眼科研究在本实验室工作的支持，杜鹃（1972）美国卫生署助理基金会，捐赠基金，香港大学专业进修学院的小项目基金（20097176185）。

1。工具显微镜，尼康立体声调查，医疗保险基金的生物科技公司 - MicroBrightField 2。准备<ol><li>任何形态分析工作之前，每个视网膜样本是在4微米厚的切片，经过H＆E染色。 </li><li>视网膜节分为4个区域 - 上周边地区，中部地区上，较低的周边地区，中部地区较低。 </li><li>后定义的区域，每个区域的图像尼康明亮的光束显微镜10x40倍率下获得。 </li><li>现在，我们已经准备好来分析视网膜的形态。 </li></ol> 3。 INL的厚度测量<ol><li>四，在每个地区的INL厚度的测量，测量。四行将来自轮廓基线垂直的INL的外部边界。 </li><li>打开称为立体声调查软件，并选择从主工具栏的40X镜头。 </li><li>选择“文件”选项卡下的“图片打开”。选择所需的视网膜区域，你想分析的图像。 </li><li>从主工具栏的下拉列表框中选择轮廓线的类型，并按下“自动移动”按钮。 </li><li>在跟踪窗口中右键单击，选择“简单的点击跟踪”。 </li><li>沿边境的INL内绘制的等高线。 </li><li>当绘制完成后，在跟踪窗口右击并选择“结束开放轮廓”。 </li><li>选择一个标记“工具栏上的标记，并在交接点添加标记。 </li><li>后加入所有的标记，在标记栏取消标记图标。 </li><li>游标放置在中心的标志之一。 </li></ol><img src="/files/ftp_upload/3377/3377fig1.jpg" alt="图1" /><ol start="11"><li>选择“快速测量角度“下的”工具“选项卡。 </li></ol><img src="/files/ftp_upload/3377/3377fig2.jpg" alt="图2" /><ol start="12"><li>在跟踪窗口中单击右键，取消选择“连续”选项。 </li><li>首先标记的中心，这是相邻线段结束选定的标记来衡量一个角度。 </li></ol><img src="/files/ftp_upload/3377/3377fig3.jpg" alt="图3" /><ol start="14"><li>然后，将鼠标移动到中心有关的标记，并再次单击。 </li><li>延长橡皮筋线的INL的外部边界。 </li><li>调整角度，直到达到90°。 </li></ol><img src="/files/ftp_upload/3377/3377fig4.jpg" alt="图4" /><ol start="17"><li>留在单击所需的位置。角度测量显示一个对话框会弹出后点击。 </li><li>按“Enter”键，同时按住鼠标仍然。 </li></ol><img src="/files/ftp_upload/3377/3377fig5.jpg" alt="图5" /><ol start="19"><li>左键点击跟踪窗口，使绘制一条线，从一端到新选定的结束。 </li><li>在跟踪窗口中右键单击并选择“结束开放轮廓”。 </li></ol><img src="/files/ftp_upload/3377/3377fig6.jpg" alt="图6" /><ol start="21"><li>在完全相同的方式如前所述，绘制在不同位置的其他线路。 </li><li>画线后，选择“编辑”模式，并相应地重命名4线段。 </li><li>按“轮廓测量”按钮，可以得到四个测量。 </li><li>选择这4个测量通过点击“复制到剪贴板并粘贴到电子表格。 </li><li>选择“另存为”选项卡下的“文件”。跟踪数据可以保存为一个数据文件。 </li></ol><ol><li>选择“文件”选项卡下的“图片打开”。选择所需的视网膜区域，你想分析的图像。 </li><li>选择轮廓线从主工具栏的下拉列表框的类型。 </li><li>沿神经纤维层（NFL）的边界绘制的等高线。 </li><li>结束追查选项“年底开放轮廓”。 </li><li>在每年年底，添加一个标记线的一角，并在尖端附近的位置。 </li><li>取消标记“工具栏上的标记图标。 </li><li>将光标定位在尖端标志的中心。 </li><li>选择“工具”选项卡下的“快速测角”。 </li><li>在跟踪窗口中单击右键，取消选择“连续”选项。 </li><li>调整橡皮筋线，并测量90°角。 </li><li>按“Enter”键后，弹出一个对话框。 </li><li>左点击ţ他跟踪窗口，使一个新的线可以垂直尖端延长。 </li><li>画出另一个新生产线的另一端​​，在完全相同的方式。 </li><li>创建两个边界内的视网膜神经节细胞的数量计算。 </li><li>选择一个边界为排除细胞接触不计入。 </li><li>选择一个标记类型和重视每一个活细胞。 </li><li>选择死细胞类型的另一个标志。 </li><li>各类型放置标记的数量将相应标记工具栏旁边的标记图标显示。 </li><li>选择编辑模式，并拿起轮廓基线。右键单击在跟踪窗口和重命名行。 </li><li>轮廓基线的长度可以按“等​​高线测量”按钮。 </li><li>细胞的数量，然后可以表示每毫米的长度。 </li><li>选择“另存为”选项卡下的“文件”。 “跟踪和标记数据可以保存为一个数据文件。 </li></ol> 5。测量的研资局的细胞的细胞大小<ol><li>选择“文件”选项卡下的“图片打开”。选择所需的视网膜区域，你想分析的图像。 </li><li>在活细胞中的变焦和选择的轮廓线，从主工具栏的下拉列表框的类型。 </li><li>沿细胞周长绘制的等高线。 </li><li>在大约最后，右击跟踪窗口，并选择“结束接近轮廓”。 </li><li>细胞的面积将显示在项目区的轮廓测量对话框。 </li></ol>

