Name: targeting alpha synuclein aggregates in cutaneous peripheral nerve fibers by free-floating immunofluorescence assay
Uploaded: 2019-06-25T13:30:00
Description: Watch this Scientific Journal Video about Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay at JoVE.com

我们感谢帕金森·施魏茨和ABREOC(恩德·奥斯佩莱罗·坎登纳莱的科学研究咨询委员会)为这项研究提供了财政支持。

该协议已获得州道德委员会的批准,所有入学的受试者都书面同意进行此项研究。
1. 皮肤活检系列
<ol><li>让合格的医生在适当的临床环境中进行皮肤活检。</li>
	<li>选择区域进行皮肤活检,用酒精拭子清洁。</li>
	<li>用1cc的利多卡因2%准备麻醉溶液。</li>
	<li>针与区域平行,注射局部麻醉皮下。</li>
	<li>检查麻醉效果后,服用3毫米一次性冲床,施加旋转和微妙的向下压力,通过表皮和?...

<ol>
	<li>McArthur, J. C., Griffin, 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=Another+Tool+for+the+neurologist's+Tollbox.">Another Tool for the neurologist's Tollbox.</a> Annals of Neurology. 57, 163-167 (2005).</li><li>Wilkinson, P. F., Millington, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Skin. , 49-50 (2009).</li><li>Weddell, 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=Nerve+endings+in+mammalian+skin.">Nerve endings in mammalian skin.</a> Biological reviews of the Cambridge Philosophical Society. 30, 159-195 (1954).</li><li>Wang, 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=Protein+gene+product+9.5-immunoreactve+nerve+fibers+and+cells+in+human+skin.">Protein gene product 9.5-immunoreactve nerve fibers and cells in human skin.</a> Cell and Tissue Research. 261 (1), 25-33 (1990).</li><li>Holland, N. R., 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=Intraepidermal+nerve+fiber+density+in+patients+with+painful+sensory+neuropathy.">Intraepidermal nerve fiber density in patients with painful sensory neuropathy.</a> Neurology. 48, 708-711 (1997).</li><li>McArthur, J. C., Stocks, E. A., Hauer, P., Cornblath, D. R., Griffin, 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=Epidermal+nerve+fiber+density:+normative+reference+range+and+diagnostic+efficiency.">Epidermal nerve fiber density: normative reference range and diagnostic efficiency.</a> Archives of Neurology. 55 (12), 1513-1520 (1998).</li><li>Lauria, 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=European+Federation+of+Neurological+Societies/Peripheral+Nerve+Society+Guideline+on+the+use+of+skin+biopsy+in+the+diagnosis+of+small+fiber+neuropathy.+Report+of+a+joint+task+force+of+the+European+Federation+of+Neurological+Societies+and+the+Peripheral+Nerve+Society.">European Federation of Neurological Societies/Peripheral Nerve Society Guideline on the use of skin biopsy in the diagnosis of small fiber neuropathy. Report of a joint task force of the European Federation of Neurological Societies and the Peripheral Nerve Society.</a> European Journal of Neurology. 17, 903-912 (2010).</li><li>Provitera, V., 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+multi-center,+multinational+age-+and+gender-adjusted+normative+dataset+for+immunofluorescent+intraepidermal+nerve+fiber+density+at+the+distal+leg.">A multi-center, multinational age- and gender-adjusted normative dataset for immunofluorescent intraepidermal nerve fiber density at the distal leg.</a> European Journal of Neurology. 23, 333-338 (2016).</li><li>Wakabayashi, K., 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=Involvement+of+the+peripheral+nervous+system+in+synucleinopathies,+tauopathies+and+other+neurodegenerative+proteinopathies+of+the+brain.">Involvement of the peripheral nervous system in synucleinopathies, tauopathies and other neurodegenerative proteinopathies of the brain.</a> Acta Neuropathology. 120, 1-12 (2010).</li><li>Ruffmann, 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=Detection+of+alpha-synuclein+conformational+variants+from+gastro-intestinal+biopsy+tissue+as+a+potential+biomarker+for+Parkinson's+disease.">Detection of alpha-synuclein conformational variants from gastro-intestinal biopsy tissue as a potential biomarker for Parkinson's disease.