作者要感谢达盖拉德的接种和后续的动物实验。这项工作是由ANSES（法语局食品，环境和职业健康安全），并从基金会法国帕金森的赠款支持。

所有的程序和涉及动物的协议是根据欧盟86/609 / EEC和来的时候，考虑在动物实验（协议11-0043）道德的法国国家委员会批准。动物饲养和照顾在ANSES的认可实验设施在里昂（许可B 69387 0801）。 1.准备小鼠<ol><li>通过腹膜内注射致死剂量的戊巴比妥钠的安乐死的小鼠。 </li><li>检索来自小鼠头盖骨的整个大脑，并放置在35毫米的塑料培养皿中在冰上直到萃取。 </li><li>提取脊髓型颈椎病。 注 :提取αS无论是从一个接矢状切片或从解剖小鼠大脑脑半部，可在表1中列出的实验之后。 </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> ExperimenŤ </td><td> 老鼠 物（相当于大脑） </td><td> 生存期 （DPI） </td><td> 平均/最大生存 （天） </td><td> αS 三维检测用ELISA / WB / IHC </td></tr><tr><td> 1 </td><td>未接种小鼠</td><td> 441±166 </td><td>七百三十六分之四百十九</td><td> 8/8 </td></tr><tr><td> 2 </td><td>接种小鼠（0.2毫克） </td><td> 150±52 </td><td> 241分之140 </td><td> 9/9 </td></tr></tbody></table> 。预防接种在6个星期进行表1中的对M83小鼠进行的实验清单实验2中的纹状体-皮质区与20μl的患病小鼠的脑匀浆（1％重量/体积的葡萄糖5％），后6周龄纯合子小鼠M83异氟烷吸入3％麻醉。 DPI:天后inocul通货膨胀。 2.αS提取脑减半<ol><li>矢状切开大脑，得到两半。每个重达半含磨球一个ribolysis管。 </li><li>制备含有50mM Tris-盐酸，pH为7.5，750 mM氯化钠，5mM的EDTA，1mM的DTT，1％磷酸和蛋白酶抑制剂鸡尾酒高盐（HS）的缓冲器。添加高盐缓冲到大脑半部，以获得20％（重量/体积）匀浆。 </li><li>制备从使用机械均化器大脑半部样品为6.0米/秒的23秒两次。前23秒均化之后，将包含第二23秒周期之前在冰上2分钟的匀浆管。 </li><li>离心样品在1000×g离心5分钟以消除未磨制脑片段。回收上清液，分成200微升的等分试样，并保持在-80℃下用于随后的ELISA分析。 </li></ol> 3.αS提取解剖脑区<ol><li>解剖全脑在35毫米的塑料培养皿在冰上与使用两个forcipes的两端放在一起解剖海马时除外低倍放大镜（8倍放大）。不要超过10分钟，以保持大脑的完整性。将大脑右侧朝上和检索下列顺序的脑区: <ol><li>使用置于仅次于灯泡镊子两个嗅球中单独之一。由向下运动的大脑分离。重复此操作，对于在第二灯泡。 </li><li>轻轻楔镊子在两皮质之间并移动其转发到促进两个皮层解离。保持大脑到位与一种钳子，使用另一个皮层从海马中分离出来。 </li><li>定位钳2毫米皮层下面。保持对钳子轻柔压力直到海马的顶部可见。剥离皮质的第一部分，并重复与第二部分。使用镊子分开的两个皮层起始于海马和移向大脑的前部。 </li><li>围绕定位海马的一个开放的镊子。关闭钳在海马的底部，然后轻轻将其删除，恢复尽可能。重复该过程的第二个海马。 </li><li>定位低于芨之一的开放镊子轻轻它从大脑中分离出来。使用镊子从纹状体删除任何剩余的皮质。重复此过程的第二纹状体。 </li><li>使用镊子至2毫米的小脑的轮廓轻踏以有利于从大脑小脑的分离。将镊子仅次于小脑和通过移动镊子向前卸下。 </li><li>使用镊子的宽部，以提高中脑，以便清楚地看到，它加入脑干。使在路口一纵切口，然后取出脑干。 </li><li>定位钳脑后面，我Ş由四个圆形结构。切开垂直直到脑已完全从剩余的脑分离。 </li></ol></li><li>准备的变量％（重量/体积）中的HS缓冲器，这取决于可用的组织的量， 即，5％的匀浆为10至30毫克的重量，为30和80毫克之间的重量的10％，和20％匀浆对于高于80毫克的重量。 <ol><li>加入的HS缓冲区足够的容量来解剖大脑区域获得匀浆的预期％。 </li><li>涡和检查组织被完全沉浸在HS缓冲区。 </li><li>均匀化从解剖大脑区域或颈髓制备用组织研磨机硼硅玻璃管和两个杵，A和B组成的样品</li><li>倾每个大脑区域直接粉碎成管。将杵进管和收回它。重复此动作大约十倍以离解的组织。然后用杵b到continue研磨组织与另外的20的运动。转移匀浆到1.5毫升管与1毫升移液管。 </li></ol></li><li>离心样品在1000×g离心5分钟，在4℃下，以消除任何未磨制脑片段。检索上清液，它们划分成200μl的等分试样，并保持在-80℃下用于随后的ELISA分析。 </li></ol> 4.检测αS的通过ELISA <ol><li>稀释涂料抗体0.01纳克/毫升。使用任一抗αS兔多克隆或单克隆的克隆42抗体在50mM的Na 2 CO 3 / 碳酸氢钠缓冲液（pH 9.6）。 </li><li>涂层的96孔板，每该涂覆溶液的孔100μl，并在4℃CO / N离开。使用抗αS兔多克隆抗体在用于使用抗体检测syn514 ELISA中的涂布液，克隆42，LB509，AS11，4D6或8A5。使用抗αS单克隆抗体克隆42作为涂布液在与抗pSer129αS检测抗体结合。 注 :如果需要的话，板可以保持在4℃下进行一周的ELISA之前进行。 </li><li>使用洗板与每孔0.05％吐温20（PBST）洗涤板五次用300μl磷酸盐缓冲盐水。从该步骤起，孵育是在室温。 </li><li>加入200μl的PBS T20阻止每口井的缓冲区。摇1小时以150rpm。用PBST洗平板五次。 </li><li>稀释脑匀浆（稀释1:100的20％匀浆，1:50 10％匀浆并在PBST中的BSA 1％5％匀浆1:25），并添加100微升到每个孔中。然后孵育2小时，在150转速下晃动。用PBST洗平板五次。 </li><li>添加不同αS检测抗体在PBST与BSA 1％的材料清单中提到的稀释液。孵育1小时以150rpm。用PBST洗平板五次。 </li><li>无论是添加抗-mouse或抗兔IgG HRP结合物稀释1:8000在PBST中补充有BSA的1％1小时以150rpm摇动。用PBST洗平板五次。 </li><li>加入100微升3,3&#39;，5,5&#39;-四甲基联苯胺（TMB）溶液至每个孔，并孵育在黑暗中15分钟以150rpm摇动。 </li><li>停止加入100微升1N HCl中，每孔然后测量450nm处的吸光度与酶标仪反应。 </li><li>进行数据分析，减去在井与除从测量每个所分析的样品的OD值的任何小鼠大脑样品（空白孔）中的所有的试剂中得到的OD值。 </li></ol> 5.表位作图<ol><li>根据由奥斯曼16中记载的方法进行表位作图。简言之，将包含在硝酸纤维素12个氨基酸的10个重叠氨基酸的人类α突触核蛋白序列的点的肽。 </li><li>用50mM的Tris / 150mM的NaCl的缓冲液1方框0含0.05％吐温20和5％奶粉。孵育抗体在阻断以每毫升2微克抗体的浓度的溶液在2-10°CO / N。 </li><li>洗膜用的50mM Tris / 150含有0.05％Tween 20的孵育与山羊抗 - 小鼠IgG HRP缀合物mM氯化钠缓冲液pH 10三次。洗用同样的缓冲液然后另一个五次染色用Western印迹TMB染色试剂盒的膜。 </li></ol> 6.统计分析<ol><li>使用R软件和NLME包进行混合效应回归模型OD。对于每一个对比，进行混合效应回归模型。使用固定的效果，从无症状群体区分症状。 </li><li>使用随机效果来反映重复的变异用于给定鼠标。查同方差通过检查残差和如果需要的话，可以使用方差函数来符合皮涅罗和贝茨1组内的错误的方差结构的模型7，设置0.05作为P的意义门槛</li></ol>

