Name: differential labeling of cell-surface and internalized proteins after antibody feeding of live cultured neurons
Uploaded: 2014-02-12T14:30:00
Description: Watch this Scientific Journal Video about Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons at JoVE.com

作者感谢Teele Palumaa与数字的援助。从国家健康和医学研究委员会，澳大利亚资助计划资助1008046。

1。大鼠海马神经元培养<ol><li>准备盖玻片（硼硅玻璃）： <ol><li>在100％乙醇洗涤。 </li><li>在紫外光照射下空气干燥。 </li><li>大衣与聚-D-赖氨酸（0.5毫克/毫升的0.15M硼酸盐缓冲液，4℃过夜°C）。 </li><li>在第二天（培养的天）的PBS洗涤3次，然后涂以层粘连蛋白（2.5微克/毫升，天然小鼠层粘连蛋白）+ 5％体积/体积的热灭活的胎牛血清（FCS）的PBS稀释为2小时，在37°C。 </li></ol></li><li>解剖胚胎18天?...

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这里所描述的技术是互补的细胞表面生物素化（由阿兰西维亚-CARCAMO 等人评论）12，它是所选择的用于保存关于内化蛋白的亚细胞定位信息的方法，提供一个合适的初级抗体，以一个胞表位是可用的。此外，蛋白质运输/内化的定量随着时间的推移可以执行（由整个活细胞温育与第一抗体固定盖玻片在不同时间），而不需要制备蛋白提取物。 染色强度或数量与泪...

在建立新发现的蛋白质的功能，有问题的蛋白质的亚细胞定位和贩运的调查可以提供有关蛋白质1,2的可能作用/ s的重要线索。显影皮层3的转录组的生物信息学分析为我们提供了基因在小鼠脑corticogenesis表现出改变的表达的列表。然后我们通过基因敲除的方法，以证实这些基因之一，sez6编码的蛋白质，具有在神经元发育的关键作用。我们观察到癫痫相关基因6，或Sez6，蛋白质位于开发树突和也存在于树突棘，在接收和集成的兴奋性信号的树突特殊结构。此外，当这种蛋白质缺乏，树突和兴奋性突触不能形成正确4。该蛋白质的可能优势亚型具有transmembran的特征Ë受体虽然，当免疫标记蛋白的亚细胞分布进行了检查，共聚焦显微镜或免疫电镜大多数，如果不是全部，信号的出现与在体树突车厢很少，或者没有，蛋白质标记在质膜上的小囊泡在细胞表面。 为了明确地表明，该推定受体与预测的大胞外结构域被贩卖到质膜上，我们使用我们已经生成的蛋白的胞外部分，以在细胞表面标记蛋白的抗血清，通过了活细胞的方法。通过组合这种“抗体馈送”的方法与差异标记的第二抗体的两个应用程序通过一个广泛的阻挡工序和透化步骤分开，我们能够确定蛋白质的区分两种不同的池通过结合到荧光标记的二级抗体轴承不同fluoresc耳鼻喉科标签。因此，我们能够区分在从蛋白质的抗体温育步骤，要么留在细胞表面上或在此期间被贩卖到表面已被内化由细胞内吞作用的蛋白质。使用这种方法，我们建立了所感兴趣的蛋白被贩卖到和从细胞表面的神经元。因此，这种相对快速和简单的技术证明比传统的免疫细胞化学方法或预嵌入免疫电镜更多的信息，尽管我们使用了相同的兔多克隆抗血清对所有这些技术的事实。该技术一般适用于任何跨膜蛋白提供了良好的抗体识别的细胞外结构域的表位是可用的。该技术已被以前用于研究谷氨酸受体的GluR1亚基5的受体转运。

