Name: high-resolution quantitative immunogold analysis of membrane receptors at retinal ribbon synapses
Uploaded: 2016-02-18T14:00:00.000+00:00
Duration: 9 min 24 s
Description: Watch this Scientific Journal Video about High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses at JoVE.com

这项工作是由神经疾病与中风国家研究所（NINDS）和国立耳聋与其他交流障碍（NIDCD），美国国立卫生研究院（NIH）的校内方案的支持。我们感谢NINDS EM设施和援助NIDCD先进的成像核心（代号＃ZIC DC 000081-03）。

保养和动物的处理均符合NIH动物护理和使用委员会的准则。产后天（P）15-21 SD大鼠，通过双边上丘用1-1.2％CTB注入，维持在12:12小时光照:黑暗周期。 1.视网膜组织固定<ol><li>组装以下材料和工具:解剖显微镜，2钳很细的技巧，剪刀，纤维素滤纸，塑料吸管和显微镜载玻片。 </li><li>麻醉大鼠与2.0毫升氟烷（吸入剂麻醉剂）的密闭室。确定由这些方法适当麻醉:缺少脚趾捏，或缺乏瞬目反射的后爪后撤回。然后用断头台斩首立即。有一对虹膜剪的并发生在在pH7.4含0.1M磷酸盐缓冲液（PB）的4％多聚甲醛的玻璃培养皿中移除眼睛。 </li><li>使用解剖显微镜，取出玉米通过切断眼球的前面EA。移除钳的内视网膜表面的晶状体和玻璃体。 </li><li>剥离与两个镊子巩膜直至视网膜被从眼罩隔离。 </li><li>立即切断视网膜为100 - 200微米厚的带用剃刀，并受pH值移固定。 </li><li>在20 pH 6.0的固定在4％低聚甲醛视网膜条在0.1M PB - 30分钟，然后在4％多聚甲醛加0.01％戊二醛在10 pH值10.5 - 在RT 20分钟。 </li><li>在PB中几次洗涤用0.15毫氯化钙 （pH为7.4，在4℃）后，加入（每次60分钟，在10％，20％，30％，然后在30％的O / N）的cryoprotect与甘油视网膜带M在0.1 PB冻结取代前。 </li></ol> 2.冻结替代注:此冷冻替代法从早期公布的协议19,20修改。此外，它是至关重要的工具是非常冷（戴手套） Øtherwise，当与文书触摸的组织可以部分解冻。所有这些步骤都在AFS室中完成，该仪器是决不允许移动腔室的边缘的上方。同样地，在AFS中使用的所有化学品的适当的冷却是必要的。 <ol><li>切入冻结在冷冻保存EM仪（CPC）的液态丙烷视网膜条在-184℃。用细刷，放置在小片双面胶带附着到去推插杆的端部的金属短截线的样品（使用刷子灯芯关闭额外缓冲液）。 </li><li>扎杆进入液体丙烷，并保持在那里大约5秒，然后转移到使用填充有液氮的小传输室中的自动冷冻替代仪（AFS）（&#39;LN 2冷却冷冻传送容器&#39;）。 </li><li>将冷冻的样品和仪器（镊子和手术刀）进入AFS后，冷却仪器在室本身veral分钟触摸组织，或通过将仪器的提示几秒钟到小型运输腔冷却之前（充满液氮（"LN 2冷却冷冻集装箱转移&#39;））。 </li><li>一旦在AFS，用细手术刀去除存根样品和磁带。另外，保持氮气流控制，TF（TF被描述为"调节器控制为LN 2汽化器&#39;）在这些过程中打开。 </li><li>之前放置冷冻组织成平嵌入持有者（即，设置在AFS之前），切细圆由透明塑料片材，将其放入保持器的底部，以便行的每个孔的底部。这使得相对容易去除聚合标本块完成时。 注意:在此之前，我们使用的另一种方法的扁平包埋持有者，并采用双赖克特+明胶胶囊（见佩特拉里亚和Wenthold，1999）&lt;SUP&gt; 20。 </li><li>使用在AFS（使用仪器术语）以下自动序列:T 1 = -90℃，32小时，S1 =增加温度由4℃/小时（11.3小时），T 2 = -45℃，50小时，S2 =增加温度由5℃/小时（9小时），T3 = 0℃下40小时（总共= 142.3小时）。它可能需要2 - 4小时放置在AFS样品（在-90℃）开始的定时序列之前。 </li><li>沉浸在甲醇1.5％乙酸双氧铀的冷冻切片在-90℃下，在与AFS&gt; 32小时。将组织（典型值）在每一个七口井在平嵌入铝制支架的两片（三级融入AFS）。 <ol><li>放置组织+磁带入孔中的乙酸双氧铀/甲醇和后来除去胶带，只是之前开始包埋介质（如Lowicryl HM20）渗透，如果太难以从磁带除去组织。 </li></ol></li><li>然后增加温度逐步提高至-45°C（+ 4°每个小时Ç;在自动序列）。 </li><li>洗样品三次在预冷的甲醇，通过使用细尖塑料吸管除去从每个平嵌入支架旧溶液，然后使用另一标准塑料吸管添加预冷新鲜甲醇。 </li><li> 1和2:然后，用同样的方法，用低温包埋树脂，例如在1包埋介质（HM20 /甲醇逐渐渗入试样1，每2小时，接着纯树脂2小时，然后改变树脂并保持O / N）。 </li><li>第二天再次改变树脂，调整树脂的水平，以达到刚好至孔的顶部边缘。 </li><li>聚合样品（-45℃至0℃下在自动序列;每小时+ 5℃）用UV光40小时。 </li><li>然后从AFS样本。通常情况下，样本块仍然显示的颜色一定的粉红色。放置靠近的荧光在化学通风橱的样品中，在室温O / N或更长，直到它们的应用耳朵完全清楚。 </li></ol> 3. Postembedding EM免疫免疫细胞化学如上所述23,24,25进行Postembedding免疫组化:注意 。 <ol><li>切1微米的部分具有超薄，染色切片用1％甲苯胺蓝，检查是否有部分定位;东方的组织块，实现切片的最佳横向平面。 </li><li>削减70纳米厚的超薄切片与超薄和收集他们对涂层的Formvar碳镍槽网格。 </li><li>洗涤栅格在蒸馏水2 O一次后跟的Tris缓冲盐水三次（TBS，0.05M的Tris缓冲液，0.7％氯化钠，pH值7.6）洗涤。 </li><li>孵育在TBS中的5％牛血清白蛋白的20微升液滴栅格30分钟，然后在含有山羊抗CTB的混合物的20微升液滴（1:3,000）和抗体的一个NMDA受体亚基（抗兔GluN2A一点50分，GluN2B 1:30），或抗兔GluA2 / 3（1:30）在TBS-的Triton（TBST，0.01％的Triton X-100，pH值7.6），用2％BSA和0.02M的NaN 3的O / N在室温。 </li><li>在TBS的三个独立的滴（20微升）（pH为7.6）为10，10和20分钟的洗涤网格，随后的TBS（pH为8.2）中5分钟。 </li><li>孵育网格包含耦合到耦合到在TBST 18nm的金颗粒为10nm的金颗粒和驴抗山羊IgG（1:20）驴抗兔IgG（1:20）的混合物中的上滴2小时（20微升） （pH值8.2），用2％BSA和0.02M的NaN 3的。 </li><li>洗网格在TBS的20微升液滴（pH为7.6）为5,5，和10分钟，然后在超纯水中，然后干燥。 </li><li>染液电网用5％的醋酸铀和0.3％的柠檬酸铅蒸馏水2 O分别为黑暗条件下8和5分钟。 </li><li>为三重标记实验，孵化网格O / N在室温用抗山羊CTB的混合物20微升滴（1:3000），抗小鼠的PSD-95（1:100），和抗 - 兔GluN2A（1 :50）。然后孵育电网为2 2小时0的IgG耦合至18，10和5纳米的金颗粒在TBST（pH值8.2），用2％BSA和0.02M的NaN 3的混合物的微升液滴。保持相同的程序作为双标记用于洗涤和之间和抗体孵育后复染。 </li><li>进行控制，其中二次抗体单独施用。 </li><li>在一个EM查看网格和数字化图像。处理在Adobe Photoshop 6.0 ​​最后数字仅用于亮度和对比度如果需要24。 </li></ol> 4.量化<ol><li>手动将内网层（IPL），而不在8,000X放大小孔或裂缝选择区域，然后随机蒙太奇IPL在25,000X放大倍率的全部深度。 </li><li>确定RGC树突在锥双极二人组合时，他们一）含有逆向转移CTB信号（大的金颗粒），B）表现出明确的膜裂，和突触后密度，C）至少包含两个小部分黄金PSD内icles，或沿突触外质膜多个小的金颗粒。 </li><li>收集45 - 55 RGC树突，根据上述条件，进行定量分析。 </li><li>测量PSD和个人RGC树突与美国国立卫生研究院的ImageJ软件的突触外质膜的长度。该膜作为10nm内计数的金颗粒膜结合的基础上，7的平均厚度- 9纳米为质膜26。 </li><li>计算颗粒密度为每线性微米（金/微米）的金颗粒的数量。测量每个金颗粒的中心，并在PSD的中间（突触位置）或PSD的边缘（对于突触外位置）之间的距离。 </li><li>通过软件进行统计分析。使用双尾t检验来比较的手段。总结意义时，P &lt;0.05。除非另有说明，报告值作为平均±SEM 18。 </li></ol>

