High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses.
High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses.
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DOI：
10.3791/53547
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发表时间：
2016-02-18
期刊：
影响因子：
--
通讯作者：
Diamond JS
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文献类型：
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作者：
Zhang J;Petralia RS;Wang YX;Diamond JS
包埋后免疫金方法是提供特定分子亚细胞定位高分辨率分析的最有效方法之一。在这里，我们描述了一种定量分析视网膜带突触谷氨酸受体的方案。视网膜神经节细胞（RGC）接收来自双极细胞的兴奋性谷氨酸能输入。 RGC 的突触兴奋由 NMDA 受体 (NMDAR) 和 AMPA 受体 (AMPAR) 在突触后介导。生理数据表明，RGC 的谷氨酸受体不仅在突触后表达，而且在突触周围或突触外膜区室中表达。然而，RGC 突触谷氨酸受体的精确解剖位置尚未确定。尽管对中央突触谷氨酸受体的高分辨率定量分析被广泛采用，但这种方法在视网膜中只取得了有限的成功。我们开发了一种用于分析膜受体的嵌入后免疫金方法，使得估计视网膜带突触处这些受体的数量、密度和变异性成为可能。在这里，我们描述了所需的工具、试剂和实际步骤：1）成功准备视网膜固定，2）冷冻替代，3）嵌入后免疫金电子显微镜（EM）免疫细胞化学，4）带状突触谷氨酸受体的定量可视化。
The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses. Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.