这项工作是支持的研究与发展，医学研究服务，退伍军人事务部办公室。这项研究是由一个VA优秀评论奖（MCL）大学和田纳西州健康科学中心多发性硬化症研究基金。

1。抗体标记（图1） <ol><li> 0.1 M NaHCO3缓冲： <ul type="circle"><li> 8.4克的NaHCO 3 </li><li> 29.2克氯化钠</li><li> 1升H 2 O </li></ul></li><li>带来的缓冲与此溶液的pH为8.4（10.6克Na 2 CO 3，29.2克NaCl，1升H 2 O）。 </li><li>加入35微升Atto550 NHS到第70微升抗hnRNPA1和500μl的NaHCO 3缓冲液，并在室温下的1小时旋转。长达一小时的旋转后，将混合物注入到一个透析器，并在2升的PBS过夜透析。 </li><li>第二天，将透析Atto550 NHS反hnRNPA1使用的旋转柱（的Amicon Ultra 0.5）集中。 </li><li>已标记的抗hnRNPA1抗体浓缩后Atto550 NHS，纳米压降样品浓度的确定。 </li></ol> 2。抗体的转染（图2） <ol><li>种子10 5细胞/孔到500微升Dulbecco的改良的Eagle培养基（DMEM/F12 +10％FBS +1％抗生素），在8孔滑动转染前24小时。细胞融合应该是至少70％。 </li><li>播种后二十四小时，放入微量离心管中加入2微升的Ab-DeliverIN试剂。 </li><li>接下来，添加2微克Atto550 NHS标记反hnRNPA1抗体（0.5微克/微升），以相同的离心管中，并在室温下孵育10-15分钟。 </li><li>在温育时，吸液媒体和394微升新鲜DMEM培养基添加到细胞中。 </li></ol> 注意：加入500μlDMEM未触动过的细胞的一室作为一个控制。 <ol start="5"><li>温育后，添加100μl的DMEM中的抗体混合物，通过移液向上和向下混合，并加至总体积为500微升的细胞在DMEM。 </li><li>更改媒体抗体交货后48小时。 </li></ol> 注：重复议定书与FITC标记RIGG作为阳性对照。 3。现场和固定影像，以决定效率<ol><li>图片细胞在48小时使用的Cy3过滤器在荧光显微镜图像转Atto550 NHS标记的抗体。 </li><li>实时成像已经完成后，吸出的媒体和修复细胞。 </li><li>抽吸介质后，将细胞用0.4％多聚甲醛在室温下的15分钟。 4×每次5分钟，用PBS洗涤细胞。然后，装入安装介质细胞的DAPI试剂和修复盖玻片。测量与Axiovision软件的固定细胞的转染效率。如果Axiovision软件是手头没有，根本从您的图片事件计数的手和插件计算结果到3.7。或者，ImageJ软件可用于这些计算。 </li><li>首先，使用“事件”测量的细胞总数在您选择的图像在放大20倍来计算。 </li><li>接下来，请使用“事件”（Events）测量细胞计数不成功转染的抗体在相同的20倍的放大倍率的图像。 </li><li>要访问在Excel风格的格式测得的“事件”，点击“属性”，点击“测量”选项卡，并单击“查看”表“。 </li><li>最后，请使用以下的计算，以确定转染效率，两相比较以前测量的事件：（ - 未转染细胞（细胞总数）/细胞总数）×100 =转染效率。 </li></ol> 4。代表性的成果未转染的实时图像（ 图3A）相比，转染（ 图3B）神经元显示，清楚地观察到细胞内（ 图3B，C，D）的标记的抗体（在每个图中的红色或绿色）。吸入后，与媒体洗涤以除去附着到细胞表面的抗体之间或标记的抗体仍然是存在于神经元（ 图3C，D）。<html>要计算的转染效率，使用本Axiovision软件来计算而无法成功转染的细胞相比，细胞总数。这些值被输入到一个转染效率公式作为细胞总数的百分比（ 图4）中获得的转染效率。 <img alt="图1" src="/files/ftp_upload/4154/4154fig1.jpg" /> ATTO 550 NHS酯用于标记抗体的程序的流程图如图1所示。其次是集中在标记的抗体需要的步骤。 <a href="http://www.jove.com/files/ftp_upload/4154/4154fig1large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/4154/4154fig2.jpg" /> 图2。标记抗体进入活细胞的培养，转染的程序流程图。arge.jpg“目标=”_blank“&gt;点击这里查看大图。 <img alt="图3" src="/files/ftp_upload/4154/4154fig3.jpg" /> 图3转染的神经细胞转染后48小时的实时图像。 <img alt="图4" src="/files/ftp_upload/4154/4154fig4.jpg" /> 图4。修正和漂洗的图像的事件计数，以确定转染效率。

