Name: quantifying the heterogeneous distribution of a synaptic protein in the mouse brain using immunofluorescence
Uploaded: 2019-01-29T13:00:00
Description: Watch this Scientific Journal Video about Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence at JoVE.com

我们感谢 irmgard weiss 提供的出色技术援助。提交人感谢 hermes pofantis 和 andoniya petkova 的支持。作者还感谢欧洲神经科学研究所使用 lsm800 和技术援助, 特别是 nils halbsteut 博士提供的技术援助。这项工作是由哥廷根大学医学中心资助的。jsv 感谢大脑纳米显微镜和分子生理学中心 (cnmpb) 的支持。

该协议不涉及对活体动物的实验。当地动物保护当局 (Tierschutzkommission der university ätsmedizin göttingen) 根据 t 10/30 号批准了涉及动物安乐死以获得大脑样本的实验。
注: 该方案使用了3例成年雄性 c57bl6 小鼠。
1. 样品制备
<ol><li>制备固定剂和 0.1 m 磷酸盐缓冲液 (pb; 见表 1)。</li>
	<li>修复动物通过经心灌注如 gage 等人所述.28</su...

<ol>
	<li>Wallrafen, R., Dresbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Presynaptic+Protein+Mover+Is+Differentially+Expressed+Across+Brain+Areas+and+Synapse+Types.">The Presynaptic Protein Mover Is Differentially Expressed Across Brain Areas and Synapse Types.</a> Frontiers in Neuroanatomy. 12, 58 (2018).</li><li>Augustin, I., Betz, A., Herrmann, C., Jo, T., Brose, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+two+novel+Munc13+proteins+in+rat+brain.">Differential expression of two novel Munc13 proteins in rat brain.</a> Biochemical Journal. 337, 363-371 (1999).</li><li>Rosenmund, 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=Differential+Control+of+Vesicle+Priming+and+Short-Term+Plasticity+by+Munc13+Isoforms.">Differential Control of Vesicle Priming and Short-Term Plasticity by Munc13 Isoforms.</a> Neuron. 33, 411-424 (2002).</li><li>Breustedt, J., 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=Munc13-2+differentially+affects+hippocampal+synaptic+transmission+and+plasticity.">Munc13-2 differentially affects hippocampal synaptic transmission and plasticity.</a> Cerebral cortex. 20, 1109-1120 (2010).</li><li>Chen, Z., Cooper, B., Kalla, S., Varoqueaux, F., Young, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Munc13+Proteins+Differentially+Regulate+Readily+Releasable+Pool+Dynamics+and+Calcium-Dependent+Recovery+at+a+Central+Synapse.">The Munc13 Proteins Differentially Regulate Readily Releasable Pool Dynamics and Calcium-Dependent Recovery at a Central Synapse.</a> The Journal of Neuroscience. 33, 8336-8351 (2013).</li><li>Taylor, S. C., Berkelman, T., Yadav, G., Hammond, 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+Defined+Methodology+for+Reliable+Quantification+of+Western+Blot+Data.">A Defined Methodology for Reliable Quantification of Western Blot Data.</a> Mol Biotechnol. , (2013).</li><li>Charette, S. J., Lambert, H., Nadeau, P. J., Landry, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+quantification+by+chemiluminescent+Western+blotting+Elimination+of+the+antibody+factor+by+dilution+series+and+calibration+curve.">Protein quantification by chemiluminescent Western blotting Elimination of the antibody factor by dilution series and calibration curve.</a> Journal of Immunological Methods. 353, 148-150 (2010).</li><li>Heidebrecht, F., Heidebrecht, A., Schulz, I., Behrens, S., Bader, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+semiquantitative+Western+blot+technique+with+increased+quantification+range.">Improved semiquantitative Western blot technique with increased quantification range.</a> Journal of Immunological Methods. 345, 40-48 (2009).