作者要感谢马修佛罗伦萨准备的海马切片培养。这项工作是由美国国家老龄问题研究所和阿尔茨海默氏症协会NZG补助。

1。固定术固定剂是致癌物质，戴手套，在通风橱中的固定剂处理。除非另有说明，所有的孵化做冰，所有的解决方案在使用前，应进行过滤。使用电子显微镜级试剂。 第1天<ol><li class="jove_content">后的实验条件下（ 例如，病毒注射剂，药物治疗），将膜与器官型海马切片，在60×15mm的聚苯乙烯培养皿中含有冰冷的0.1M的磷酸盐缓冲液（索伦森的磷酸盐缓冲液），pH值为7.3。加入1 - 2毫升0.1M磷酸盐缓冲液直接在顶部的切片，以保持它们的冷。 </li></ol> 关键的步骤： 请务必使用新鲜配制的缓冲和固定剂。确保的缓冲液的pH值是在所需的范围之内。一个不这样做可能会破坏细胞结构。 <ol start="2"><li>片CA1区的隔离，使用一次性解剖刀轻轻横切旁边切片到DG，CA1区细胞层（ 图1）平行。然后切剩下的片垂直删除CA3区和下托。切的切片，以帮助识别组织的顶表面上的一个角落。 </li></ol> 注：在没有病毒交付兴趣蛋白质的情况下是必需的，可以是固定的整个组织切片，用温和的冰冷的缓冲液冲洗后，除去介质。然后这之后是通过切出的CA1。 关键的步骤： 跟踪的上部的组织，以适当的网格切片上。 <ol start="3"><li>小心地取出的组织，从膜中使用的背面侧的手术刀。使用巴斯德吸液管轻轻地组织转移到含12孔板的0.1M磷酸盐缓冲液。 </li><li>在井中取出缓冲从板，并添加500米ü;升 - 1毫升冰冷的固定剂（pH 7.3）的组成如下：0.1％苦味酸，1％多聚甲醛和2.5％戊二醛在0.1M磷酸盐缓冲液。孵育2小时，在4℃下</li></ol> 注：苦味酸干燥时可能会爆炸。保持湿润​​密闭容器中的水。应贮存在干燥，通风良好的地方。 <ol start="5"><li>移除固定液从井和洗涤样品在0.1M磷酸盐缓冲液中的3次（每次20分钟）。 </li><li>孵育40分钟，在1％的单宁酸（重量/体积），在0.1M马来酸缓冲液，pH6.0。 </li><li>马来酸盐缓冲液中冲洗两次（每次20分钟）。 </li><li>在1％乙酸双氧铀（重量/体积）在黑暗中马来酸盐缓冲液中孵育40分钟。醋酸铀对光很敏感，是放射性的。量用Parafilm的烧杯中溶解时，在4℃下存放任何未使用的缓冲液在黑暗</li><li>马来酸盐缓冲液中冲洗两次（每次20分钟）。 </li><li>孵育20分钟，在0.5％铂氯碱IDE（重量/体积）马来酸盐缓冲液中。 </li><li>马来酸盐缓冲液中冲洗两次（每次20分钟）。将样品储存在4℃下直到他们准备好以供进一步处理。 </li></ol> 2。脱水和嵌入环氧丙烷是致癌物质。避免蒸气，在通风橱中工作。使用绝对的，100％纯乙醇，不包含任何水迹。这绝对乙醇稀释到不同浓度的脱水。 第2天<ol><li>孵育5分钟，在50％乙醇中，然后在70％乙醇中的5分钟。 </li><li>在新鲜制备的1％，在70％乙醇中的对苯二胺（PPD）孵育15分钟。 </li><li>在70％乙醇中冲洗三次。 </li><li>孵育5分钟，在80％乙醇中，然后在95％乙醇中的5分钟。 </li><li>在100％的乙醇中温育两次每次5分钟。 </li><li>准备带螺丝帽的玻璃小瓶，并确保它们是干净和完全干燥。将切片，玻璃瓶和简单而清楚地标示每小瓶。 </li><li>孵育5分钟，在1:1的乙醇：氧化丙烯。 </li><li>孵育在100％氧化丙烯两次每次5分钟。 </li><li>加入树脂（EPON），每个管形瓶中，使与环氧丙烷的1:1混合物。轻轻搅拌2小时。不要摇晃瓶太严格，以防止泡沫，可能会干扰嵌入。 </li><li>将树脂以3:1的混合物与环氧丙烷，和混合2小时。 </li><li>转移到100％的树脂，并培育O / N。 </li></ol>第3天<ol start="12"><li>夹层之间的采样条状的的ACLAR塑料和固化24小时在60℃下</li></ol> 3。切割和安装在网格第4天<ol><li>剪切半薄（0.5微米）的部分，和染色，用1％甲苯胺蓝+ 1％硼砂，以确定样本的正确方向。 </li><li>切割超薄切片（60纳米）和安装在镍网格，每个网格的一个部分。 </li></ol>4。免疫组化所有的缓冲液和水在使用前，应进行过滤。 第5天<ol><li>将下降（〜50微升）的1％Tween-20/phosphate缓冲液（T / PB），pH为7.5，在平坦的表面上一块干净的封口膜。用干净的镊子轻轻拿起电网的边缘，浮动缓冲区，节下来，并在室温下孵育10分钟。 </li><li>沥干多余的液体从电网通过载置部侧上的滤纸，然后浮于50mM甘氨酸在T / PB 15分钟，在RT。 </li></ol> 关键步骤：为了避免过度的“进位”在孵育期间溶液液滴从一个到下一个的解决方案中，排出多余的液体通过轻轻触摸网格的边缘到＃1滤纸每个解决方案之间变化。同时删除任何解决方案被困在两臂之间的EM钳轻轻一小块滤纸之间的钳子武器排汗电网，同时可确保响的部分不完全干燥。 <ol start="3"><li>用滤纸擦干电网，然后漂浮在封闭液含2.5％BSA和2.5％血清的30分钟，在RT下的二次抗体在T / PB从该动物。 </li><li>孵育在4℃下与第一抗体（在T / PB）在RT或O / N的最佳时间和保温温度，以及抗体的浓度，需要实验确定。 </li><li>网格洗净3次（每次2分钟）与T / PB。 </li><li>在RT下的1小时的二次抗体（1:20抗兔或抗小鼠耦合到10 nm的金）孵育。 </li><li> T / PB洗3次（每次2分钟）。 </li><li> Postfix和2％戊二醛在T / PB 5分钟。 </li><li> T / PB，然后三次（2分钟），用过滤水清洗3次（每次2分钟）。 </li><li>孵育在水中10分钟，用2％乙酸双氧铀。 </li></ol>而电网染色，准备一个CO 2室placin嘎一块石蜡膜的玻璃陪替氏培养皿的中心。然后放置在盘4-6的NaOH粒料周围封口膜在空气中的CO 2的吸收。保持顶部封闭了几分钟。使用巴斯德吸管，并迅速转移雷诺的柠檬酸铅溶液的封口膜在玻璃陪替氏培养皿中的一个小体积的。打开顶盖，就足以插入巴斯德吸管，以减少CO 2的重新引入到室内。 注：为了使雷诺的柠檬酸铅溶液，煮沸100毫升去离子水在微波炉中，在一个密闭的容器中，放凉。在与止动件50毫升容量瓶中，混合1.33克硝酸铅，1.76克柠檬酸钠和30毫升的开水剧烈振摇1分钟。然后摇间歇30分钟。这浑浊的溶液中，加入8毫升的1 M氢氧化钠烧瓶中，慢慢地倒置了几次。解决方案将变得清晰起来。使溶液至50毫升与煮娃之三。紧紧密封存储解决方案。如果沉淀物出现，丢弃，使一个新的。 <ol start="11"><li>在三个小烧杯准备温暖，刚煮沸的去离子水。洗掉的醋酸铀浸在电网中的第一个烧杯中，轻轻摇动30秒左右。重复此步骤，在其他两个烧杯中。 </li><li>打开的玻璃陪替氏培养皿的顶部刚好够放置在网格上的下拉雷诺柠檬酸铅溶液。如果可能的话，要戴上口罩，而这样做是为了避免呼吸柠檬酸铅和防止沉淀物的形成。孵育10分钟。 </li><li>打开顶盖，就足以消除电网。避免吸入柠檬酸铅。三次浸渍，依次在三个烧杯用温水，刚煮沸的蒸馏水洗净电网。在一张滤纸，条达，让电网干燥的。现在已准备好用于电子显微镜的样品。 </li></ol>