<ol>
	<li>Goedert, M., Spillantini, M. G., Del Tredici, K., Braak, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=100+years+of+Lewy+pathology.">100 years of Lewy pathology.</a> Nat Rev Neurol. 9, 13-24 (2013).</li><li>Waxman, E. A., Giasson, B. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+and+regulation+of+casein+kinase-mediated+phosphorylation+of+alpha-synuclein.">Specificity and regulation of casein kinase-mediated phosphorylation of alpha-synuclein.</a> J Neuropathol Exp Neurol. 67, 402-416 (2008).</li><li>Anderson, J. P., 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=Phosphorylation+of+Ser-129+is+the+dominant+pathological+modification+of+alpha-synuclein+in+familial+and+sporadic+Lewy+body+disease.">Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease.</a> J Biol Chem. 281, 29739-29752 (2006).</li><li>Mougenot, A. L., 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=Prion-like+acceleration+of+a+synucleinopathy+in+a+transgenic+mouse+model.">Prion-like acceleration of a synucleinopathy in a transgenic mouse model.</a> Neurobiol Aging. 33, 2225-2228 (2012).</li><li>Luk, K. 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=Intracerebral+inoculation+of+pathological+alpha-synuclein+initiates+a+rapidly+progressive+neurodegenerative+alpha-synucleinopathy+in+mice.">Intracerebral inoculation of pathological alpha-synuclein initiates a rapidly progressive neurodegenerative alpha-synucleinopathy in mice.</a> J Exp Med. 209, 975-986 (2012).</li><li>Giasson, B. I., 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=Neuronal+alpha-synucleinopathy+with+severe+movement+disorder+in+mice+expressing+A53T+human+alpha-synuclein.">Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein.</a> Neuron. 34, 521-533 (2002).</li><li>Luk, K. 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=Pathological+alpha-synuclein+transmission+initiates+Parkinson-like+neurodegeneration+in+nontransgenic+mice.">Pathological alpha-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice.</a> Science. 338, 949-953 (2012).</li><li>Masuda-Suzukake, 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=Prion-like+spreading+of+pathological+alpha-synuclein+in+brain.">Prion-like spreading of pathological alpha-synuclein in brain.</a> Brain : a journal of neurology. 136, 1128-1138 (2013).</li><li>Sacino, A. 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=Amyloidogenic+alpha-synuclein+seeds+do+not+invariably+induce+rapid,+widespread+pathology+in+mice.">Amyloidogenic alpha-synuclein seeds do not invariably induce rapid, widespread pathology in mice.</a> Acta neuropathologica. 127, 645-665 (2014).</li><li>Recasens, 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=Lewy+body+extracts+from+parkinson's+disease+brains+trigger+alpha-synuclein+pathology+and+neurodegeneration+in+mice+and+monkeys.">Lewy body extracts from parkinson's disease brains trigger alpha-synuclein pathology and neurodegeneration in mice and monkeys.</a> Ann Neurol. , (2013).</li><li>Foulds, P. 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=Phosphorylated+alpha-synuclein+can+be+detected+in+blood+plasma+and+is+potentially+a+useful+biomarker+for+Parkinson's+disease.">Phosphorylated alpha-synuclein can be detected in blood plasma and is potentially a useful biomarker for Parkinson's disease.</a> FASEB J. 25, 4127-4137 (2011).</li><li>El-Agnaf, O. 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=Detection+of+oligomeric+forms+of+alpha-synuclein+protein+in+human+plasma+as+a+potential+biomarker+for+Parkinson's+disease.">Detection of oligomeric forms of alpha-synuclein protein in human plasma as a potential biomarker for Parkinson's disease.</a> Faseb J. 20, 419-425 (2006).</li><li>Lee, H. 