一种改进的视网膜分离工作流程和组织样本储存方案，这对于研究RGCs是有用和实用的。 视网膜是脆弱和折叠的，这限制了制作整个视网膜贴片时的实验研究。此外，所有视网膜都不需要立即免疫染色。有时，额外的视网膜样本需要保存以备进一步使用。我们详细介绍了一种可靠且技术上简单的整体视网膜染色方法，该方法强调了适当的准备和长期储存?...

视频: 作者聚焦:用于研究视网膜神经节细胞的基于甲醇的整装制剂  

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

科研

神经科学

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

整装免疫荧光染色新生小鼠视网膜血管生成调查<em&gt;在体内</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

在体内的器官型小鼠视网膜Wholemount文化

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

生物学

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

从健康和视网膜疾病特异性人诱导的多能干细胞中生成视网膜类器官

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative ...

发育生物学

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

视网膜神经节细胞定义子集的单细胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

视网膜平片准备成像和免疫组化优化方案

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

<meta charset="utf8"></head><body>使用体积电子显微镜在 3D 中可视化视网膜突触结构

Preparation of Retinal Samples for Volume Electron Microscopy

Volume electron microscopy (Volume EM) has emerged as a powerful tool for visualizing the 3D structure of cells and tissues with nanometer-level precision. Within the retina, various types of neurons establish synaptic connections in the inner and outer plexiform layers. While conventional EM techniques have yielded valuable insights into retinal subcellular organelles, their limitation lies in providing 2D image data, which can hinder accurate measurements. For instance, quantifying the size of three distinct synaptic vesicle pools, crucial for synaptic transmission, is challenging in 2D. Volume EM offers a solution by providing large-scale, high-resolution 3D data. It is worth noting that sample preparation is a critical step in Volume EM, significantly impacting image clarity and contrast. In this context, we outline a sample preparation protocol for the 3D reconstruction of photoreceptor axon terminals in the retina. This protocol includes three key steps: retina dissection and fixation, sample embedding processes, and selection of the area of interest.

<meta charset="utf8"></head><body>作者聚焦：使用体电子显微镜进行视网膜神经科学研究

This protocol describes the detailed steps for preparing retinal samples for volume electron microscopy, focusing on the structural features of retinal photoreceptor terminals.

用于体电子显微镜检查的视网膜样品的制备

Volume electron microscopy (Volume EM) has emerged as a powerful tool for visualizing the 3D structure of cells and tissues with nanometer-level ...

<meta charset="utf8"></head><body>小鼠视网膜中的突触回路分析和蛋白质定位

Investigating Retinal Circuits and Molecular Localization by Pre&#45;Embedding Immunoelectron Microscopy

The retina comprises numerous cells forming diverse neuronal circuits, which constitute the first stage of the visual pathway. Each circuit is characterized by unique features and distinct neurotransmitters, determining its role and functional significance. Given the intricate cell types within its structure, the complexity of neuronal circuits in the retina poses challenges for exploration. To better investigate retinal circuits and cross-talk, such as the link between cone and rod pathways, and precise molecular localization (neurotransmitters or neuropeptides), such as the presence of substance P-like immunoreactivity in the mouse retina, we employed a pre-embedding immunoelectron microscopy (immuno-EM) method to explore synaptic connections and organization. This approach enables us to pinpoint specific intercellular synaptic connections and precise molecular localization and could play a guiding role in exploring its function. This article describes the protocol, reagents used, and detailed steps, including (1) retina fixation preparation, (2) pre-embedding immunostaining, and (3) post-fixation and embedding.

<meta charset="utf8"></head><body>作者聚焦：了解小鼠视网膜突触连接和神经递质定位的超微结构基础

This protocol outlines the detailed steps of pre-embedding immunoelectron microscopy, with a focus on exploring synaptic circuits and protein localization in the retina.

基于甲醇的全支架制备用于视网膜神经节细胞的研究

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此视频中的章节

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

整装免疫荧光染色新生小鼠视网膜血管生成调查

在体内的器官型小鼠视网膜Wholemount文化

从健康和视网膜疾病特异性人诱导的多能干细胞中生成视网膜类器官

视网膜神经节细胞定义子集的单细胞RNA-Seq

视网膜平片准备成像和免疫组化优化方案

用于体电子显微镜检查的视网膜样品的制备

用于体电子显微镜检查的视网膜样品的制备

用于体电子显微镜检查的视网膜样品的制备

通过预包埋免疫电子显微镜研究视网膜回路和分子定位