<ol>
	<li>Faulk, W. P., Taylor, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immunocolloid+method+for+the+electron+microscope.">An immunocolloid method for the electron microscope.</a> Immunochemistry. 8, 1081-1083 (1971).</li><li>Stirling, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immuno-+and+affinity+probes+for+electron+microscopy:+a+review+of+labeling+and+preparation+techniques.">Immuno- and affinity probes for electron microscopy: a review of labeling and preparation techniques.</a> J. Histochem. Cytochem. 38, 145-157 (1990).</li><li>Phend, K. D., Weinberg, R. J., Rustioni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+to+optimize+post-embedding+single+and+double+staining+for+amino+acid+neurotransmitters.">Techniques to optimize post-embedding single and double staining for amino acid neurotransmitters.</a> Journal of Histochemistry &amp; Cytochemistry. 40, 1011-1020 (1992).</li><li>Berryman, 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=Effects+of+tannic+acid+on+antigenicity+and+membrane+contrast+in+ultrastructural+immunocytochemistry.">Effects of tannic acid on antigenicity and membrane contrast in ultrastructural immunocytochemistry.</a> J. Histochem. Cytochem. 40, 845-857 (1992).</li><li>Akagi, T., 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=Improved+methods+for+ultracryotomy+of+CNS+tissue+for+ultrastructural+and+immunogold+analyses.">Improved methods for ultracryotomy of CNS tissue for ultrastructural and immunogold analyses.</a> J. Neurosci. Methods. 153, 276-282 (2006).</li><li>Stirling, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructual+localization+of+lysozyme+in+human+colon+eosinophils+using+the+protein+A-gold+technique:+effects+of+processing+on+probe+distribution.">Ultrastructual localization of lysozyme in human colon eosinophils using the protein A-gold technique: effects of processing on probe distribution.</a> J. Histochem. Cytochem. 37, 709-714 (1989).</li><li>Phend, K. D., Rustioni, A., Weinberg, 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=An+osmium-free+method+of+epon+embedment+that+preserves+both+ultrastructure+and+antigenicity+for+post-embedding+immunocytochemistry.">An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry.</a> Journal of Histochemistry &amp; Cytochemistry. 43, 283-292 (1995).</li><li>Zhong, L., Cherry, T., Bies, C. E., Florence, M. A., Gerges, N. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogranin+enhances+synaptic+strength+through+its+interaction+with+calmodulin.">Neurogranin enhances synaptic strength through its interaction with calmodulin.</a> Embo J. 28, 3027-3039 (2009).</li><li>Zhong, L., Kaleka, K. S., Gerges, N. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogranin+phosphorylation+fine-tunes+long-term+potentiation.">Neurogranin phosphorylation fine-tunes long-term potentiation.</a> Eur. J. Neurosci. 33, 244-250 (2011).</li><li>Zhong, L., Gerges, N. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogranin+targets+calmodulin+and+lowers+the+threshold+for+the+induction+of+long-term+potentiation.">Neurogranin targets calmodulin and lowers the threshold for the induction of long-term potentiation.</a> PloS one. 7, e41275 (2012).</li><li>Horisberger, M., Vauthey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labelling+of+colloidal+gold+with+protein.+A+quantitative+study+using+beta-lactoglobulin.">Labelling of colloidal gold with protein. A quantitative study using beta-lactoglobulin.</a> Histochemistry. 80, 13-18 (1984).