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=Enzyme-linked+immunosorbent+assays+for+alpha-synuclein+with+species+and+multimeric+state+specificities.">Enzyme-linked immunosorbent assays for alpha-synuclein with species and multimeric state specificities.</a> J Neruosci Meth. 199, 249-257 (2011).</li><li>Unterberger, U., 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=Detection+of+disease-associated+alpha-synuclein+in+the+cerebrospinal+fluid:+a+feasibility+study.">Detection of disease-associated alpha-synuclein in the cerebrospinal fluid: a feasibility study.</a> Clin Neuropathol. 33, 329-334 (2014).</li><li>Betemps, 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=Alpha-synuclein+spreading+in+M83+mice+brain+revealed+by+detection+of+pathological+alpha-synuclein+by+enhanced+ELISA.">Alpha-synuclein spreading in M83 mice brain revealed by detection of pathological alpha-synuclein by enhanced ELISA.</a> Acta Neuropathol. (Berl). 2, 29 (2014).</li><li>Osman, A. 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=A+monoclonal+antibody+that+recognizes+a+potential+coeliac-toxic+repetitive+pentapeptide+epitope+in+gliadins).">A monoclonal antibody that recognizes a potential coeliac-toxic repetitive pentapeptide epitope in gliadins).</a> Eur J Gastroenterol Hepatol. 13, 1189-1193 (2001).</li><li>Pinheiro, J. C., Bates, D. M., Chambers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+5.">Ch. 5.</a> Mixed-Effects Models in S and S-PLUS. 5, 206-225 (2000).</li><li>Mougenot, A. L., 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=Production+of+a+monoclonal+antibody,+against+human+alpha-synuclein,+in+a+subpopulation+of+C57BL/6J+mice,+presenting+a+deletion+of+the+alpha-synuclein+locus.">Production of a monoclonal antibody, against human alpha-synuclein, in a subpopulation of C57BL/6J mice, presenting a deletion of the alpha-synuclein locus.</a> J Neruosci Meth. 192, 268-276 (2010).</li><li>Specht, C. G., Schoepfer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+the+alpha-synuclein+locus+in+a+subpopulation+of+C57BL/6J+inbred+mice.">Deletion of the alpha-synuclein locus in a subpopulation of C57BL/6J inbred mice.</a> BMC Neurosci. 2, 11 (2001).</li><li>Lee, B. R., Matsuo, Y., Cashikar, A. G., Kamitani, T. <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+Ser129+phosphorylation+of+alpha-synuclein+in+melanoma+cells.">Role of Ser129 phosphorylation of alpha-synuclein in melanoma cells.</a> J Cell Sci. 126, 696-704 (2013).</li><li>Perrin, R. 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=Epitope+mapping+and+specificity+of+the+anti-alpha-synuclein+monoclonal+antibody+Syn-1+in+mouse+brain+and+cultured+cell+lines.">Epitope mapping and specificity of the anti-alpha-synuclein monoclonal antibody Syn-1 in mouse brain and cultured cell lines.</a> Neurosci Lett. 349, 133-135 (2003).</li><li>Emmanouilidou, E., 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=Assessment+of+alpha-synuclein+secretion+in+mouse+and+human+brain+parenchyma.">Assessment of alpha-synuclein secretion in mouse and human brain parenchyma.</a> PLoS One. 6, e22225 (2011).</li><li>Mougenot, A. L., 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=Transmission+of+prion+strains+in+a+transgenic+mouse+models+overexpressing+human+A53T+mutated+alpha-synuclein.">Transmission of prion strains in a transgenic mouse models overexpressing human A53T mutated alpha-synuclein.</a> J Neuropathol Exp Neurol. 70, 377-385 (2011).</li><li>Foulds, P. 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=A+longitudinal+study+on+alpha-synuclein+in+blood+plasma+as+a+biomarker+for+Parkinson's+disease.">A longitudinal study on alpha-synuclein in blood plasma as a biomarker for Parkinson's disease.</a> Sci Rep. 3, 2540 (2013).</li></ol>