</a> Neuropathology and Applied Neurobiology. 44 (7), 722-736 (2018).</li><li>Lee, J. 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=The+search+for+a+peripheral+biopsy+indicator+of+alpha-synuclein+pathology+for+Parkinson+Disease.">The search for a peripheral biopsy indicator of alpha-synuclein pathology for Parkinson Disease.</a> Journal of Neuropathology &amp; Experimental Neurology. 76, 2-15 (2017).</li><li>Donadio, V., 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=Skin+nerve+misfolded+alpha-synuclein+in+pure+autonomic+failure+and+Parkinson+disease.">Skin nerve misfolded alpha-synuclein in pure autonomic failure and Parkinson disease.</a> Annals of Neurology. 79, 306-316 (2016).</li><li>Doppler, K., 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=Cutaneous+neuropathy+in+Parkinson's+disease:+a+window+into+brain+pathology.">Cutaneous neuropathy in Parkinson's disease: a window into brain pathology.</a> Acta Neuropathologica. 128, 99-109 (2014).</li><li>Zange, L., Noack, C., Hahn, K., Stenzel, W., Lipp, A. <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+in+skin+nerve+fibres+differentiates+Parkinson's+disease+from+multiple+system+atrophy.">Phosphorylated alpha-synuclein in skin nerve fibres differentiates Parkinson's disease from multiple system atrophy.</a> Brain. 138, 2310-2321 (2015).</li><li>Braak, H., 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=Staging+of+brain+pathology+related+to+sporadic+Parkinson's+disease.">Staging of brain pathology related to sporadic Parkinson's disease.</a> Neurobiology of Aging. 24, 197-211 (2003).</li><li>Doppler, K., 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=Dermal+phospho-alpha-synuclein+deposits+confirm+REM+sleep+behaviour+disorder+as+prodromal+Parkinson's+disease.">Dermal phospho-alpha-synuclein deposits confirm REM sleep behaviour disorder as prodromal Parkinson's disease.</a> Acta Neuropathologica. 133 (4), 535-545 (2017).</li><li>Antelmi, 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=Skin+nerve+phosphorylated+&#945;-synuclein+deposits+in+idiopathic+REM+sleep+behavior+disorder.">Skin nerve phosphorylated &#945;-synuclein deposits in idiopathic REM sleep behavior disorder.</a> Neurology. 88 (22), 2128-2131 (2017).</li><li>Nolano, 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=Small+fiber+pathology+parallels+disease+progression+in+Parkinson+disease:+a+longitudinal+study.">Small fiber pathology parallels disease progression in Parkinson disease: a longitudinal study.</a> Acta Neuropathologica. , (2018).</li><li>Melli, 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=Cervical+skin+denervation+associates+with+alpha-synuclein+aggregates+in+Parkinson+disease.">Cervical skin denervation associates with alpha-synuclein aggregates in Parkinson disease.</a> Annals of Clinical and Translational Neurology. 5, 1394-1407 (2018).</li><li>Kovacs, G. 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=Intracellular+processing+of+disease-associated+alpha-synuclein+in+the+human+brain+suggests+prion-like+cell-to-cell+spread.">Intracellular processing of disease-associated alpha-synuclein in the human brain suggests prion-like cell-to-cell spread.</a> Neurobiology Disease. 69, 76-92 (2014).</li><li>Kovacs, G. 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=An+antibody+with+high+reactivity+for+disease-associated+alpha-synuclein+reveals+extensive+brain+pathology.">An antibody with high reactivity for disease-associated alpha-synuclein reveals extensive brain pathology.</a> Acta Neuropathologica. 124, 37-50 (2012).</li></ol>