</li><li>Toki, M. I., Cecchi, F., Hembrough, T., Syrigos, K. N., Rimm, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proof+of+the+quantitative+potential+of+immunofluorescence+by+mass+spectrometry.">Proof of the quantitative potential of immunofluorescence by mass spectrometry.</a> Laboratory Investigation. 97, 329-334 (2017).</li><li>Wilhelm, B. G., 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=Composition+of+isolated+synaptic+boutons+reveals+the+amounts+of+vesicle+trafficking+proteins.">Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins.</a> Science. 344, 1023-1028 (2014).</li><li>Navone, F., 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=Protein+p38:+An+integral+membrane+protein+specific+for+small+vesicles+of+neurons+and+neuroendocrine+cells.">Protein p38: An integral membrane protein specific for small vesicles of neurons and neuroendocrine cells.</a> Journal of Cell Biology. 103, 2511-2527 (1986).</li><li>Gundelfinger, E. D., Reissner, C., Garner, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Bassoon+and+Piccolo+in+Assembly+and+Molecular+Organization+of+the+Active+Zone.">Role of Bassoon and Piccolo in Assembly and Molecular Organization of the Active Zone.</a> Frontiers in Synaptic Neuroscience. 7, (2016).</li><li>Ziegler, D. R., Cullinan, W. E., Herman, J. P. <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+vesicular+glutamate+transporter+mRNA+in+rat+hypothalamus.">Distribution of vesicular glutamate transporter mRNA in rat hypothalamus.</a> Journal of Comparative Neurology. 448, 217-229 (2002).</li><li>Chaudhry, F. A., 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=The+vesicular+GABA+transporter,+VGAT,+localizes+to+synaptic+vesicles+in+sets+of+glycinergic+as+well+as+GABAergic+neurons.">The vesicular GABA transporter, VGAT, localizes to synaptic vesicles in sets of glycinergic as well as GABAergic neurons.</a> Journal of Neuroscience. 18, 9733-9750 (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+SAP-97+at+Postsynaptic,+Presynaptic,+and+Nonsynaptic+Sites+of+Adult+and+Neonatal+Rat+Visual+Cortex.">Electron Microscopic Immunocytochemical SAP-97 at Postsynaptic, Presynaptic, and Nonsynaptic Sites of Adult and Neonatal Rat Visual Cortex.</a> Synapse. 257, 239-257 (2001).</li><li>Valtschanoff, J. G., 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=Expression+of+NR2+Receptor+Subunit+in+Rat+Somatic+Sensory+Cortex+:+Synaptic+Distribution+and+Colocalization+With+NR1+and+PSD-95.">Expression of NR2 Receptor Subunit in Rat Somatic Sensory Cortex : Synaptic Distribution and Colocalization With NR1 and PSD-95.</a> Journal of Comparative Neurology. 611, 599-611 (1999).</li><li>Hunt, A., Schenker, L. J., Kennedy, 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> Journal of Neuroscience. 76, 1380-1388 (1996).</li><li>Luscher, B., Fuchs, T., Kilpatrick, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+A+Receptor+Trafficking-Mediated+Plasticity+of+Inhibitory+Synapses.">GABA A Receptor Trafficking-Mediated Plasticity of Inhibitory Synapses.</a> Neuron. 70, 385-409 (2011).</li><li>Kirby, 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> Understanding the molecular diversity of GABAergic synapses. 5, 1-12 (2011).</li><li>Harris, K. M., Weinberg, R. J., Verrall, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+Synapses+in+the+Mammalian+Brain.">Ultrastructure of Synapses in the Mammalian Brain.</a> Cold Spring Harbor Perspectives in Biology. , 1-30 (2012).