The authors have no competing interests to disclose.

采用ELISA检测法被证明特别是从小鼠脑组织匀浆检测αSÐ直接的病在M83转基因小鼠模型中。事实上，这ELISA可以很容易地分辨健康M83小鼠在高盐缓冲只使用全脑组织匀浆病鼠M83。 使用这种ELISA成功的结果的最重要的步骤是:正确地制定必要的手巧，以防止在切除损坏解剖老鼠大脑的不同区域;执行样本稀释只在HS缓冲区;和抗体的选择，因为并非所有的抗体将工作在ELISA格式...

Most current methods for detecting disease-associated &#945;-synuclein (&#945;SD) in experimental models of Parkinson's disease (PD), such as immunohistochemistry or Western blot, are time-consuming and not quantitative. This neurodegenerative disease is characterized by alpha-synuclein aggregation mainly in the form of inclusions containing an aggregated form of the normally soluble presynaptic protein &#945;S1,2 (Lewy bodies and Lewy neurites). Normally only marginally phosphorylated, &#945;S is hyperphosphorylated at its serine 129 residue in these inclusions3 and can be monitored by antibodies specifically directed against Ser129 phosphorylated &#945;S, thus providing a reliable marker of the pathology. 
Recent research suggests that a &#8220;prion-like&#8221; mechanism could be involved in the propagation of &#945;S aggregation within the nervous system of an affected patient4,5. These studies reported the acceleration of a synucleinopathy by inoculating brain extracts containing &#945;SD into a transgenic mouse model (M83) expressing an A53T mutated human &#945;S protein associated with a severe motor impairment occurring as the mice age6. In the same manner, intra-cerebral inoculation of aggregated recombinant &#945;S in the same M83 mouse model confirmed the acceleration of aggregation5. The induction of deposits of phosphorylated &#945;S has also been reported after inoculation of C57Bl/6 wild-type mice with either fibrillar recombinant &#945;S or brain extracts from human DLB patients7,8. Sacino et al.9 recently pointed out that after injection of fibrillar human &#945;S, a widespread and progressive cerebral &#945;S inclusion formation could be induced in M83 mice, but not in E46K transgenic mice or non-transgenic mice in which induced &#945;S inclusions were transient, and mainly restricted to the site of injection. Recent studies on monkeys confirmed propagation of &#945;S aggregates after inoculation of PD-derived extracts in species closer to humans10.
The link between &#945;S alterations and Parkinson&#8217;s disease suggest that &#945;SD is a potential biomarker for Parkinson&#8217;s disease11. A recent study showed the detection of oligomeric soluble aggregates of &#945;-synuclein in human cerebro-spinal fluid (CSF) and plasma as a potential biomarker for Parkinson&#8217;s disease based on a conventional sandwich system ELISA using the same antibody to capture and detect &#945;S12. Based on the same method, multimeric proteins were recognized in biological samples, including the brain, because there are multiple copies of epitopes present in the assembled forms13. Very recently, pathological &#945;S in the CSF of patients with a proven Lewy body pathology was detected using both an ELISA kit with a highly specific antibody against &#945;SD (5G4) and an immunoprecipitation assay14. These methods could differentiate patients with PD/DLB from other types of dementia.
The &#8220;prion-like&#8221; propagation of &#945;S aggregation was further studied in transgenic mouse model M83 using an ELISA approach that was designed to specifically identify &#945;SD15. In this study, we report the detailed ELISA protocol used to quantitatively detect &#945;SD in sick mice (whether or not inoculated with &#945;SD from sick M83 mice) and more especially in the brain regions specifically targeted by the pathological process in this M83 transgenic mouse model4.