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	<li>Copenhagen, D. R., Jahr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+endogenous+excitatory+amino+acids+from+turtle+photoreceptors.">Release of endogenous excitatory amino acids from turtle photoreceptors.</a> Nature. 341, 536-539 (1989).</li><li>W&#228;ssle, H., Boycott, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+architecture+of+the+mammalian+retina.">Functional architecture of the mammalian retina.</a> Physiol. Rev. 71, 447-480 (1991).</li><li>Mittman, S., Taylor, W. R., Copenhagen, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concomitant+activation+of+two+types+of+glutamate+receptor+mediates+excitation+of+salamander+retinal+ganglion+cells.">Concomitant activation of two types of glutamate receptor mediates excitation of salamander retinal ganglion cells.</a> J. Physiol. 428, 175-197 (1990).</li><li>Matsui, K., Hosoi, N., Tachibana, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitatory+synaptic+transmission+in+the+inner+retina:+paired+recordings+of+bipolar+cells+and+neurons+of+the+ganglion+cell+layer.">Excitatory synaptic transmission in the inner retina: paired recordings of bipolar cells and neurons of the ganglion cell layer.</a> J. Neurosci. 18, 4500-4510 (1998).</li><li>Chen, S., Diamond, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptically+released+glutamate+activates+extrasynaptic+NMDA+receptors+on+cells+in+the+ganglion+cell+layer+of+rat+retina.">Synaptically released glutamate activates extrasynaptic NMDA receptors on cells in the ganglion cell layer of rat retina.</a> J. Neurosci. 22, 2165-2173 (2002).</li><li>Diamond, J. S., Copenhagen, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+NMDA+and+non-NMDA+receptors+to+the+light-evoked+input-output+characteristics+of+retinal+ganglion+cells.">The contribution of NMDA and non-NMDA receptors to the light-evoked input-output characteristics of retinal ganglion cells.</a> Neuron. 11, 725-738 (1993).</li><li>Lukasiewicz, P. D., Wilson, J. A., Lawrence, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA-preferring+receptors+mediate+excitatory+synaptic+inputs+to+retinal+ganglion+cells.">AMPA-preferring receptors mediate excitatory synaptic inputs to retinal ganglion cells.</a> J. Neurophysiol. 77, 57-64 (1997).</li><li>Higgs, M. H., Lukasiewicz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+uptake+limits+synaptic+excitation+of+retinal+ganglion+cells.">Glutamate uptake limits synaptic excitation of retinal ganglion cells.</a> J. Neurosci. 19, 3691-3700 (1999).</li><li>Taylor, W. R., Chen, E., Copenhagen, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+spontaneous+excitatory+synaptic+currents+in+salamander+retinal+ganglion+cells.">Characterization of spontaneous excitatory synaptic currents in salamander retinal ganglion cells.</a> J. Physiol. 486, 207-221 (1995).</li><li>Kennedy, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+of+PDZ+(DHR,+GLGF)+domains.">Origin of PDZ (DHR, GLGF) domains.</a> Trends. Biochem. Sci. 20, 350 (1995).</li><li>Kim, E., Sheng, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDZ+domain+proteins+of+synapses.">PDZ domain proteins of synapses.</a> Nat. Rev. Neurosci. 5, 771-781 (2004).</li><li>Migaud, M., 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=Enhanced+long-term+potentiation+and+impaired+learning+in+mice+with+mutant+postsynaptic+density-95+protein.">Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein.</a> Nature. 396, 433-439 (1998).</li><li>Aoki, C., 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=Electron+microscopic+immunocytochemical+detection+of+PSD-95,+PSD-93,+SAP-102,+and+SAP-97+at+postsynaptic,+presynaptic,+and+nonsynaptic+sites+of+adult+and+neonatal+rat+visual+cortex.">Electron microscopic immunocytochemical detection of PSD-95, PSD-93, SAP-102, and SAP-97 at postsynaptic, presynaptic, and nonsynaptic sites of adult and neonatal rat visual cortex.