</li><li>Heise, 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=Selective+Localization+of+Shanks+to+VGLUT1-Positive+Excitatory+Synapses+in+the+Mouse+Hippocampus.">Selective Localization of Shanks to VGLUT1-Positive Excitatory Synapses in the Mouse Hippocampus.</a> Frontiers in Cellular Neuroscience. 10, (2016).</li><li>Peters, P. J., Yuan, L. C., Biology, C., Branch, M. <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+TGN38+to+the+trans-Golgi+network:+involvement+of+a+cytoplasmic+tyrosine-containing+sequence.">Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence.</a> Journal of Cell Biology. 120, 1123-1135 (1993).</li><li>Antonini, D., 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=Tprg,+a+gene+predominantly+expressed+in+skin,+is+a+direct+target+of+the+transcription+factor+p63.">Tprg, a gene predominantly expressed in skin, is a direct target of the transcription factor p63.</a> Journal of Investigative Dermatology. 128, 1676-1685 (2008).</li><li>Burre, J., 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=Synaptic+vesicle+proteins+under+conditions+of+rest+and+activation:+Analysis+by+2-D+difference+gel+electrophoresis.">Synaptic vesicle proteins under conditions of rest and activation: Analysis by 2-D difference gel electrophoresis.</a> Electrophoresis. 27, 3488-3496 (2006).</li><li>Kremer, 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=Mover+is+a+novel+vertebrate-specific+presynaptic+protein+with+differential+distribution+at+subsets+of+CNS+synapses.">Mover is a novel vertebrate-specific presynaptic protein with differential distribution at subsets of CNS synapses.</a> FEBS Letters. 581, 4727-4733 (2007).</li><li>Ahmed, 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=Mover+Is+a+Homomeric+Phospho-Protein+Present+on+Synaptic+Vesicles.">Mover Is a Homomeric Phospho-Protein Present on Synaptic Vesicles.</a> PLoS ONE. 8, (2013).</li><li>K&#246;rber, 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=Modulation+of+Presynaptic+Release+Probability+by+the+Vertebrate-Specific+Protein+Mover.">Modulation of Presynaptic Release Probability by the Vertebrate-Specific Protein Mover.</a> Neuron. 87, 521-533 (2015).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Animal+Perfusion+Fixation+for+Rodents.">Whole Animal Perfusion Fixation for Rodents.</a> Journal of Visualized Experiments. , 1-9 (2012).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9, (2012).</li><li>Schneider Gasser, E. 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=Immunofluorescence+in+brain+sections:+simultaneous+detection+of+presynaptic+and+postsynaptic+proteins+in+identified+neurons.">Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified neurons.</a> Nature Protocols. 1, 1887-1897 (2006).</li><li>Paxinos, G., Franklin, K. B. J. <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 Mouse Brain in Stereotaxic Coordinates. , (2001).</li><li>Sun, Y., Ip, P., Chakrabartty, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+Elimination+of+Background+Fluorescence+in+Formalin-Fixed+Human+Brain+Tissue+for+Immunofluorescence+Microscopy.">Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy.</a> Journal of Visualized Experiments. , 1-6 (2017).</li><li>Poole, A. R., Dingle, J. T., Mallia, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+localization+of+retinol-binding+protein+in+rat+liver+by+immunofluorescence+microscopy.">The localization of retinol-binding protein in rat liver by immunofluorescence microscopy.</a> Journal of Cell Science. 394, 379-394 (1975).</li></ol>