<table><tbody><tr><td>LB509</td><td>Abcam</td><td>ab27766</td><td>Detection antibody 1/2,000</td></tr><tr><td>AS11</td><td>Produced at Anses</td><td></td><td>Detection antibody 1/1,000</td></tr><tr><td>4D6</td><td>Abcam</td><td>ab1903</td><td>Detection antibody 1/2,000</td></tr><tr><td>PSer129</td><td>Abcam</td><td>ab59264</td><td>Detection antibody 1/3,000</td></tr><tr><td>PSer129&amp;nbsp;EP1536Y</td><td>Abcam</td><td>ab51253</td><td>Detection antibody 1/1,000</td></tr><tr><td>syn514</td><td>Abcam</td><td>ab24717</td><td>Detection antibody 1/500</td></tr><tr><td>clone 42</td><td>BD Biosciences</td><td>610787</td><td>Coating and detection antibody (1/2,000)</td></tr><tr><td>8A5</td><td>Provided by Dr. Anderson</td><td></td><td>Detection antibody 1/2,000</td></tr><tr><td>polyclonal anti-&amp;alpha;syn antibody</td><td>Millipore</td><td>AB5038P</td><td>Coating antibody</td></tr><tr><td>Anti-mouse IgG HRP conjugate</td><td>Southern Biotech</td><td>1010-05</td><td></td></tr><tr><td>Anti-rabbit IgG HRP conjugate</td><td>Southern Biotech</td><td>4010-05</td><td></td></tr><tr><td>Goat anti-mouse IgG HRP conjugate</td><td>Dianova</td><td>115-035-164</td><td></td></tr><tr><td>&lt;strong&gt;HS buffer&lt;/strong&gt;</td><td></td><td></td><td>Adjust at pH 7.5 and keep at 4 &amp;#176;C</td></tr><tr><td>&lt;ul&gt;
&lt;li&gt;Tris-HCl 50&amp;nbsp;mM &lt;/li&gt;
&lt;/ul&gt;</td><td>Euromedex</td><td>26-128-3094-B</td><td></td></tr><tr><td>&lt;ul&gt;
&lt;li&gt;NaCl 750 mM &lt;/li&gt;
&lt;/ul&gt;</td><td>Euromedex</td><td>1112-A</td><td></td></tr><tr><td>&lt;ul&gt;
&lt;li&gt;EDTA 5 mM &lt;/li&gt;
&lt;/ul&gt;</td><td>Euromedex</td><td>EU0007-B</td><td></td></tr><tr><td>&lt;ul&gt;
&lt;li&gt;DTT 1 mM &lt;/li&gt;
&lt;/ul&gt;</td><td>Sigma</td><td>43815</td><td></td></tr><tr><td>&lt;strong&gt;PBS&lt;/strong&gt;</td><td></td><td></td><td>Adjust at pH 7.5</td></tr><tr><td>&lt;ul&gt;
&lt;li&gt;Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4 &lt;/sub&gt;1&amp;nbsp;mM &lt;/li&gt;
&lt;/ul&gt;</td><td>Euromedex</td><td>1309</td><td></td></tr><tr><td>&lt;ul&gt;
&lt;li&gt;KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4 &lt;/sub&gt;1.5&amp;nbsp;mM &lt;/li&gt;
&lt;/ul&gt;</td><td>Euromedex</td><td>2018</td><td></td></tr><tr><td>&lt;ul&gt;
&lt;li&gt;NaCl&amp;nbsp; 137 mM &lt;/li&gt;
&lt;/ul&gt;</td><td>Euromedex</td><td>1112-A</td><td></td></tr><tr><td>&lt;ul&gt;
&lt;li&gt;KCl 2.7 mM &lt;/li&gt;
&lt;/ul&gt;</td><td>Euromedex</td><td>P017</td><td></td></tr><tr><td>Tween 20</td><td>Euromedex</td><td>2001-C</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>DTT 1 mM</td><td>Sigma</td><td>43815</td><td>Stock solution 100 mM, toxic</td></tr><tr><td>1% phosphatase cocktail</td><td>Pierce</td><td>78428</td><td></td></tr><tr><td>1% protease inhibitor cocktail</td><td>Roche</td><td>04&amp;nbsp;693&amp;nbsp;132&amp;nbsp;001</td><td>50x concentrated</td></tr><tr><td>Microplate MaxiSorpTM</td><td>Thermo Scientific</td><td>442404</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Tampon carbonate 50 mM pH 9.6&lt;/strong&gt;</td></tr><tr><td>&lt;ul&gt;
&lt;li&gt;Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;, 10H&lt;sub&gt;2&lt;/sub&gt;O &lt;/li&gt;
&lt;/ul&gt;</td><td>Sigma</td><td>71360</td><td>2.86 g/L</td></tr><tr><td>&lt;ul&gt;
&lt;li&gt;NaHCO&lt;sub&gt;3&lt;/sub&gt; &lt;/li&gt;
&lt;/ul&gt;</td><td>Merk</td><td>6329</td><td>3.36 g/L, pH&amp;nbsp;9.6</td></tr><tr><td>Superblock T20 PBS blocking buffer</td><td>Pierce</td><td>E6423H</td><td>10x concentrated</td></tr><tr><td>TMB</td><td>Sigma</td><td>T0440</td><td>Used for ELISA</td></tr><tr><td>TMB</td><td>Analytik Jena AG</td><td>847-0104200302</td><td>Used for epitope mapping</td></tr><tr><td>HCl 1&amp;nbsp;N</td><td>Chimie plus</td><td>40030</td><td></td></tr><tr><td>Ribolyser</td><td>Thermo</td><td>Fast prep FP120</td><td>keep on ice at this step</td></tr><tr><td>Grinding tubes</td><td>Biorad</td><td>355-1197</td><td></td></tr><tr><td>Plate washer</td><td>Tecan</td><td>Columbus Pro</td><td></td></tr><tr><td>Plate reader</td><td>Biorad</td><td>Model 680</td><td></td></tr><tr><td>Low power magnifier&amp;nbsp;</td><td>VWR</td><td>630-1062</td><td>8X magnification</td></tr><tr><td>Forceps Dumont#7</td><td>WPI</td><td>14097</td><td>For dissection steps</td></tr><tr><td>Transfer pipette 1ml Samso</td><td>Samso</td><td>043231</td><td></td></tr><tr><td>1.5 ml tubes</td><td>Dutscher</td><td>033290</td><td></td></tr></tbody></table>