</li><li>Bendayan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+potential+and+versatility+of+colloidal+gold+cytochemical+labeling+for+molecular+morphology.">A review of the potential and versatility of colloidal gold cytochemical labeling for molecular morphology.</a> Biotech. Histochem. 75, 203-242 (2000).</li><li>Racz, B., Weinberg, 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=Spatial+organization+of+coflin+in+dendritic+spines.">Spatial organization of coflin in dendritic spines.</a> J. Neuroscience. 138 (2), 447-456 (2006).</li><li>Posthuma, G., Slot, J. W., Geuze, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+the+immunogold+technique+in+quantitation+of+a+soluble+protein+in+ultra-thin+sections.">Usefulness of the immunogold technique in quantitation of a soluble protein in ultra-thin sections.</a> J. Histochem. Cytochem. 35, 405-410 (1987).</li><li>Howell, K. E., Reuter-Carlson, U., Devaney, E., Luzio, J. P., Fuller, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+antigen,+one+gold?+A+quantitative+analysis+of+immunogold+labeling+of+plasma+membrane+5'-nucleotidase+in+frozen+thin+sections.">One antigen, one gold? A quantitative analysis of immunogold labeling of plasma membrane 5'-nucleotidase in frozen thin sections.</a> Eur. J. Cell Biol. 44, 318-327 .</li><li>Yokota, E., Kawaguchi, T., Kusaba, T., Niho, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Immunologic+analysis+of+families+with+complement+receptor+deficiency].">[Immunologic analysis of families with complement receptor deficiency].</a> Nihon. Rinsho. 46, 2040-2045 (1988).</li><li>Kehle, T., Herzog, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+protein-gold+complexes+and+cell+surfaces:+a+method+for+precise+quantitation.">Interactions between protein-gold complexes and cell surfaces: a method for precise quantitation.</a> Eur. J. Cell Biol. 45, 80-87 (1987).</li><li>Bozzola, J. R., Lonnie, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+2.">Ch. 2.</a> Electron Microscopy. 30, (1992).</li><li>Cho, K. O., Hunt, C. A., 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=The+rat+brain+postsynaptic+density+fraction+contains+a+homolog+of+the+Drosophila+discs-large+tumor+suppressor+protein.">The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein.</a> Neuron. 9, 929-942 (1992).</li><li>Hunt, C. A., Schenker, L. J., 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=PSD-95+is+associated+with+the+postsynaptic+density+and+not+with+the+presynaptic+membrane+at+forebrain+synapses.">PSD-95 is associated with the postsynaptic density and not with the presynaptic membrane at forebrain synapses.</a> J. Neurosci. 16, 1380-1388 (1996).</li><li>Gispen, W. H., Leunissen, J. L., Oestreicher, A. B., Verkleij, A. J., Zwiers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+localization+of+B-50+phosphoprotein:+the+(ACTH)-sensitive+protein+kinase+substrate+involved+in+rat+brain+polyphosphoinositide+metabolism.">Presynaptic localization of B-50 phosphoprotein: the (ACTH)-sensitive protein kinase substrate involved in rat brain polyphosphoinositide metabolism.</a> Brain Research. 328, 381-385 (1985).</li><li>Biewenga, J. E., Schrama, L. H., Gispen, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+phosphoprotein+B-50/GAP-43+in+neuronal+and+synaptic+plasticity.">Presynaptic phosphoprotein B-50/GAP-43 in neuronal and synaptic plasticity.</a> Acta Biochim. Pol. 43, 327-338 (1996).</li><li>Maunsbach, A. A., Bjorn, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+16.">Ch. 16.</a> Biomedical Electron Microscopy Illustrated Methods and Interpretations. , 383-426 (1999).</li></ol>