这里提出的方法是量化感兴趣的蛋白质相对于已知分布的标记蛋白的丰度的分布。免疫荧光染色可显示不同切片之间染色强度的高变异性。这里描述的量化方法通过确定整个半球感兴趣的蛋白质与平均值的比率来规避这个问题。因此, 不同切片的染色强度被取消, 并允许定量描述。
与每一个免疫荧光协议一样, 定性或定量, 有几个因素会影响成功, 从而混淆分析。因此, 应特别?...

神经元之间的通信发生在称为突触的特殊接触点。突触包含无数不同的蛋白质, 这些蛋白质协调着突触的传递。其中一些蛋白质在整个神经系统中表现出异质分布, 并不是在每个突触1中都存在。这种蛋白质的一个例子是 munc13, 它参与了突触囊泡的启动过程。munc13 有不同的等形, 它们在整个大脑中分布不均, 而特定等形的存在或不存在会影响短期突触可塑性和突触囊动力学3,4 个,5. 因此, 能够识别大脑各区域存在不同的突触蛋白至关重要。
突触蛋白的定量选择方法--到目前为止--是质谱和西方印迹, 而不是免疫组织化学 6,7,8,9。在某些情况下, 有几种方法相互补充, 以评估特定蛋白质的数量和定位 (即wilhelm等人).10). 我们在这里描述的方法允许对感兴趣的蛋白质进行定位和定量, 而无需使用任何生化方法, 只需使用免疫荧光污渍。这里的另一个优点是, 量化可以在比其他方法实现的更小的区域上进行, 因此也可以更具体。然而, 人们必须考虑到, 需要一种可靠的参考蛋白质来评估感兴趣的蛋白质的分布。
免疫组织化学的荧光染色使我们能够例行识别蛋白质在大脑区域以及不同神经元隔间内的定位。若要标识不同的隔间, 请使用特定标记。通常情况下, 抗突触素和突触素11的抗体可用于标记突触囊泡, 而针对巴松突的抗体标记突触前端子 12的活动区。水泡转运体, 如水泡谷氨酸转运体 (vglot) 或水泡 gaba 转运体 (vgat), 分别用于标记兴奋13 和抑制性 14前突触前端子。在突触后, 针对荷马蛋白的抗体可用于标记突触后端子, 而针对突触后密度蛋白 95 (psd95)15、16、17或 gephyrin18的抗体可用于标记突触后端子。,19,20可分别标记兴奋或抑制突触后端子。通过对感兴趣的蛋白质和标记 (如上面描述的) 使用抗体, 可以确定这种蛋白质的定位。迄今为止, 许多研究都以定性的方式做到了这一点。然而, 要可靠地确定特定突触蛋白的差异分布, 不仅必须确定其存在或缺失, 还必须确定其相对浓度。突触的大小和密度的异质性使得建立突触标记和感兴趣的蛋白质之间的比例变得很重要。否则, 突触丰富的区域, 如海马的非锥体层和小脑的分子层, 将显示突触蛋白的高密度, 这只是由于突触的密度较高, 但不是由于该蛋白的强大存在每个突触 (例如, wallrafen 和 dresbach1)。另一方面, 由于神经元细胞体浓度高, 神经元 soma 中的蛋白质 (例如tgn38 22) 通常会在海马锥体细胞层或海马或小脑颗粒层中显示出强烈的存在在这些地区。因此, 这种结构的非均匀分布, 在这种情况下是突触, 会导致对感兴趣的蛋白质本身的分布的错误估计。此外, 免疫组织化学污渍样品的染色强度存在内在差异。这里描述的协议考虑到了这一点, 避免了这种偏见, 以及免疫组织化学方法引起的其他警告。
在我们最近的研究中, 我们用这种方法来描述 mof (也称为 tprgl23或 sap3024) 在16个不同大脑区域1的差异表达。m接管是一种脊椎动物特异性突触蛋白, 可与突触囊泡有关, 并影响神经递质释放25,26,27.我们有相关的运动表达突触囊的丰富, 通过染色突触素作为突触囊参考标记。我们发现了高水平的 mof, 特别是在隔膜核, 腹侧苍白, 和杏仁核。在海马区内, 我们发现了一个均匀分布的 mof, 与海马内计算相关的层数较高, 在输入和输出层的水平较低。