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			<td height="21" style="height:21px;width:383px;">PBS with Ca and Mg</td>
			<td style="width:269px;">Invitrogen</td>
			<td style="width:119px;">14040182</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Neurobasal medium</td>
			<td style="width:269px;">Invitrogen</td>
			<td style="width:119px;">21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">B27 supplement</td>
			<td style="width:269px;">Invitrogen</td>
			<td style="width:119px;">17504-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">L-glutamine</td>
			<td style="width:269px;">Invitrogen</td>
			<td style="width:119px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Papain Dissociation System</td>
			<td style="width:269px;">Worthington Biochemical Corporation</td>
			<td style="width:119px;">PDS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Bovine Serum Albumin&#160;</td>
			<td style="width:269px;">Sigma Aldrich Australia</td>
			<td style="width:119px;">A9418</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:383px;">Hank&#39;s balanced salt solution without calcium, magnesium, phenol red</td>
			<td style="width:269px;">Invitrogen (Gibco)</td>
			<td style="width:119px;">14175-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Poly-D-Lysine</td>
			<td style="width:269px;">Sigma Aldrich Australia</td>
			<td style="width:119px;">P0899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Natural mouse laminin</td>
			<td style="width:269px;">Invitrogen</td>
			<td style="width:119px;">23017-015</td>
			<td>Thaw on ice prior to making aliquots</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Fetal bovine serum HyClone</td>
			<td style="width:269px;">Thermo Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">fluorodeoxyuridine</td>
			<td style="width:269px;">Sigma Aldrich Australia</td>
			<td style="width:119px;">F0503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">uridine</td>
			<td style="width:269px;">Sigma Aldrich Australia</td>
			<td style="width:119px;">U3003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">18 mm round glass coverslips</td>
			<td style="width:269px;">Menzel Gl&#228;ser</td>
			<td>CB00180RA1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">VECTASHIELD aqueous mounting medium</td>
			<td style="width:269px;">Vector Laboratories</td>
			<td style="width:119px;">H1400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey anti-rabbit Dylight 649&#160;</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td style="width:119px;">711-495-152</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">AffiniPure Fab fragment Goat anti-Rabbit 1gG (H+L)&#160;</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>111-007-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Paraformaldehyde</td>
			<td style="width:269px;">Sigma Aldrich Australia</td>
			<td style="width:119px;">P6148</td>
			<td>TOXIC - handle in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Triton-X-100</td>
			<td style="width:269px;">Sigma Aldrich Australia</td>
			<td style="width:119px;">T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexafluor 488-conjugated donkey anti-rabbit 2&#176; antibody</td>
			<td style="width:269px;">Invitrogen - Molecular Probes</td>
			<td style="width:119px;">A21206</td>
			<td></td>
		</tr>
	</tbody>
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differential labeling of cell-surface and internalized proteins after antibody feeding of live cultured neurons

为了证明在培养的神经元的推定的跨膜受体的细胞表面定位，我们标记的蛋白活神经元的表面上与针对该蛋白的细胞外部分中的特定一抗。鉴于受体被贩卖到和从该表面，如果细胞被透化固定后那么这两个细胞表面和内部蛋白质将用相同的标记的第二抗体来检测。在这里，我们适于用来研究蛋白质运输（“抗体馈送”）进行差分时的抗体温育步骤和蛋白质，要么留在细胞表面上或在此期间被贩卖到表面已被内化通过内吞作用标签蛋白的方法。区分蛋白质的这两个池的能力成为可能通过阻断过夜与步高浓度未标记的二抗的初始incubatio后注册成立Ñ​​unpermeabilized神经元用荧光标记的第二抗体。经过封闭步骤，通透允许检测的内在池与标有不同的荧光团的荧光二抗神经元。使用这种技术，我们能够获得关于这一推定受体的亚细胞定位的重要信息，揭示它，的确，贩卖到细胞表面的神经元。这种技术广泛适用于各种细胞类型和细胞表面蛋白，从而提供一个合适的抗体的细胞外表位是可用的。

这里提出的双色荧光免疫染色技术是在活细胞标记的跨膜蛋白的细胞外结构域可使用（ 图1中示意性地示出）。在潜伏期，免疫球蛋白结合抗原表位访问和蛋白质分子的人口比例，与抗体结合在一起，被内吞。此外，新合成的蛋白质可通过向前贩卖到达细胞表面，并回收蛋白质分子可能返回至质膜10。 这种方法已被优化，两个不同的蛋白池，细胞表面的检...

Watch this Scientific Journal Video about Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons at JoVE.com

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

我们描述了一种方法，以使用特定的多克隆抗体对表位的细胞外活神经元的表面上的标记蛋白。蛋白结合的抗体在细胞表面上，并随后通过胞吞作用内在化可以从蛋白质上残留进行区分，或在培养过程中贩卖到，该表面。

细胞表面和内蛋白质的现场培养神经细胞的抗体后喂养差异化标记

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Research

JoVE Journal

Neuroscience

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

从啮齿动物的神经元和非人类灵长类动物的脑片DiOLISTIC标签

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

科研

神经科学

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

漂白后荧光标记蛋白的荧光恢复（FRAP）在培养海马神经元的树突棘

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

在培养的中枢神经系统的神经元树突棘形态分析

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Live Imaging of Dorsal Root Axons after Rhizotomy

实时成像背根轴突后根切断术

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

诱导培养的交感神经元的树突状增长

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

从查明的神经元和取食行为的钙信号的同时记录果蝇</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

膜下丘脑腹内侧神经元的电位染料成像从成年小鼠为研究葡萄糖传感

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...