我们描述了皮肤活检的免费浮动免疫荧光测定,用于诊断PD:它利用抗PGP9.5抗体的双重免疫染色剂、泛化标记物和抗5G4,一种识别聚合形式的构象特异性抗体*Syn。
在PD和可能在其他蛋白质构象障碍中,皮肤活检用于诊断目的的巨大优点是:1) 通过轻度侵入性技术直接进入容易患病的神经组织,从而具有预期的更好的测定与血液或CSF等生物流体相比,对βSyn聚集物的敏感性;2) 有机会检测?...

皮肤活检作为神经疾病领域的诊断和研究工具,已经变得越来越重要。事实上,表皮和真皮含有丰富的体性感觉神经纤维(骨髓和未骨髓),感知自由神经末梢,感觉受体和汗腺、血管、皮脂腺和肌肉呼吸体毛质的自主内侧2.
在20世纪中叶,PGP9.5抗体的免疫组织化学设置为人类表皮哺乳动物皮肤的广泛内生提供了证据。PGP9.5 是一种沿中枢和周围神经系统 (PNS) 的轴子均匀分布的碳化物终端水解酶。这种抗体的可得性不仅澄清了皮肤中PNS的形态和解剖学,还实现了与PNS相关的疾病的研究3,4。皮肤活检有助于定义一个新的临床实体:小纤维神经病变。几个国际团体通过皮肤活检分析证明了皮内神经纤维的丧失与小纤维神经病变5的症状/体征之间的联系,并为神经活检和神经活检提供了标准化的规程规范参考值用于临床实践6,7,8。
最近大量证据表明,神经退行性疾病,其特点是中枢神经系统中蛋白质积累错误,是多系统疾病。事实上,PD的特点是在基斯坦尼格拉的多巴胺能神经元中积累βSyn,但已经证明,βSyn及其病理形式磷酸化βSyn(P-αSyn)也可以在外围组织中检测到。胃肠粘液10,唾液腺11,皮肤自主纤维周围的汗腺和皮洛运动肌肉12,13,14,显示免疫反应的致病形式βSyn,在根据布拉克的假说,耐人寻味的假设,_Syn病理学可能开始在PNS提前,在它在大脑积累15。此外,p-syn 的存在已在 REM 行为障碍患者的皮肤神经中得到证明,这些患者被视为前体 PD16,17因此皮肤病理学 _Syn 可被视为有前途的早期外周核糖核酸组织病理学标记。
小纤维神经病变在PD的关联已经证明之前,并已发现,皮内神经纤维密度反映疾病进展18,19。因此,皮肤活检是研究PD神经退化和建立疾病的验尸前组织病理学诊断的有用工具。事实上,皮肤活检具有一个很大的优势,是一个容易接近和微创的程序,允许分析容易发生病理的神经组织。最后,在同一患者的随访过程中重复皮肤活检的可能性允许研究与疾病进展的纵向相关性。
在我们的实验室中,利用PGP9.5和构象特异性单克隆5G4抗体的双重免疫染色,识别疾病特定形式的βSyn,包括小聚合体20,21,我们能够显示在皮肤神经中存在βSyn聚集,具有有前途的高诊断效率19。结合蛋白质聚集物的检测和体内神经退化的测量,对构象性疾病的皮肤活检进行免疫荧光分析,是生物标志物的一个有前途的来源。下面,我们演示了处理皮肤活检和执行自由浮动免疫荧光染色以检测 _Syn 聚合物的简单而通用的协议。此外,该协议可以适应针对皮肤PNS表达的任何其他感兴趣的蛋白质。
以下研究方案已用于评估在PDPNS中通过皮肤活检19聚合+Syn分析的诊断效用。PD的纳入标准是:根据英国脑库诊断标准进行明确的临床诊断,疾病持续时间至少3年,无家族史,历史上无重大认知障碍或主要失学症状。排除标准是已知神经病变的原因(糖化血红蛋白、肌氨酸、维生素B12、TSH、血清免疫修复、艾滋病毒、丙肝病毒、梅毒和结节)。每个受试者在三个解剖部位(颈部在C8皮肤水平,大腿在膝盖上方10厘米,腿在侧侧10厘米以上)进行3毫米直径的皮肤活检,这在临床上受到的影响更大。一般来说,以下方案是关于处理皮肤活检和执行自由浮动免疫荧光染色和分析。因此,它可以被调整和用于检测皮肤组织中感兴趣的其他蛋白质。

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:264px;">5G4 (anti human &#945;Syneclein 5G4)</td>
			<td style="width:161px;">Analytik Jena Roboscreen</td>
			<td style="width:344px;">847-0102004001</td>
			<td style="width:195px;">Mouse monoclonal&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AlexaFluor 488 Goat anti Rabbit IgG&#160;</td>
			<td>Invitrogen</td>
			<td>1971418</td>
			<td>2mg/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AlexaFluor 594 Goat anti Mouse IgG&#160;</td>
			<td>Invitrogen</td>
			<td>1922849</td>
			<td>2mg/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Disodium hydrogen phosphate solution</td>
			<td>Merk Millipore</td>
			<td>106586</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ethylene Glycol</td>
			<td>Sigma-Aldrich</td>
			<td>324558</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G7757</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Lysine monohydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>L5626</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paraformaldehyde</td>
			<td>Aldrich Chemistry</td>
			<td>441244</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:264px;">PGP9.5</td>
			<td>Abcam</td>
			<td>ab15503</td>
			<td>Rabbit polyclonal</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium Chloride</td>
			<td>Sigma&#160;</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium Dihydrogen Phosphate Monohydrate</td>
			<td>Merck Millipore</td>
			<td>106346</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium (meta)periodate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S1878</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trizma Base</td>
			<td>Sigma&#160;</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tryton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vectashield&#160;</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>Mounting medium</td>
		</tr>
	</tbody>
</table>

targeting alpha synuclein aggregates in cutaneous peripheral nerve fibers by free-floating immunofluorescence assay