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			<td height="40" style="height:40px;width:216px;">1.5 mL reaction tubes</td>
			<td style="width:167px;">Eppendorf</td>
			<td align="right" style="width:187px;">30120094</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;">50 mL reaction tubes</td>
			<td>Greiner Bio-One</td>
			<td align="right">227261</td>
			<td></td>
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			<td height="40" style="height:40px;">multiwell 24 well</td>
			<td>Fisher Scientific</td>
			<td colspan="2">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 087721H</td>
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			<td height="40" style="height:40px;">plastic pipette (disposable)</td>
			<td>Sarstedt</td>
			<td align="right">861,176</td>
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			<td height="40" style="height:40px;width:216px;">1000 mL pipette</td>
			<td style="width:167px;">Rainin</td>
			<td style="width:187px;">&#160;17014382</td>
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			<td height="40" style="height:40px;width:216px;">2 ml pipette</td>
			<td style="width:167px;">Eppendorf</td>
			<td align="right">3123000012</td>
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			<td height="40" style="height:40px;">Vortex Genius 3&#160;</td>
			<td>IKA</td>
			<td align="right">3340001</td>
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			<td height="40" style="height:40px;">Menzel microscope slides</td>
			<td>Fisher Scientific</td>
			<td colspan="2">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 10144633CF</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Stereoscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">LSM800</td>
			<td>Zeiss</td>
			<td></td>
			<td>Confocal microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">freezing microtome</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">PBS (10X)</td>
			<td style="width:167px;">Roche</td>
			<td style="width:187px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 11666789001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PFA</td>
			<td>Sigma</td>
			<td colspan="2">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; P6148-1kg</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NaCl</td>
			<td>BioFroxx</td>
			<td>1394KG001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">sucrose</td>
			<td>neoFroxx</td>
			<td>1104KG001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue Tek</td>
			<td>Sakura&#160;</td>
			<td align="right">4583</td>
			<td>OCT</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Na2HPO4</td>
			<td>BioFroxx</td>
			<td>5155KG001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NaH2PO4</td>
			<td>Merck</td>
			<td align="right">1,063,460,500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">normal goat serum</td>
			<td>Merck Millipore</td>
			<td>S26-100ML</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">normal donkey serum</td>
			<td>Merck</td>
			<td>S30-100ML</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Triton X-100</td>
			<td>Merck</td>
			<td align="right">1,086,031,000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">rabbit anti-Mover</td>
			<td>Synaptic Systems</td>
			<td>RRID: AB_10804285</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">guinea pig anti-Synaptophysin</td>
			<td>Synaptic Systems</td>
			<td>RRID: AB_1210382</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">donkey anti-rabbit AF647</td>
			<td>Jackson ImmunoResearch</td>
			<td>RRID: AB_2492288</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">goat anti-mouse AF488</td>
			<td>Jackson ImmunoResearch</td>
			<td>RRID: AB_2337438</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Mowiol4-88</td>
			<td style="width:167px;">Calbiochem</td>
			<td style="width:187px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 475904</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">ZEN2 blue software</td>
			<td style="width:167px;">Zeiss</td>
			<td style="width:187px;"></td>
			<td>Microscopy software</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">FIJI</td>
			<td style="width:167px;">ImageJ</td>
			<td style="width:187px;"></td>
			<td>Analysis software</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying the heterogeneous distribution of a synaptic protein in the mouse brain using immunofluorescence

特定突触蛋白的存在、缺失或水平会严重影响突触的传播。除了阐明蛋白质的功能外, 还必须确定其分布。在这里, 我们描述了一个使用免疫荧光、共聚焦显微镜和基于计算机的分析来确定突触蛋白 mof (也称为 tprgl 或 synaptic) 的分布的协议。我们比较了切除物与突触泡蛋白突触素的分布, 从而定量地确定了运动物相对于突触囊泡丰度的分布。值得注意的是, 这种方法有可能被采用, 以便利用不同的抗体或显微镜或在不同的研究中比较蛋白质的分布。我们的方法通过产生比而不是绝对荧光水平来规避免疫荧光污渍的固有变异性。此外, 我们描述的方法使研究人员能够分析蛋白质在不同层次上的分布: 从整个大脑切片到大脑区域, 再到大脑区域的不同次区域, 如海马或感觉的不同层皮层。马剂是一种脊椎动物特有的蛋白质, 与突触囊泡有关。通过这种方法, 我们表明, 蒙特是异质分布在大脑的各个区域, 在腹侧苍白, 隔膜核, 杏仁核的高水平, 也在单个大脑区域, 如海马的不同层。