我们什么都没有透露。

在这个协议中，我们采取了Phend和Weinberg方法，对大脑和脊髓组织，研究在大鼠海马脑片培养的树突棘。在海马CA3-CA1区树突棘是脆弱的结构，包含一个庞大的多种蛋白质中发挥重要作用，在调节神经功能。所提出的方法提供了一个平衡的方法，用于实现增强的抗原性，同时保持良好的的超微结构保存（图2A），允许合理的标记效率和良好的再现性，可用于脊柱内的特定抗原的分布特征?...

<table border="1">
 <tbody>
 <tr>
 <td>NAME OF REAGENT</td>
 <td>COMPANY</td>
 <td>CATALOG NUMBER</td>
 <td></td>
 </tr>
 <tr>
 <td>60 x 15 mm polystyrene Petri dish</td>
 <td>Falcon</td>
 <td>351007</td>
 <td></td>
 </tr>
 <tr>
 <td>Disposable scalpel</td>
 <td>EXELINT</td>
 <td>29552</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell culture inserts</td>
 <td>Millipore</td>
 <td>PICM03050</td>
 <td></td>
 </tr>
 <tr>
 <td>10 nm Goat-anti-rabbit gold</td>
 <td>Electron Microscopy Sciences</td>
 <td>25108</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Neurogranin antibody</td>
 <td>Millipore</td>
 <td>AB5620</td>
 <td></td>
 </tr>
 <tr>
 <td>100% Picric acid</td>
 <td>Electron Microscopy Sciences</td>
 <td>19550</td>
 <td></td>
 </tr>
 <tr>
 <td>96% Paraformaldehyde</td>
 <td>Acros Organics</td>
 <td>AC41678-0030</td>
 <td></td>
 </tr>
 <tr>
 <td>25% Glutaraldehyde (EM grade)</td>
 <td>Sigma</td>
 <td>G5882</td>
 <td></td>
 </tr>
 <tr>
 <td>Uranyl acetate</td>
 <td>Electron Microscopy Sciences</td>
 <td>22400</td>
 <td></td>
 </tr>
 <tr>
 <td>p-Phenylenediamine</td>
 <td>Sigma</td>
 <td>P6001</td>
 <td></td>
 </tr>
 <tr>
 <td>Platinum (IV) chloride</td>
 <td>Sigma</td>
 <td>379840</td>
 <td></td>
 </tr>
 <tr>
 <td>Tannic acid</td>
 <td>Electron Microscopy Sciences</td>
 <td>21710</td>
 <td></td>
 </tr>
 </tbody>
</table>

post-embedding immunogold labeling of synaptic proteins in hippocampal slice cultures