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没有利益冲突的声明。

为了测试假设特定的分子和它们的表达，基因和其他载体转染到细胞中。此过程中的独特的方面是能够容易地转染到靶细胞的抗体。我们进行了这种方法在5个不同的实验中，各一式两份。在所有的实验中，转染效率的结果是相似的。更重要的是，该方法使进入细胞的抗体的条目而不改变抗体的结构或功能。另外，用荧光显微镜的活神经元的使用，我们能够确定，通常超过55％的转染效率。这允许?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>荧光显微镜AxioObserver A1 </td><td>蔡司</td><td></td></tr><tr><td> Axiovision 4.2版</td><td>蔡司</td><td></td></tr><tr><td>反rhnRNPA1 </td><td> Abcam公司</td><td> Ab4791 </td></tr><tr><td> FITC标记的抗RIGG </td><td> OZ生物科技公司</td><td> AI20100 </td></tr><tr><td> Atto550 NHS酯</td><td>西格玛</td><td> 92835 </td></tr><tr><td> AB-DeliverIN的</td><td> OZ生物科技公司</td><td> AI20100 </td></tr><tr><td> DMEM/F12 +10％FBS +1％抗生素</td><td> Gibco公司</td><td> 11320-033 </td></tr><tr><td>聚-D-赖氨酸的8孔文化相片数</td><td> BD </td><td> 354632 </td></tr><tr><td>多功能肩</td><td>科技INDUSTRIES公司</td><td>型号151 </td></tr><tr><td> DAPI安装介质</td><td>密理博</td><td> S7113 </td></tr><tr><td> PBS </td><td> Ambion公司</td><td> 9626 </td></tr><tr><td>透析</td><td>赛默飞世尔科技</td><td> 66380 </td></tr><tr><td>的Amicon Ultra-0.5 </td><td>密理博</td><td> UFC501096 </td></tr><tr><td>多聚甲醛（4％溶液） </td><td>电子显微镜科学</td><td></td></tr><tr><td>纳米掉落</td><td>赛默飞世尔科技</td><td></td></tr></tbody></table>

antibody transfection into neurons as a tool to study disease pathogenesis

抗体的能力，获得新的洞察发生在生命系统的各种事件。产生非常具体的目标蛋白的抗体，可以非常精确的生物学问题的能力加以解决。更重要的是，抗体的发病机制中的数量的人类疾病包括系统性红斑狼疮（SLE），类风湿性关节炎（RA），副肿瘤综合征，多发性硬化症（MS）和人T-淋巴病毒1型（HTLV-1）有牵连相关性脊髓病/热带痉挛性瘫痪（HAM / TSP）1-9。如何抗体，引起病是一种正在进行的调查，数据表明，抗体和各种细胞内的分子在炎症之间的相互作用，改变细胞的信息传递，细胞凋亡10。它已被证明，MS和HAM / TSP患者产生自身抗体，细胞内的RNA结合蛋白异构核糖核酸蛋白甲1（中hnRNP A1）3，5-7，9，11。最近的数据表明，内神经元和表面抗原的抗体是致病3，5-9，11。因此，一个程序，允许细胞内抗体的研究蛋白质相互作用，将给予很大的洞察疾病的发病机制。 基因通常在培养的原代细胞和细胞系转染，但转染进入细胞的抗体已被阻碍，通过改变抗体的结构或转染效率差12。其它转染方法包括基于阳离子脂质体（由DOTAP / DOPE）和聚乙烯亚胺（PEI）的抗体的转染，这两种抗体转染在10倍的减少相比，控制12。在我们的研究中进行的方法是类似的阳离子脂质介导的方法，并使用的脂质为基础的机制，以形成非共价复合物与抗体通过静电和hydrophobic的相互作用13。我们利用的Ab-DeliverIN试剂，这是能够捕获抗体通过非共价键的静电和疏水相互作用，并提供它们在细胞内的脂质制剂。因此，化学和遗传联轴器交付到活细胞的功能性抗体是没有必要的。此方法使我们能够执行各种抗体跟踪和蛋白定位的实验，以及作为细胞内抗体的分子后果：蛋白质相互作用9分析。 在这个协议中，我们将展示如何为神经元转染抗体快速，重复性好，具有高度的转染效率。作为一个例子，我们将使用抗核糖核蛋白A1和抗IgG抗体。对于容易量化的转染效率，我们使用抗核糖核蛋白A1与ATTO-550-NHS和FITC标记的IgG抗体标记。 Atto550 NHS是一个新的标签与的高分子吸收和量子易领域。激发源和荧光过滤器Atto550是类似的Cy3标记（例如：556 EM 578）。 Atto550此外，具有高的耐光性。 FITC标记的IgG作为对照表明，该方法是多才多艺的，不染依赖。这种方法产生的数据将有助于了解人类疾病的发病机制中可能发挥的作用，细胞内的靶抗原的抗体。