图 1显示了不同标记的代表性染色模式。模式因蛋白质的分布而异。五个星冠水平的例子在 (a)-(e) 列中显示。第一行显示了具有代表性的 dapi 染色: dapi 粘附于细胞的 dna 上, 因此细胞核被染色。这将导致点状图案。具有高细胞密度的区域比细胞密度较低的区域更明亮。在第二行可以看到异质分布蛋白的一个例子。马弗染色显?...

Watch this Scientific Journal Video about Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence at JoVE.com

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

在这里, 我们描述了一种定量的方法来确定突触蛋白相对于标记蛋白使用免疫荧光染色, 共聚焦显微镜, 和基于计算机的分析。

免疫荧光定量小鼠脑突触蛋白的异种分布

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

在这里，我们描述了一个利用免疫荧光、共体显微镜和基于计算机的分析来确定突触蛋白相对于参考蛋白的分布的协议。我们的方法通过使用比而不是绝对荧光水平来规避免疫荧光染色的固有变异性。此外，通过这种方法，您可以分析不同水平上蛋白质分布的异质性。从整个大脑切片到大脑区域，甚至亚区域，如海马的不同层。这种技术可以适应，以确定在大脑和模型系统以外的不同组织中，除了小鼠的蛋白质分布。首先清洗以前分离的固定小鼠大脑，如手稿中所述。然后将大脑转移到15毫升的反应管与30%蔗糖0.1摩尔PB。低温保护大脑在4摄氏度下孵育48小时或直到它下沉。之后，使用锋利的刀片修剪低温保护的大脑，这取决于感兴趣的区域。然后将大脑放在低温模具中，并添加最佳切割温度化合物，使其嵌入，确保避免气泡并正确定位大脑，以便有一个切削的日冕平面。将低温模具放在零下80摄氏度的冰柜中，直到冷冻固体。将冷冻组织安装在低温微原子上，并等待至少 15 分钟，然后切入，以平衡其与微原子温度。开始将大脑切成25微米厚的日冕片。小心地使用玻璃钩到第一片的 OCT，不要接触脑组织。十进制将坚持在玻璃钩上。使用玻璃钩将大脑切片转移到第一个井中。在充满 0.1 摩尔 PB 的 24 井板中收集每井三个相邻切片。将切片存放在摄氏四度，直到染色长达两周。要准备免疫染色的切片，请使用塑料移液器从一个井中去除PB溶液，而无需在脑片中绘制。然后使用1000微升移液器添加250微升新鲜PB洗涤多余的10月。此时对每一个井重复这种清洗，以避免将切片晾干。然后，使用塑料移液器将 PB 溶液从第一个井中拆下。使用 1000 微升移液器，每井添加 250 微升的阻塞缓冲器，再次很好地工作。在室温下在摇床上孵育板三个小时。在孵育过程中，每井向反应管中加入250微升抗体缓冲液。然后使用2微升移液器将适当数量的抗体直接加入溶液中，然后轻轻地上下移液几次混合。然后涡流这种稀释的抗体不确定正确的混合。通过用塑料移液器去除阻塞缓冲液，每井添加250微升原抗体溶液，工作良好。在四摄氏度的摇床上孵育板。第二天，用塑料移液器去除抗体溶液。在室温下，每井用300微升的洗涤缓冲液清洗切片，每井洗10分钟，每次洗涤。在洗涤过程中，在黑暗中工作，在反应管中稀释氟化物，产生几秒抗体，与以前使用原抗体时相同。洗涤完成后，用塑料移液器取出洗涤液，每井加入250微升的二级抗体溶液。在室温下在黑暗中孵育90分钟。完成孵育后，用塑料移液器去除抗体溶液。用洗涤缓冲液二用洗涤缓冲器二次洗涤三次，与用洗涤缓冲液一相同的方式清洗。在洗涤过程中稀释DAPI污渍在0.1摩尔PB达到1至2000浓度。从板上取出洗涤缓冲液后，加入 250 微升 DAPI 溶液，并在摇床的室温下孵育 5 分钟。