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?...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#8805;99.5%</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>SecheVite</td>
			<td></td>
			<td>Sealing agent</td>
		</tr>
		<tr>
			<td>Needles&#160;</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&#160;</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RG...

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

Research

JoVE Journal

Neuroscience

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

2.9K 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

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

成人整装准备果蝇巨纤维染料注射腹侧神经索

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

全山神经肌肉交界分析的 横向解剖阿布多米尼斯 肌肉

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

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 ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...