这项工作得到了中国国家自然科学基金 (U1503223, 81673407, 21475131) 的资助, 浙江省非营利技术应用研究项目 (2016C37110), 宁波国际科技合作项目 (2014D10019)、宁波市生命科学与健康创新团队 (2015C110026)、宁波市科技 & 科技项目共同财富 (2017C50042)、李家和业杨厝港钦利海洋生物制药发展基金,宁波大学的肯塔基州金麦格纳基金。

1. 解决方案准备
注: 有关试剂来源, 请参阅材料表。
<ol><li>在100毫升的双蒸馏水中加入5克 bsa, 制备5% 牛血清白蛋白 (bsa) 溶液。涡流他们完全混合。将解决方案存储在摄氏4摄氏度, 最多1月。</li>
	<li>在 5% BSA 溶液的10毫升中加入10µL 抗体储存液, 制备抗低聚物抗体 A11 溶液 (1:1, 000)。涡流他们完全混合。将解决方案存储在摄氏4摄氏度, 最多1月。</li>
	<li>在10毫升 5% BSA 溶液中加入10µL 抗体库溶液, 制备抗 Aβ抗体6E10 溶液 (1:1, 000)。完全混合涡流。将解决方案存储在摄氏4摄氏度, 最多1月。</li>
	<li>制备抗纤维寡聚物抗体 OC 溶液 (1:1, 000), 添加10µL 抗体库溶液到10毫升 5% BSA 溶液。完全混合涡流。将解决方案存储在摄氏4摄氏度, 最多1月。</li>
	<li>在1000毫升的双蒸馏水中加入24克的三基和88克氯化钠, 制备三缓冲盐水 (TBS) 溶液。将 pH 值调整为7.4。将解决方案存储在摄氏4摄氏度, 长达3月。</li>
	<li>通过增加1毫升的 Tween-20 到100毫升的 Tween-20 溶液和900毫升的双蒸馏水, 准备一个三缓冲盐水和 TBST 溶液。</li>
	<li>通过添加10µL 辣根过氧化物酶 (HRP) 山羊抗兔 IgG (H + L) 到10毫升的 TBST 溶液, 制备二级抗体溶液。涡流他们完全混合。将解决方案存储在摄氏4摄氏度, 最多1月。</li>
	<li>在1毫升二甲基亚砜 (亚砜) 中溶解3.68 克姜黄素, 形成10毫米姜黄素溶液。</li>
	<li>溶解5毫克合成 Aβ1-42在2毫升 11, 13, 33-氟, 2-丙醇 (HFIP), 形成一个2.5 毫克/毫升 Aβ单体溶液。把它放在室温下 (25 °c) 20 分钟, 使100µL 整除数, 并存储在-20 摄氏度, 长达6月。 注意: 此过程应尽可能快地运行。应切断吸管的提示, 以确保准确的吹打。</li>
	<li>将 ecl 流体 a 和 B 混合, 制备出电化学发光 (ecl) 流体, 体积比为1:1。此解决方案应在使用前准备就绪。</li>
</ol>2. 样品准备
注: 在斑点印迹分析前2天进行样品准备。
<ol><li>将900µL 的双蒸馏水添加到 Aβ1-42单体溶液的管中;Aβ1-42的浓度将为0.25 毫克/毫升。将混合物室温放置20分钟。</li>
	<li>用高纯度的氮气蒸发溶液, 直到其体积约为850µL。Aβ1-42解决方案的浓度将约为0.29 毫克/毫升。 注意: 该解决方案应不时动摇, 以确保 HFIP 完全蒸发。通常, 在蒸发30分钟后, 剩余溶液的体积约为850µL。</li>
	<li>用双蒸馏水稀释姜黄素溶液, 以姜黄素工作溶液 (0.2 和2µM)。将 Aβ1-42解决方案和姜黄素工作解决方案混合在一起, 体积比为1:1。姜黄素的最终浓度将是0.1 和1µM。 注: 如果需要, 潜在的低聚物抑制剂可与 Aβ1-42解决方案混合使用。</li>
	<li>在磁力搅拌器中连续摇动溶液 (请参阅材料表)。<ol>
<li>将塑料分隔盒固定在磁力搅拌器上。在盒子的2个角落放置2个磁力搅拌棒, 将样品管放在盒子的中心。</li>
		<li>在室温下摇动盒子 (25 °c), 48 小时。 注: 磁力搅拌器的速度大约是 60 rpm。</li>
	</ol>
</li>
	<li>离心管15分钟在4°c 和 1.8万 x g. 收集上清。</li>
</ol>3. 斑点印迹分析
注: 所有孵化均在卧式振动筛上执行。
<ol><li>将硝化棉膜切成1厘米宽的长条。 注: 硝化棉膜的宽度可根据实验需要进行调整。</li>
	<li>2-µL 样品均匀地在2条 (条1和 2) 与每个点间隔在 0.5 cm。</li>
	<li>将小条放在室温下5分钟, 直到带上的水滴干燥。</li>
	<li>用 5% BSA 溶液在室温下孵化出30分钟的小条。</li>
	<li>在室温下用 TBST 溶液冲洗带5分钟。</li>
	<li>吸入 TBST 溶液。在室温下1小时, 孵育1的抗寡聚物抗体 A11 溶液和带2的抗 Aβ抗体6E10 溶液。</li>
	<li>用 TBST 溶液冲洗带三次, 每次室温5分钟。</li>
	<li>吸入 TBST 溶液。在室温下用二次抗体溶液孵化带40分钟。</li>
	<li>用 TBST 溶液冲洗带三次, 每次室温5分钟。</li>
	<li>将混合的 ECL 流体均匀地应用于带钢表面。在自动化学发光成像系统中暴露条纹 (请参阅材料表)。 注: 正常情况下, 300 µL 的 ECL 流体足以用于一膜。膜在暴露时必须保持湿润。曝光时间由成像系统自动计算。</li>
	<li>使用 ImageJ (国立卫生研究院) 或其他图像处理软件进行灰度分析, 以获得半定量结果。</li>
</ol>