迄今为止,对于大多数神经退行性疾病,只有死后组织病理学的明确诊断可用。对于帕金森病 (PD),诊断仍然只依赖于运动参与的临床迹象,这些症状在疾病过程中出现,当时大多数多巴胺能神经元已经丢失。因此,我们强烈需要一种生物标志物,能够识别疾病开始时的患者或有患疾病的风险。在过去几年中,皮肤活检已被证明是一个优秀的研究和诊断工具,为周围神经疾病,如小纤维神经病变。有趣的是,一种小纤维神经病变和α核糖核酸(βSyn)神经沉积已经通过PD患者的皮肤活检显示。事实上,皮肤活检具有一种易于接近、微创和无痛的手术,能够分析易发生病理学的周围神经组织。此外,在同一患者的随访过程中重复皮肤活检的可能性允许研究与疾病进展的纵向相关性。我们建立了一个标准化的可靠协议,以研究PD患者皮肤神经纤维中是否存在βSyn聚集体。该协议涉及几个短的固定步骤,一个冷冻体切片,然后一个自由浮动免疫荧光双染色与两个特定的抗体:抗蛋白基因产品9.5(PGP9.5),以标记皮神经纤维和抗5G4检测*Syn 聚合。它是一种多功能、敏感且易于执行的协议,也可用于靶向皮肤神经中感兴趣的其他蛋白质。标记 _Syn 聚合体的能力是使用皮肤活检作为建立 PD 的验尸前组织病理学诊断的工具的又一进步。

按照所述程序(图1),我们在PD患者的皮神经裂解内化自主结构中检测到带有5G4抗体标记的βSyn聚合体。α-核素沉积的形态以皮肤神经的斧子上的虚线信号出现(图2)。事实上,在19个PD患者和3个解剖部位(宫颈、大腿和远端腿)的皮肤活检中利用此协议,我们发现5G4具有81%的敏感性和86%的特异性,对健康的控制和P-+Syn有56%的灵敏度和100%的特异性19。</su...

Watch this Scientific Journal Video about Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay at JoVE.com

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

在这里,我们提出了一个协议,在皮肤活检部分自由浮动间接免疫荧光测定,允许识别疾病特异性形态变异的阿尔法核糖核酸与多种蛋白质外围神经系统。

通过自由浮动免疫荧光测定,在皮外神经纤维中定位阿尔法核糖核酸聚合体

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...