</a> Synapse. 40, 239-257 (2001).</li><li>Davies, C., Tingley, D., Kachar, B., Wenthold, R. J., Petralia, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+members+of+the+PSD-95+family+of+MAGUK+proteins+at+the+synaptic+region+of+inner+and+outer+hair+cells+of+the+guinea+pig+cochlea.">Distribution of members of the PSD-95 family of MAGUK proteins at the synaptic region of inner and outer hair cells of the guinea pig cochlea.</a> Synapse. 40, 258-268 (2001).</li><li>Hartveit, E., Brandst&#228;tter, J. H., Sasso&#232;-Pognetto, M., Laurie, D. J., Seeburg, P. H., W&#228;ssle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+and+developmental+expression+of+the+NMDA+receptor+subunit+NR2A+in+the+mammalian+retina.">Localization and developmental expression of the NMDA receptor subunit NR2A in the mammalian retina.</a> J. Comp. Neurol. 348, 570-582 (1994).</li><li>Fletcher, E. L., Hack, I., Brandst&#228;tter, J. H., W&#228;ssle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+localization+of+NMDA+receptor+subunits+in+the+rat+retina.">Synaptic localization of NMDA receptor subunits in the rat retina.</a> J. Comp. Neurol. 420, 98-112 (2000).</li><li>Pourcho, R. G., Qin, P., Goebel, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+subcellular+distribution+of+NMDA+receptor+subunit+NR2B+in+the+retina.">Cellular and subcellular distribution of NMDA receptor subunit NR2B in the retina.</a> J. Comp. Neurol. 433, 75-85 (2001).</li><li>Ottersen, O. P., Landsend, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+glutamate+receptors+at+the+synapse.">Organization of glutamate receptors at the synapse.</a> Eur. J. Neurosci. 9, 2219-2224 (1997).</li><li>Petralia, R. S., Wenthold, R. J., Ariano, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+receptor+antibodies:+Production+and+immunocytochemistry.">Glutamate receptor antibodies: Production and immunocytochemistry.</a> Receptor Localization: Laboratory Methods and Procedures. , 46-74 (1998).</li><li>Petralia, R. S., Wenthold, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemistry+of+NMDA+receptors.">Immunocytochemistry of NMDA receptors.</a> Methods. Mol. Biol. 128, 73-92 (1999).</li><li>Nusser, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+and+NMDA+receptors:+similarities+and+differences+in+their+synaptic+distribution.">AMPA and NMDA receptors: similarities and differences in their synaptic distribution.</a> Curr. Opin. Neurobiol. 10, 337-341 (2000).</li><li>Qin, P., Pourcho, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+AMPA-selective+glutamate+receptor+subunits+in+the+cat+retina:+a+light-+and+electron-microscopic+study.">Localization of AMPA-selective glutamate receptor subunits in the cat retina: a light- and electron-microscopic study.</a> Vis. Neurosci. 16, 169-177 (1999).</li><li>Zhang, J., Wang, H. H., Yang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+organization+of+GABAergic+amacrine+cells+in+salamander+retina.">Synaptic organization of GABAergic amacrine cells in salamander retina.</a> Visual. Neurosci. 21, 817-825 (2004).</li><li>Zhang, J., Diamond, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+perisynaptic+and+synaptic+localization+of+NMDA+and+AMPA+receptors+on+ganglion+cells+in+rat+retina.">Distinct perisynaptic and synaptic localization of NMDA and AMPA receptors on ganglion cells in rat retina.</a> J. Comp. Neurol. 498, 810-820 (2006).</li><li>Zhang, J., Diamond, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit-+and+Pathway-Specific+Localization+of+NMDA+Receptors+and+Scaffolding+Proteins+at+Ganglion+Cell+Synapses+in+Rat+Retina.">Subunit- and Pathway-Specific Localization of NMDA Receptors and Scaffolding Proteins at Ganglion Cell Synapses in Rat Retina.