<ol>
	<li>Lam, T. T., Abler, A. S., Tso, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Methyl-D-Aspartate+(NMDA)â&#x20AC;&#x201C;Induced+Apoptosis+in+Rat+Retina.">N-Methyl-D-Aspartate (NMDA)â&#x20AC;&#x201C;Induced Apoptosis in Rat Retina.</a> Investigative Ophthalmology &amp; Visual Science. 40, 2391-2397 (1999).</li><li>Lam, T. T., Abler, A. S., Tso, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+and+caspases+after+ischemia-reperfusion+injury+in+rat+retina.">Apoptosis and caspases after ischemia-reperfusion injury in rat retina.</a> Invest. Ophthalmol. Vis. Sci. 40, 967-975 (1999).</li><li>Luo, X. G., Chiu, K., Lau, H. S., Lee, V. W. H., Yung, K. K. L., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Selective+Vulnerability+of+Retinal+Ganglion+Cells+in+Rat+Chronic+Ocular+Hypertension+Model+at+Early+Phase.">The Selective Vulnerability of Retinal Ganglion Cells in Rat Chronic Ocular Hypertension Model at Early Phase.</a> Cellular and Molecular Neurobiology. 29 (8), 1143-1151 (2009).</li><li>Han, Y. S., Chung, I. Y., Park, J. M., Yu, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effect+of+citicoline+on+retinal+cell+damage+induced+by+kainic+acid+in+rats.">Neuroprotective effect of citicoline on retinal cell damage induced by kainic acid in rats.</a> Korean J. Ophthalmol. 19, 219-226 (2005).</li><li>Hernandez, M., Urcola, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+ganglion+cell+neuroprotection+in+a+rat+model+of+glaucoma+following+brimonidine,+latanoprost+or+combined+treatments.">Retinal ganglion cell neuroprotection in a rat model of glaucoma following brimonidine, latanoprost or combined treatments.</a> Exp. Eye Res. 86, 798-806 (2008).</li><li>Chan, H. C., Chang, R. C. C., Ip, A. K. C., Chiu, K., Yuen, W. H., Zee, S. Y., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effects+of+Lycium+barbarum+Lynn+on+protecting+retinal+ganglion+cells+in+an+ocular+hypertension+model+of+glaucoma.">Neuroprotective effects of Lycium barbarum Lynn on protecting retinal ganglion cells in an ocular hypertension model of glaucoma.</a> Experimental Neurology. 203, 269-273 (2007).</li></ol>

作者没有透露。

1。如何获得更精确​​的厚度测量？ 您可以放大图像，通过点击“放大”按钮，并留在跟踪窗口点击。 INL的边界可以清楚地看到。如果线的一角，是不是在外部边界的INL的，我们可以调整行的编辑模式下。有关行采摘后，右击跟踪窗口，选择“插入点在选定的轮廓”。有一点可以添加外部边界，而加点应该是在同一直线上。然后，你必须删除原有的点和线可以得到一个更精?...

A correction was made to <a href="http://www.jove.com/video/3377">Morphometric Analyses of Retinal Sections</a>. There was an error with an author's name.

Erratum: Morphometric Analyses of Retinal Sections

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 类型 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>立体声调查</td><td>医疗保险基金生命科学分析软件</td><td> MicroBright场</td><td></td></tr><tr><td>显微镜</td><td>奥林巴斯BX51 </td><td>奥林巴斯公司</td><td> BX51 </td></tr></tbody></table>