detection of disease-associated &#945;-synuclein by enhanced elisa in the brain of transgenic mice overexpressing human a53t mutated &#945;-synuclein

除了 ​​像Western印迹确定的方法，需要新的方法来快速并容易地量化疾病相关α突触核蛋白（αSD）在突触核蛋白病的实验模型。转基因小鼠线（M83）过表达人类A53TαS和自发发展8和22个月的年龄，特点是症状包括体重下降，虚脱，和严重运动功能障碍之间的戏剧性的临床表型，被用于这项研究。为αSD（疾病相关αS）在这些小鼠中的分子分析，将ELISA被设计成特异性量化αS中的D患病小鼠。中枢神经系统在该小鼠模型的分析表明αSD的存在主要是在尾部脑区域和脊髓。有不同的实验条件之间αS 二维分布无显着差异，导致临床疾病， 即在uninoculated和正常老化的转基因小鼠在接种与患病小鼠大脑提取物的小鼠。的αSð使用免疫抗体对抗Ser129的特异性检测磷酸化ELISAαS与Western blot和免疫组化基本得到相关。出乎意料的是，类似的结果，观察对αS的C-末端部分的几个其它抗体。 αSD的传播，这表明了"朊病毒样"机制的参与，因此，可以容易地监测和定量用ELISA方法该小鼠模型。

在这项研究中，所用的ELISA中具体鉴定疾病相关αS（αSD）在从患病M83小鼠高盐缓冲液中制备脑匀浆。使用抗体特异性识别pSer129αS（p值= 0.0074），酶联免疫吸附容易从年轻（2-5个月大），健康M83小鼠（ 图1）区分老，病小鼠（&gt; 8月龄）。其他几个抗体显示类似的高信号（&gt; 0.6 OD）仅在从患病老鼠脑组织匀浆。这是129-140的情况下为4D6（p值= 0.01），LB509（p值= 0.0047）和8A5（P &lt;...

Watch this Scientific Journal Video about Detection of Disease-associated ÃŽÂ±-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated ÃŽÂ±-synuclein at JoVE.com

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

发现疾病相关的α突触核蛋白通过增强ELISA在转基因小鼠表达人A53T的突变脑α突触核蛋白

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

detection-disease-associated-synuclein-enhanced-elisa-brain

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

10.6K 次观看.  ANSES - French Agency for Food, Environmental and Occupational Health & Safety. An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated α-synuclein (αSD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated αS form or the C-terminal part of the protein.

视频: Detection of Disease-associated α-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated α-synuclein