</a> J. Neurosci. 29, 4274-4286 (2009).</li><li>Peters, A., Palay, S., Webster, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The fine structure of the nervous system: neurons and their supporting cells, 3rd ed. , (1991).</li><li>Baude, A., Nusser, Z., Roberts, J. D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabotropic+glutamate+receptor+(mGluR1)+is+concentrated+at+perisynaptic+membrane+of+neuronal+subpopulations+as+detected+by+immunogold+reaction.">The metabotropic glutamate receptor (mGluR1) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction.</a> Neuron. 11, 771-787 (1993).</li><li>Van Lookeren Campagne, M., Oestreicher, A. B., van der Krift, T. P., Gispen, W. H., Verkleij, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze-substitution+and+Lowicryl+HM20+embedding+of+fixed+rat+brain:+Suitability+for+immunogold+ultrastructural+localization+of+neural+antigens.">Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: Suitability for immunogold ultrastructural localization of neural antigens.</a> J. Hist. Cyt. 39, 1267-1279 (1991).</li><li>Matsubara, A., Laake, J. H., Davanger, S., Usami, S., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+AMPA+receptor+subunits+at+a+glutamate+synapse:+A+quantitative+immunogold+analysis+of+hair+cell+synapses+in+the+rat+organ+of+Corti.">Organization of AMPA receptor subunits at a glutamate synapse: A quantitative immunogold analysis of hair cell synapses in the rat organ of Corti.</a> J. Neurosci. 16, 4457-4467 (1996).</li><li>Petralia, R. S., 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=Organization+of+NMDA+receptors+at+extrasynaptic+locations.">Organization of NMDA receptors at extrasynaptic locations.</a> Neuroscience. 167, 68-87 (2010).</li><li>Merighi, A., Polak, J. M., Priestley, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-embedding+electron+microscopic+immunocytochemistry.">Post-embedding electron microscopic immunocytochemistry.</a> Electron Microscopic Immunocytochemistry: Principles and Practice. , 51-87 (1992).</li><li>Ottersen, O. P., Takumi, Y., Matsubara, A., Landsend, A. S., Laake, J. H., Usami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+organization+of+a+type+of+peripheral+glutamate+synapse:+the+afferent+synapses+of+hair+cells+in+the+inner+ear.">Molecular organization of a type of peripheral glutamate synapse: the afferent synapses of hair cells in the inner ear.</a> Prog. Neurobiol. 54, 127-148 (1998).</li><li>Nusser, Z., Lujan, R., Laube, G., Roberts, J. D., Molnar, E., Somogyi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type+and+pathway+dependence+of+synaptic+AMPA+receptor+number+and+variability+in+the+hippocampus.">Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus.</a> Neuron. 21, 545-559 (1998).</li><li>Takumi, Y., Ramirez-Leon, V., Laake, P., Rinvik, E., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+modes+of+expression+of+AMPA+and+NMDA+receptors+in+hippocampal+synapses.">Different modes of expression of AMPA and NMDA receptors in hippocampal synapses.</a> Nat. Neurosci. 2, 618-624 (1999).</li><li>Grimes, W. N., 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=Complex+inhibitory+microcircuitry+regulates+retinal+signaling+near+visual+threshold.">Complex inhibitory microcircuitry regulates retinal signaling near visual threshold.</a> J. Neurophysiol. 114, 341-353 (2015).</li><li>Sassoe-Pognetto, M., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+ionotropic+glutamate+receptors+at+dendrodendritic+synapses+in+the+rat+olfactory+bulb.">Organization of ionotropic glutamate receptors at dendrodendritic synapses in the rat olfactory bulb.</a> J. Neurosci. 20, 2192-2201 (2000).</li></ol>