morphometric analyses of retinal sections

已被用于检查视网膜疾病视网膜节的形态分析。举例来说，神经细胞均显着失去了在大鼠模型中视网膜神经节细胞层（RGCL）与N-甲基-D-天冬氨酸（NMDA）受体诱导的兴奋1，视网膜缺血再灌注损伤的2和3青光眼。 INL和内网状层（IPL），厚度减少了与胞二磷胆碱治疗老鼠的眼睛红藻酸介导的谷氨酸4逆转。修改RGC的密度和体躯大小不同的药物治疗，升高眼内压3,5,6眼睛观察。因此，有客观分析视网膜morphometries的方法可能是在评价视网膜病变和治疗策略的有效性具有十分重要的意义。 视网膜结构是多层次和几种不同类型的神经元EXIST在视网膜上。视网膜细胞数量，细胞的大小和不同层的厚度，如形态参数是比细胞培养系统更复杂。在早期，这些参数可以使用其他的商业成像软件检测。该值通常是相对值，和不断变化的精确值，可能需要进一步的精确计算。此外，细胞大小和形态的跟踪可能不准确，灵敏的统计分析不够，特别是在慢性青光眼模型。在本协议所使用的测量提供了一个更精确和更容易的方式。与细胞的大小和行的绝对长度可以直接和容易被复制到其他文件。例如，我们追踪内层和最外层的INL在核边缘，形成一条线，然后使用该软件绘制成90度角来衡量的厚度。虽然没有软件的帮助，也许斜行和视网膜厚度的变化可能个别观察员之间不重复的。此外，视网膜神经节细胞的数量和密度也可以量化。该协议成功的减少在定量的视网膜功能的变异，增加检测微小变化的敏感性。 这部影片将展示三种类型的视网膜节的形态分析。它们包括测量的INL厚度，定量视网膜神经节细胞的数量和绝对值测量视网膜神经节细胞的大小。这三种分析进行与立体声调查（医疗保险基金生命科学 - 公司MicroBrightField）。该技术可以提供一个简单，但科学的形态分析的平台。

Watch this Scientific Journal Video about Morphometric Analyses of Retinal Sections at JoVE.com

Morphometric Analyses of Retinal Sections

该视频演示了三种类型，其中包括视网膜内的核层厚度测量，定量视网膜神经节细胞（RGCs）的数量，并测量视网膜神经节细胞的大小，形态分析。该技术可以提供一个简单，但科学的形态分析的平台。

视网膜节的形态分析

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

morphometric-analyses-of-retinal-sections

Research

JoVE Journal

Neuroscience

10.2K 次观看.  The University of Hong Kong. 该视频演示了三种类型，其中包括视网膜内的核层厚度测量，定量视网膜神经节细胞（RGCs）的数量，并测量视网膜神经节细胞的大小，形态分析。该技术可以提供一个简单，但科学的形态分析的平台。

视频: Morphometric Analyses of Retinal Sections

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

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

协议星形胶质细胞的三维共聚焦形态分析

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

对于神经化学标记用biocytin填充和加工部分的免疫染色

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

Microstate and Omega Complexity Analyses of the Resting-state Electroencephalography

Microstate and omega complexity are two reference-free electroencephalography (EEG) measures that can represent the temporal and spatial complexities of EEG data and have been widely used to investigate the neural mechanisms in some brain disorders. The goal of this article is to describe the protocol underlying EEG microstate and omega complexity analyses step by step. The main advantage of these two measures is that they could eliminate the reference-dependent problem inherent to traditional spectrum analysis. In addition, microstate analysis makes good use of high time resolution of resting-state EEG, and the four obtained microstate classes could match the corresponding resting-state networks respectively. The omega complexity characterizes the spatial complexity of the whole brain or specific brain regions, which has obvious advantage compared with traditional complexity measures focusing on the signal complexity in a single channel. These two EEG measures could complement each other to investigate the brain complexity from the temporal and spatial domain respectively.

This article describes the protocol underlying electroencephalography (EEG) microstate analysis and omega complexity analysis, which are two reference-free EEG measures and highly valuable to explore the neural mechanisms of brain disorders.

静止状态脑电图的微状态和欧米茄复杂性分析

Microstate and omega complexity are two reference-free electroencephalography (EEG) measures that can represent the temporal and spatial complexities of ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

小鼠视网膜冷冻节的制备,用于免疫性植物化学

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

We describe procedures for processing newborn rat brain tissue to obtain high-resolution electron micrographs for morphometric analysis of synaptic vesicle distribution at nerve terminals. The micrographs obtained with these methods can also be used to study the morphology of a number of other cellular components and their dimensional structural relationships.

新生大鼠脑组织在神经端子突形圆锥花序分布超微结构形态分析中的制备

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

用于下游分析的冷冻啮齿动物大脑区域集合

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

用于神经肌肉分析的大鼠声带的制备

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

单骨骼肌肉纤维解剖，用于全山神经肌肉结的免疫荧光和形态分析

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Quantification of Immunostained Caspase-9 in Retinal Tissue

The family of caspases is known to mediate many cellular pathways beyond cell death, including cell differentiation, axonal pathfinding, and proliferation. Since the identification of the family of cell death proteases, there has been a search for tools to identify and expand the function of specific family members in development, health, and disease states. However, many of the currently commercially available caspase tools that are widely used are not specific for the targeted caspase. In this report, we delineate the approach we have used to identify, validate, and target caspase-9 in the nervous system using a novel inhibitor and genetic approaches with immunohistochemical read-outs. Specifically, we used the retinal neuronal tissue as a model to identify and validate the presence and function of caspases. This approach enables the interrogation of cell-type specific apoptotic and non-apoptotic caspase-9 functions and can be applied to other complex tissues and caspases of interest. Understanding the functions of caspases can help to expand current knowledge in cell biology, and can also be advantageous to identify potential therapeutic targets due to their involvement in disease.

Presented here is a detailed immunohistochemistry protocol to identify, validate, and target functionally relevant caspases in complex tissues.