Watch this Scientific Journal Video about Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis at JoVE.com

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

一种快速的方法，调查相关的抗体的存在下，在细胞内环境中的分子机制的相互作用和效果进行说明。该方法涉及的抗体的转染到活细胞，使用的基础上的脂质制剂的非共价复合物的形成。该技术是适应的永生化细胞系和原代细胞。

抗体转染神经元作为一种工具来研究疾病的发病机制

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

antibody-transfection-into-neurons-as-tool-to-study-disease

Research

JoVE Journal

Neuroscience

10.7K 次观看.  Veterans Administration Medical Center, Memphis, TN. 一种快速的方法，调查相关的抗体的存在下，在细胞内环境中的分子机制的相互作用和效果进行说明。该方法涉及的抗体的转染到活细胞，使用的基础上的脂质制剂的非共价复合物的形成。该技术是适应的永生化细胞系和原代细胞。

视频: Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

科研

神经科学

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.
Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.
Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas7, 8,13, and can be used to study coupling between other retinal neurons, as described here.

An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.

剪切载荷：一个有用​​的工具，用于检查视网膜神经细胞之间的缝隙连接示踪剂耦合程度

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

原代海马神经元的磷酸钙转染

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

作为一种工具来描绘分子途径参与神经毒性和神经保护神经元细胞在线实时阻抗为基础的细胞分析仪

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

肌肉注射沿运动终板：微创方法来接送示踪剂直接为运动神经元

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

条纹分析来研究蛋白质底物使用游离海马神经元的吸引力或令人反感的活动

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

微粒相关α synuclein 聚合体对原代神经元的外源性管理--纤维形成的强大细胞模型

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Cell-based Assay to Study Antibody-mediated Tau Clearance by Microglia

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing neuronal dysfunction which eventually leads to cognitive decline. Hyperphosphorylated tau aggregates in the neuron are believed to cause most of the pathology associated with AD. These aggregates are assumed to be released into the extracellular compartment and taken up by adjacent healthy neurons where they induce further tau aggregation. This &#34;prion-like&#34; spreading can be interrupted by antibodies capable of binding and &#34;neutralizing&#34; extracellular tau aggregates as shown in preclinical mouse models of AD. One of the proposed mechanisms by which therapeutic antibodies reduce pathology is antibody-mediated uptake and clearance of pathological aggregated forms of tau by microglia. Here, we describe a quantitative cell-based assay to assess tau uptake by microglia. This assay uses the mouse microglial cell line BV-2, allows for high specificity, low variability and medium throughput. Data generated with this assay can contribute to a better characterization of anti-tau antibody effector functions.

Here we describe a cell-based assay to quantitatively assess tau uptake by microglia with the aim of creating an investigational tool to better characterize the mechanisms of action of anti-tau antibodies.

微胶质细胞抗体介导的陶氏清除研究

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative condition in which aggregated tau and amyloid proteins accumulate in the brain causing ...