使用塑料移液器去除 DAPI 溶液后，使用 1000 微升移液器每井添加 500 微升 0.1 摩尔 PB。将显微镜幻灯片放在立体镜下。使用精细画笔在幻灯片上添加三个单独的 0.1 摩尔 PB 滴。使用画笔在幻灯片上放置每一滴一个切片，然后拼合并定向切片。正确定位所有切片后，使用纸纸巾去除多余的 PB 并仔细干燥，而无需完全干燥切片。然后将80微升嵌入介质添加到幻灯片上，然后用盖板小心地盖住它，以嵌入大脑切片。盖住幻灯片，避免光线照射，让它们干燥在烟罩中一到两个小时，然后将它们存放在四摄氏度的显微镜幻灯片盒中，直到准备好进行共和显微镜。获取整个大脑切片的虚拟组织后，如手稿中描述的所述，通过单击文件然后打开，将所有单个通道或一个图像加载到 FIJI 中。然后使用自由手选择工具在 DAPI 通道中描绘一个半球。单击编辑，然后选择，然后创建蒙版以创建所选区域的蒙版。然后点击分析，然后测量颗粒，以确定单个通道的均度荧光强度。确保选择单个通道以确定每个通道的均度荧光强度值。之后，将单个通道的均荧光强度复制到电子表格中。要确定感兴趣区域中单个通道的均荧光强度，请使用自由手选择工具划定该区域。用DAPI染色后，染色核脑部分具有双核形态，细胞密度高的区域比细胞密度低的区域更亮。与 Mover 抗体的染色揭示了异构分布与明亮的热点区域和整个大脑的变暗区域，其中 Synapto 物理揭示了更均匀的分布。图像叠加显示红色荧光的差分分布，指示移动蛋白与绿色荧光相比，绿色荧光表示 Synapto 物理。量化分析后，对于 Mover 和 Synapto 物理，已确定跨半球和感兴趣区域不同通道的荧光强度值。移动器荧光值与 Synapto 物理荧光值的比率显示移动器的异构分布，相对于 Synaptic 囊泡而言，移动器水平高至低。相对 Mover 丰度与整个半球的感兴趣区域的比率相比，并转换为百分比显示一个感兴趣区域相对于平均值存在多少 Mover。相对移动丰度确定为海马区子领域的不同层。通过比较相应图层中的比率与相应半球的比率来量化三个感兴趣区域。此过程可以很容易地适应，并结合不同的成像技术，如超分辨率显微镜，进一步确定感兴趣的蛋白质的亚细胞分布。该技术也可以应用于不同的模型系统，如转基因或药物治疗的动物。这允许对不同的实验条件甚至物种进行比较。

Research

JoVE Journal

Neuroscience

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

量化的活动顺</em在小鼠视网膜>调控元件外植体电穿孔

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

科研

神经科学

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

监测变化的细胞内钙离子浓度和突触效能的软体动物<em&gt;海兔</em

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

室管膜纤毛在小鼠脑侧脑室实时成像

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

影像学检查HT能纤维在小鼠脊髓使用CLARITY / CUBIC技术

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain

量化龙脑中的亚细胞泛基蛋白-蛋白酶体活性

The ubiquitin-proteasome system is a key regulator of protein degradation and a variety of other cellular processes in eukaryotes. In the brain, increases ...