The authors have no disclosures.

我们已经描述了四种技术对于成功的定量后包埋免疫EM:1）短和弱固定，2）冷冻取代，3）后包埋免疫染色，以及4）定量。 EM免疫允许在超薄组织切片的特定蛋白质的检测。标记有金颗粒的抗体可以用EM直接可视化。而在检测细胞膜受体的subsynaptic本地化功能强大，EM免疫可以技术上具有挑战性，并要求组织固定和加工方法严谨的优化。 罢工膜的完整性和?...

谷氨酸是在视网膜1中的主要兴奋性神经递质。视网膜神经节细胞（RGC）中，从双极电池 2接收的谷氨酸突触输入，是视网膜的输出神经元发送视觉信息到大脑。生理学研究表明，视网膜神经节细胞突触激励是通过NMDA受体（NMDA受体）和AMPA受体（的AMPARs）3,4,5突触后介导的。虽然在视网膜神经节细胞兴奋性突触后电流（EPSCS）通过的AMPARs NMDA受体和介导视网膜神经节细胞-1,3,5,6,7,8-，自发微型EPSCS（mEPSCs）只呈现一个的AMPARs介导的分量4,5,9。然而，减少摄取谷氨酸自发EPSCS 5透露出一股NMDA受体成分，这表明在研资局树突NMDA受体可能位于兴奋性突触之外。膜相关鸟苷酸激酶（MAGUKs）如PSD-95该集群神经递质受体，包括谷氨酸盐受体和离子通道■在突触部位，也表现出明显的subsynaptic表达模式10,11,12,13,14。 近几十年来，共焦免疫组织化学和预嵌入型电子显微镜（EM）的免疫组化已被用于研究膜受体的表达。虽然共焦免疫染色揭示受体表达的宽图案，其较低的分辨率使得不可能使用来区分的亚细胞位置。在哺乳动物视网膜预嵌入-EM研究表明NMDA受体亚基存在于视锥双极细胞带状突触15,16,17突触后的元件。这是明显的对比，以生理学证据。然而，反应产物的扩散是在预嵌入免疫过氧化物酶方法的公知的构件。因此，这种方法通常不会给统计上可靠的数据和可以排除国产化的区分突触膜与膜突触外18,19,20,21。上另一方面，生理和解剖数据与的AMPARs对视网膜神经节细胞3,5,7,9,22突触定位相一致。因此，谷氨酸受体和MAGUKs在视网膜色带突触本地化不仅对突触后也给perisynaptic或突触外膜隔室。然而，仍然需要在视网膜色带突触这些膜蛋白的高分辨率定量分析。 在这里，我们开发了一种postembedding EM免疫技术检测​​NMDA受体亚基，AMPAR亚基和PSD-95接着在突触到大鼠视网膜神经节细胞估算这些蛋白的数量，密度和可变性的subsynaptic定位用霍乱毒素B亚基标记的（CTB）逆行追踪方法。

<table><tbody><tr><td>Paraformaldehyde</td><td>EMS</td><td>15710</td><td></td></tr><tr><td>Glutaraldehyde</td><td>EMS</td><td>16019</td><td></td></tr><tr><td>NaH2PO4</td><td>Sigma</td><td>S9638</td><td></td></tr><tr><td>Na2HPO4</td><td>Sigma</td><td>7782-85-6</td><td></td></tr><tr><td>CaCl2</td><td>Sigma</td><td>C-8106</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A-7030</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T-8787</td><td></td></tr><tr><td>NaOH</td><td>Sigma</td><td>221465</td><td></td></tr><tr><td>NaN3</td><td>JT Baker</td><td>V015-05</td><td></td></tr><tr><td>Glycerol</td><td>Gibco BRL</td><td>15514-011</td><td></td></tr><tr><td>Lowicryl HM 20</td><td>Polysciences</td><td>15924-1</td><td></td></tr><tr><td>Tris-Base</td><td>Fisher</td><td>BP151-500</td><td></td></tr><tr><td>Tris</td><td>Fisher</td><td>04997-100</td><td></td></tr><tr><td>Anti-GluN2A</td><td>Millipore</td><td>AB1555P</td><td>Dilution 1/50</td></tr><tr><td>Anti-GluN2B</td><td>Millipore</td><td>AB1557P</td><td>Dilution 1/30</td></tr><tr><td>Anti-GluA2/3</td><td>Millipore</td><td>AB1506</td><td>Dilution 1/30</td></tr><tr><td>Anti-PSD-95</td><td>Millipore</td><td>MA1&amp;ndash;046</td><td>Dilution 1/100</td></tr><tr><td>Donkey anti-rabbit IgG-10 nm gold particles</td><td>EMS</td><td>25704</td><td>Dilution 1/20</td></tr><tr><td>Donkey anti-mouse IgG-10 nm gold particles</td><td>EMS</td><td>25814</td><td>Dilution 1/20</td></tr><tr><td>Donkey anti-mouse IgG-5 nm gold particles</td><td>EMS</td><td>25812</td><td>Dilution 1/20</td></tr><tr><td>Donkey anti-goat IgG-18 nm gold particles</td><td>Jackson ImmunoResearch</td><td>705-215-147</td><td>Dilution 1/20</td></tr><tr><td>Formvar-Carbon coated nickel-slot grids.</td><td>EMS</td><td>FCF2010-Ni</td><td></td></tr><tr><td>Uranyl acetate</td><td>EMS</td><td>22400-1</td><td></td></tr><tr><td>Methanol</td><td>EMS</td><td>67-56-1</td><td></td></tr><tr><td>Lead citrate</td><td>Leica</td><td></td><td></td></tr><tr><td>Leica EM AFS</td><td>Leica</td><td></td><td></td></tr><tr><td>Leica EM CPC</td><td>Leica</td><td></td><td></td></tr><tr><td>Ultramicrotome</td><td>Leica</td><td></td><td></td></tr><tr><td>JEOL 1200 EM</td><td>JEOL</td><td></td><td></td></tr><tr><td>liquid nitrogen&amp;nbsp;</td><td>Roberts Oxygen</td><td></td><td></td></tr><tr><td>Propane</td><td>Roberts Oxygen</td><td></td><td></td></tr><tr><td>CTB</td><td>List Biological Laboratories</td><td>104</td><td>1 - 1.2%</td></tr><tr><td>Anti-CTB</td><td>List Biological Laboratories</td><td>703</td><td>Dilution 1/4,000</td></tr></tbody></table>