在亚细胞水平研究生物分子免疫电镜是一个功能强大的工具。耦合到电子致密如胶体金标记的抗体，可以揭示在各种组织中的特定的抗原1的定位和分布。两个最广泛使用的技术是预嵌入和嵌入后的技术。在预嵌入免疫金电子显微镜（EM）技术中，组织必须透性以允许抗体渗透之前，它被嵌入。这些技术是理想的保护结构，而且渗透力差的抗体（通常只有几微米）是一个很大的缺点2。后嵌入标记方法可以避免这个问题，因为标签上发生的部分固定的组织抗原更容易获得。多年来，多次修改，完善后的嵌入方法，以提高免疫，并保留超微结构<suP&gt; 3-5。 组织固定EM研究的重要组成部分。固定剂的化学交联的大分子锁定的组织结构。固色剂的选择不仅影响结构保存，但也抗原性和对比度。几十年来，四氧化锇（OSO 4），甲醛，和戊二醛已被标准的固定剂，包括中枢神经系统（CNS）的组织，尤其是在化学和物理处理过程中容易产生结构损伤。不幸的是，OSO 4是反应性高的，并已被证明，以掩盖抗原6，从而导致在穷人和不足的标签。替代方法来避免化学固定，包括冻结的组织。但是，这些技术是难以执行，并需要昂贵的仪器。为了解决这些问题中的某些问题，并提高CNS组织标签，Phend 等 。 OSO 4醋酸铀（UA）和单宁的ACI代替D（TA），并成功地引入了额外的修改，以提高抗原检测的敏感性和结构保存在大脑和脊髓组织7。我们都采用了这种锇后埋线法对大鼠脑组织和优化的探测和研究突触蛋白的免疫胶体金标记技术。 我们在座的方法来确定大鼠海马CA1锥体神经元突触蛋白的超微结构定位。我们使用海马切片培养的切片。这些切片保持的trisynaptic的的电路的海马，从而学习突触可塑性是特别有用的，一个机制，人们普遍认为，学习和记忆的基础。如前所述8，可以制备从出生后的第5和6天小鼠/大鼠幼仔器官型海马切片，以及急性击倒或过度表达的外源蛋白是特别有用的。我们以前使用过该协议的通道aracterize的NG（NG），神经元特异性蛋白的关键作用，在调节突触功能8,9。我们还用它来 ​​描述的超微结构定位钙调素（CAM）和Ca 2 + /钙调素依赖性蛋白激酶II（CaMKII）10。所示的结果，该协议允许的树突棘超微结构的保存和高效的标签，吴，，帮助刻画其分布在脊柱8。此外，这里所描述的过程可以有广泛的适用性，在研究参与神经元功能的许多其他蛋白质。

图2B示出的一个例子的内源性伍分子分布在海马CA1区锥体神经元树突棘。与超薄（60 nm）的组织中的海马CA1区域的（如在图2A中可见）的镍网格覆盖在1％T / PB，50mM甘氨酸，然后，用2.5％BSA和2.5％血清阻塞之前，用抗孵育-吴抗体。洗涤后，使用T / PB，网格，然后覆盖在耦合到10nm的金的抗兔第二抗体。最后，网格，用T / PB后缀为2％戊二醛2％醋酸铀和雷诺的柠檬酸铅染色，?...

Watch this Scientific Journal Video about Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures at JoVE.com

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

蛋白质的定位和分布提供了重要的信息，了解他们的细胞功能。可以使用电子显微镜（EM）的优越的空间分辨率，以确定一个给定的抗原后免疫组化的亚细胞定位。对于组织的中枢神经系统（CNS），保持结构完整性的同时保持抗原性已经在EM研究特别困难。在这里，我们采用的程序已经被用来保存在中枢神经系统的研究和定性大鼠海马CA1锥体神经元突触蛋白的结构和抗原。

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 ...

post-embedding-immunogold-labeling-synaptic-proteins-hippocampal

Research

JoVE Journal

Neuroscience

17.0K 次观看.  Medical College of Wisconsin. 蛋白质的定位和分布提供了重要的信息，了解他们的细胞功能。可以使用电子显微镜（EM）的优越的空间分辨率，以确定一个给定的抗原后免疫组化的亚细胞定位。对于组织的中枢神经系统（CNS），保持结构完整性的同时保持抗原性已经在EM研究特别困难。在这里，我们采用的程序已经被用来保存在中枢神经系统的研究和定性大鼠海马CA1锥体神经元突触蛋白的?...

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

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

低聚β-淀粉样蛋白的制备 1-42和突触可塑性损伤诱导海马脑片

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

科研

神经科学

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

海马脑片的器官型检查神经元损伤的文化模式

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

浦肯野细胞树突形态的器官切片培养分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

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

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

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 ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

神经血管重塑的分析Entorhino，海马脑切片培养

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

使用急性海马脑片啮齿动物突触标记/捕获和交叉捕获的研究

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

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.

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 ...

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. ...