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

鱼藤酮灌胃和图像分析α- synuclein的夹杂物在肠道神经系统的一个使用口服

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

科研

神经科学

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

可溶和不可溶α-突触核蛋白从帕金森脑连续提取

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

通过重组腺相关病毒载体注射立体的α-突触核蛋白基于大鼠模型为帕金森氏病的发展

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

医学

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

在转基因小鼠神经炎症的生物发光成像的α-共核蛋白纤丝的外围接种后

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

微粒相关α synuclein 聚合体对原代神经元的外源性管理--纤维形成的强大细胞模型

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

生物学

Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &#945;-Synuclein

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, consequently, the progressive loss of control of voluntary movements. This neurodegenerative process is triggered by the deposition of protein aggregates in the brain, which are mainly constituted of &#945;-synuclein. Several studies have indicated that neuroinflammation is required to develop the neurodegeneration associated with Parkinson&#39;s disease. Notably, the neuroinflammatory process involves microglial activation as well as the infiltration of peripheral T cells into the substantia nigra (SN). This work analyzes a mouse model of Parkinson&#39;s disease that recapitulates microglial activation, T-cell infiltration into the SN, the neurodegeneration of nigral dopaminergic neurons, and motor impairment. This mouse model of Parkinson&#39;s disease is induced by the stereotaxic delivery of adeno-associated viral vectors encoding the human wild-type &#945;-synuclein (AAV-h&#945;Syn) into the SN. The correct delivery of viral vectors into the SN was confirmed using control vectors encoding green fluorescent protein (GFP). Afterward, how the dose of AAV-h&#945;Syn administered in the SN affected the extent of h&#945;Syn expression, the loss of nigral dopaminergic neurons, and motor impairment were evaluated. Moreover, the dynamics of h&#945;Syn expression, microglial activation, and T-cell infiltration were determined throughout the time course of disease development. Thus, this study provides critical time points that may be useful for targeting synuclein pathology and neuroinflammation in this preclinical model of Parkinson&#39;s disease.

This work analyzes the vector dose and exposure time required to induce neuroinflammation, neurodegeneration, and motor impairment in this preclinical model of Parkinson&#39;s disease. These vectors encoding the human &#945;-synuclein are delivered into the substantia nigra&#160;to recapitulate the synuclein pathology associated with Parkinson&#39;s disease.

分析编码人类α突触核蛋白的腺相关病毒载体诱导的帕金森病小鼠模型

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, ...

A Gravity-Fed Transcardial Perfusion Method for Histologic Analysis of the Mouse Central Nervous System

The histologic analysis of brain and spinal cord specimens isolated from mice is common practice for the assessment of pathology in this model system. To maintain the morphology of these delicate tissues, it is routine to administer a chemical fixative such as paraformaldehyde via cannulation of the heart in anesthetized animals (transcardial perfusion). Transcardial perfusion of the mouse heart has traditionally relied on the use of peristaltic pumps or air pressure to deliver both the saline and fixative solutions necessary for this process. As an easily accessible alternative to these methods, this work demonstrates the use of a gravity-fed method of perfusate delivery that uses materials available in most hardware stores. 
To validate this new perfusion method, this work demonstrates all the subsequent steps necessary for the sensitive detection of phosphorylated &#945;-synuclein in both the brain and spinal cord. Included in these steps are the dissection of the fixed brain and spinal cord tissues, rapid freezing/embedding and cryosectioning of the tissues, and immunofluorescent staining. As this method results in whole-body delivery of the fixative, it may also be used to prepare other non-neuronal tissues for histologic analysis.

A convenient gravity-fed perfusion method for histological analysis of the mouse central nervous system is presented. Immunofluorescent detection of phosphorylated &#945;-synuclein is demonstrated in a mouse model of Parkinson's disease. This work also comprehensively describes the transcardial perfusion, dissection, tissue freezing/embedding, and frozen sectioning steps.

一种重力喂养的经心灌注方法，用于小鼠中枢神经系统的组织学分析

The histologic analysis of brain and spinal cord specimens isolated from mice is common practice for the assessment of pathology in this model system. To ...

Induction of Neuroinflammation in Mice via Injection of Alpha-Synuclein Fibrils

Source: Breid, S. et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55503">Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils.</a> J. Vis. Exp. (2017).
This video demonstrates the injection of alpha-synuclein fibrils into a mouse to induce neuroinflammation. The fibrils are injected intraperitoneally into a transgenic mouse expressing mutated alpha-synuclein proteins. Upon reaching the central nervous system, the fibrils enter neurons and induce aggregation of the mutated proteins. The aggregates spread to neighboring neurons, microglia, and astrocytes. Recognition of the aggregates triggers microglia to induce neuroinflammation, damaging the neurons.

通过注射 α-突触核蛋白原纤维诱导小鼠神经炎症

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160; ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

Studying Alpha-Synuclein Accumulation in Primary Embryonic Mouse Dopamine Neurons

Source: Er, S. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/61118">Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons.</a>&#160;J. Vis. Exp. (2020).
This video demonstrates the process of quantifying alpha-synuclein accumulation in embryonic mouse dopamine neurons pre-treated with alpha-synuclein pre-formed fibrils. It outlines the steps for fixing, staining, and quantifying the dopamine neurons containing alpha-synuclein aggregates using immunofluorescence.

研究原代胚胎小鼠多巴胺神经元中的 α-突触核蛋白积累

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1.&#160;Preparation

	Prepare ...