high-resolution quantitative immunogold analysis of membrane 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.

此处呈现的结果表明对大鼠视网膜RGC树突GluA 2/3的显着的不同subsynaptic本地化模式和NMDA受体，如先前24,25说明。在RGC树突型材GluA 2/3免疫颗粒的77％分别位于所述PSD（图1A）内，类似于大多数中央突触。然而，NMDA受体都位于突触两种或extrasynaptically。 GluN2A免疫颗粒的83％在PSD分别定位（图1C，2A，2B），优先在关突触，而金颗粒更?...

Watch this Scientific Journal Video about High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses at JoVE.com

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

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.

高分辨率定量免疫膜受体在视网膜带状突触分析

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

high-resolution-quantitative-immunogold-analysis-membrane-receptors

Research

JoVE Journal

Neuroscience

7.5K 次观看.  National Institutes of Health. 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.

视频: High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

突触量化：基于一个免疫细胞化学检测，以量化突触数量

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

科研

神经科学

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

TIRF显微镜视网膜双极细胞的影像胞外分泌

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

生物学

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

嵌入后突触蛋白的免疫标记海马切片培养

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

在Wholemount大鼠视网膜免疫组化和钙成像方法

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

结合光遗传学和冷冻断裂免疫标记副本研究在老鼠杏仁核谷氨酸受体的特定投入安排

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

成像人类免疫学突触

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

免疫与感染

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 ...

<meta charset="utf8"></head><body>TEM 视网膜类器官样品制备的优化方案

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.

<meta charset="utf8"></head><body>作者聚焦:使用 TEM 分析成熟视网膜类器官的突触超微结构

This protocol provides an optimized and elaborate preparation procedure for retinal organoid samples for transmission electron microscopy. It is suitable for applications that involve the analysis of synapses in mature retinal organoids.

制备用于透射电子显微镜的视网膜类器官样品

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem ...

高分辨率定量免疫膜受体在视网膜带状突触分析

摘要

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此视频中的章节

突触量化：基于一个免疫细胞化学检测，以量化突触数量

TIRF显微镜视网膜双极细胞的影像胞外分泌

嵌入后突触蛋白的免疫标记海马切片培养

在Wholemount大鼠视网膜免疫组化和钙成像方法

结合光遗传学和冷冻断裂免疫标记副本研究在老鼠杏仁核谷氨酸受体的特定投入安排

成像人类免疫学突触

视网膜平片准备成像和免疫组化优化方案

制备用于透射电子显微镜的视网膜类器官样品

制备用于透射电子显微镜的视网膜类器官样品

制备用于透射电子显微镜的视网膜类器官样品