<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

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+diversity+in+the+retina.">Neuronal diversity in the retina.</a> Curr Opin Neurobiol. 11 (4), 431-436 (2001).</li><li>Svara, F. N., Kornfeld, J., Denk, W., Bollmann, 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=Volume+EM+reconstruction+of+spinal+cord+reveals+wiring+specificity+in+speed-related+motor+circuits.">Volume EM reconstruction of spinal cord reveals wiring specificity in speed-related motor circuits.</a> Cell Rep. 23 (10), 2942-2954 (2018).</li><li>Kubota, Y., Sohn, J., Kawaguchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+volume+electron+microscopy+and+neural+microcircuit+analysis.">Large volume electron microscopy and neural microcircuit analysis.</a> Front Neural Circuits. 12, 98 (2018).</li><li>Guo, J., 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=An+optimized+approach+using+cryofixation+for+high-resolution+3D+analysis+by+FIB-SEM.">An optimized approach using cryofixation for high-resolution 3D analysis by FIB-SEM.</a> J Struct Biol. 212 (1), 107600 (2020).</li><li>Seligman, A. 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<table><tbody><tr><td>2,2,2-Tribromoethanol</td><td>Sigma-Aldrich</td><td>T48402</td><td></td></tr><tr><td>Acetone</td><td>Electron Microscopy Science</td><td>10000</td><td></td></tr><tr><td>Amira 6.8</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C-2661</td><td></td></tr><tr><td>Embedding mold</td><td>Beijing Zhongjingkeyi Technology</td><td>GP10590</td><td></td></tr><tr><td>Epon resin</td><td>Electron Microscopy Science</td><td>14900</td><td></td></tr><tr><td>Ethanol</td><td>Sigma</td><td>64-17-5</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Electron Microscopy Science</td><td>16020</td><td></td></tr><tr><td>Helios NanoLab 600i dual-beam SEM</td><td>FEI</td><td></td><td></td></tr><tr><td>L-aspartic acid</td><td>Sigma</td><td>56-84-8</td><td></td></tr><tr><td>Lead nitrate</td><td>Sigma</td><td>10099-74-8</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;.12H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>71650</td><td>A component of phosphate buffer</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;.H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>71507</td><td>A component of phosphate buffer</td></tr><tr><td>OsO&lt;sub&gt;4&lt;/sub&gt;</td><td>TED PELLA</td><td>4008-160501</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Science</td><td>157-8</td><td></td></tr><tr><td>Potassium ferrocyanide</td><td>Sigma</td><td>14459-95-1</td><td></td></tr><tr><td>Sodium cacodylate</td><td>Sigma</td><td>6131-99-3</td><td></td></tr><tr><td>Sputter coater</td><td>Leica</td><td>ACE200</td><td></td></tr><tr><td>Thiocarbohydrazide</td><td>Sigma</td><td>2231-57-4</td><td></td></tr><tr><td>Uranyl acetate</td><td>TED PELLA</td><td>CA96049</td><td></td></tr></tbody></table>

preparation of retinal samples for volume electron microscopy

<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

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 ...

preparation-of-retinal-samples-for-volume-electron-microscopy

Research

JoVE Journal

Neuroscience

Preparation of Retinal Samples for Volume Electron Microscopy

523 次观看.  Wenzhou Medical University. 我们专注于检查小鼠视网膜中感光器末端的纳米级 3D 结构特征。我们的主要目标是设计适用于小体积生物样品的 3D EM 样品制备方法，我们使用 FIB-SEM 重建了视网膜光感受器的末端结构。使用视网膜 3D EM 样品和 OTO 方法，我们分割和重建了四种类型的感光器末端的 3D 结构，随后分析了它们成分之间的关系。我们的视网膜样本变异方案易于使用，...

视频: 作者聚焦：使用体电子显微镜进行视网膜神经科学研究

<meta charset="utf8"></head><body>使用组织透明化和荧光标记进行视网膜周细胞可视化

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining 

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor &#946; to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.

<meta charset="utf8"></head><body>通过免疫染色对小鼠视网膜血管周细胞进行 3D 可视化

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor &#946; after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

通过免疫染色对小鼠视网膜血管周细胞进行 3D 可视化 

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

科研

生物化学

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method&#39;s versatility. This protocol was developed for imaging Ca2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca2+ or metabolites in M&#252;ller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca2+, and energy homeostasis.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types.

从成年斑马鱼中制备新鲜视网膜切片用于前体成像实验

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

神经科学

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 ...

生物学

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of&#160;unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

<meta charset="utf8"></head><body>显微外科解剖和组织透明化，用于高分辨率完整全视网膜和玻璃体成像

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

显微外科解剖和组织透明化，用于高分辨率完整全视网膜和玻璃体成像

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

两种剥离方法，用于分离小鼠视网膜中的光感受器细胞区室以进行蛋白质分析

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

非增殖性和增殖性视网膜病变小鼠整个视网膜血管参数的定量

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

研究小鼠分裂视网膜中的双极和水平神经元细胞

Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by photoreceptors. They perform fundamental operations such as light adaptation, contrast sensitivity, and spatial and color opponency. A complete understanding of the precise circuitry and biochemical mechanisms that govern their behavior will advance visual neuroscience research and ophthalmological medicine. However, current preparations for examining bipolar and horizontal cells (retinal whole mounts and vertical slices) are limited in their capacity to capture the anatomy and physiology of these cells. In this work, we present a method for removing photoreceptor cell bodies from live, flatmount mouse retinas, providing enhanced access to bipolar and horizontal cells for efficient patch clamping and rapid immunolabeling. Split retinas are prepared by sandwiching an isolated mouse retina between two pieces of nitrocellulose, then gently peeling them apart. The separation splits the retina just above the outer plexiform layer to yield two pieces of nitrocellulose, one containing the photoreceptor cell bodies and another containing the remaining inner retina. Unlike vertical retina slices, the split retina preparation does not sever the dendritic processes of inner retinal neurons, allowing for recordings from bipolar and horizontal cells that integrate the contributions of gap junction-coupled networks and wide-field amacrine cells. This work demonstrates the versatility of this preparation for the study of horizontal and bipolar cells in electrophysiology, immunohistochemistry, and in situ hybridization experiments.

作者聚焦：使用 Split Retina 技术增强访问和加速实验 

This work presents an alternative flatmount retina preparation in which the removal of photoreceptor cell bodies enables faster antibody diffusion and improved patch pipette access to inner retinal neurons for immunohistochemistry, in situ hybridization, and electrophysiology experiments.

用于体电子显微镜检查的视网膜样品的制备

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通过免疫染色对小鼠视网膜血管周细胞进行 3D 可视化

通过免疫染色对小鼠视网膜血管周细胞进行 3D 可视化

通过免疫染色对小鼠视网膜血管周细胞进行 3D 可视化

从成年斑马鱼中制备新鲜视网膜切片用于前体成像实验

视网膜平片准备成像和免疫组化优化方案

显微外科解剖和组织透明化，用于高分辨率完整全视网膜和玻璃体成像

显微外科解剖和组织透明化，用于高分辨率完整全视网膜和玻璃体成像

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