帕金森病的诊断仍然依赖于在疾病过程中后来出现的运动参与的临床迹象，当时大多数的亚斯坦蒂纳的神经元已经丢失。该协议允许皮肤活检的皮肤神经分析，代表该疾病的潜在新生物标志物，用于临床试验。皮肤活检是一种易于评估的、不侵入性的程序，可以分析容易患病理学的周围神经组织。通过皮肤活检检测的α突核素聚合检测可用于诊断目的，可能在早期阶段，当潜在的治疗是最有效的。在同一患者中进行后续皮肤活检，可以对α合成素聚合在人类皮肤神经系统中传播进行时间和特殊过程分析。这些可以揭示疾病的发病机制。若要开始此过程，请准备新的 PLP 固定解决方案，如文本协议表 1 中所述。收集皮肤活检后，立即将皮肤淹没在含有10毫升固定溶液的管子中。在摄氏四度下孵育过夜。第二天，在烟气罩中，轻轻取出 PLP 固定。在同一管中，每次洗涤使用5毫升0.1摩尔索伦森溶液洗活检三次，每次洗涤五分钟。丢弃索伦森的溶液，并添加五毫升的低温保护溶液。在摄氏四度下孵育过夜。第二天，如果在一周内进行低温切割，活检将储存在摄氏四度。首先，将低温设置为零下20摄氏度。拿一个低温，并填补它与低温嵌入介质。使用钳子，将活检浸入低温嵌入介质中，纵向轴平行于低温的底部。用液氮捕捉样品，获得含有正确方向活检的低温嵌入介质的固体立方体。将样品放入低温恒温器中，等待 30 分钟，以便活检适应。然后，将样品固定到低温恒温器上，切成 50 微米部分。使用小刷子将低温部分转移到每个井中包含 200 微升防冻液的 96 井板中。在此之后，将盘子存放在零下20摄氏度。首先，用100微升的洗涤液填充一个新的96孔板的一些水井。将要分析的部分从存储板转移到包含洗涤溶液的储物板。在室温下将部分留在洗涤液中 10 分钟。然后，将每个部分转移到包含相同洗涤溶液的空井中，然后重复洗涤。接下来，将洗涤液添加到各部分，并将其转移到包含 100 微升的阻块溶液的新井中。在室温下孵育至少90分钟，最多4小时。在工作溶液中稀释原抗体、抗PGP9.5和抗5G4。将各部分转移到含有100微升原抗体工作溶液的新井中，并在室温下孵育过夜。第二天，洗净井中含有洗涤溶液的部分，如前所述。稀释二级抗体，山羊抗兔子检测PGP9.5，山羊抗小鼠检测5G4，在工作溶液中。将洗涤的部分转移到含有100微升二级抗体工作溶液的新井中。用铝箔盖住板，避免氟磷与二次抗体结合的漂白，并在室温下孵育90分钟。在此之后，如前所述，在洗涤溶液中洗涤两次。在室温下将洗净的部分转移到含有 100 微升 DAPI 的新井中，为期 5 分钟。如前所述，在洗涤液中清洗部分两次。然后，将幻灯片上的部分安装到正确的位置，避免错误折叠。在幻灯片中添加几滴安装介质，然后用盖滑盖住该部分。让幻灯片在一夜之间变干。第二天，将幻灯片存放在4摄氏度的适当盒子里，确保避免光线照射。使用倒置荧光显微镜或共和显微镜，查看各部分。通过连接到显微镜的摄像机获取图像。然后，使用荧光显微镜或共和显微镜，根据文本协议中概述的信号的空间分布和强度来分析各部分的正信号。按照所述方法，在PD患者皮肤神经分膜内注射自主结构中检测标有5G4抗体的α核素聚合体。阿尔法突核素沉积的形态显示为沿着皮肤神经轴突的虚线信号。在19例PD患者和3个解剖点的皮肤活检中，有代表性地使用此协议表明，与健康对症相比，5G4的灵敏度为81%，特异性为86%。磷酸化α突核素的灵敏度为56%，特异性为100%5G4，磷酸化α突触阳性沉积物主要在汗腺周围发现，但也存在于肌肉反应器皮利、小血管、亚皮末和皮肤丛中，但从未在内皮神经纤维中发现。一般来说，在汗腺流明中，可以观察到可能被误解为5G4或磷酸化阿尔法突触正向的非特异性信号。这种类型的信号是点状的，球形的，它最有可能是由于在发光内自发光材料，如在没有初级抗体和二级抗体的技术控制证明。使用 PGP9.5 进行结肠化，该纤维在形态学上是丝丝和拉长的，有助于识别正确的信号。因此，5G4信号的特异性通过带气管标记的双重免疫着色而得到高度提高。活检的准确固定对于产生高质量的免疫荧光染色和荧光信号的可靠解释是必要的。如果未正确准备固定剂，或者发生了过度固定，则结果将是高自荧光，将遮盖神经纤维穿过皮肤-表皮结或内侧主皮结构的信号。这个过程最关键的步骤是组织固定。注意PLP固定溶液的pH值和孵育时间，避免自荧光和特定信号。

Research

JoVE Journal

Neuroscience

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions 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

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

禽流感胚胎听神经听觉神经纤维的选择性跟踪

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

嗅鞘细胞移植周围神经损伤后，以评估功能恢复

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

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

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, ...

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

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 ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

创新 RNA原位检测技术在 D1-Alpha 鼠伏隔核中多巴胺受体的鉴定

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

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

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 ...

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

从单体和体内使用的Α-syr核 in 预成型纤维的产生

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

来自成人大脑的短期自由浮动切片培养

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

Assessing Early Stage Open-Angle Glaucoma in Patients by Isolated-Check Visual Evoked Potential

通过隔离检查视觉诱发潜力评估患者早期开放角青光眼

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and ...