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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=In+vitro+amyloid+aggregate+forming+ability+of+TGFBI+mutants+that+cause+corneal+dystrophies.">In vitro amyloid aggregate forming ability of TGFBI mutants that cause corneal dystrophies.</a> Investigative Ophthalmology &amp; Visual Science. 53 (9), 5890-5898 (2012).</li><li>Tomaselli, 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=The+alpha-to-beta+conformational+transition+of+Alzheimer's+A+beta-(1-42)+peptide+in+aqueous+media+is+reversible:+A+step+by+step+conformational+analysis+suggests+the+location+of+beta+conformation+seeding.">The alpha-to-beta conformational transition of Alzheimer's A beta-(1-42) peptide in aqueous media is reversible: A step by step conformational analysis suggests the location of beta conformation seeding.</a> ChemBioChem. 7 (2), 257-267 (2006).</li><li>Shigemitsu, Y., 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=Nuclear+magnetic+resonance+evidence+for+the+dimer+formation+of+beta+amyloid+peptide+1-42+in+1,1,1,3,3,3-hexafluoro-2-propanol.">Nuclear magnetic resonance evidence for the dimer formation of beta amyloid peptide 1-42 in 1,1,1,3,3,3-hexafluoro-2-propanol.</a> Analytical Biochemistry. 498, 59-67 (2016).</li><li>Khan, M. 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作者没有什么可透露的。

在本协议中, 我们报告了一种方法, 用于准备包含 Aβ1-42低聚物的样本, 并通过斑点印迹分析来分析 A11-positive Aβ1-42寡聚的数量。虽然我们的制备 Aβ1-42富聚物样品的方法非常简单、可靠、重现性好, 但仍有一些值得注意的点。首先, HFIP 用于溶解合成 Aβ1-42肽。聚合 Aβ1-42肽可以在 HFIP 溶液中分解成单体。但是, HFIP 容易挥发, HFIP 的粘度很低。因此, Aβ1-42</s...

阿尔茨海默病 (AD) 是影响全球老年人的最重要的神经退行性疾病之一1。普遍认为β淀粉样蛋白 (Aβ) 的异常聚集可能是 AD 的主要病理因子。Aβ骨料是老年斑块的主要成分, 是 AD 患者大脑中的生物学标志物之一。此外, Aβ聚集物, 特别是寡聚物, 产生强大的神经毒性, 这可能是导致神经元死亡的原因。因此, 抑制 Aβ寡聚物的形成可以防止变性, Aβ寡聚物抑制剂可作为 AD 的病害修饰处理。许多研究使用合成 Aβ肽在体外形成寡聚物, 探讨人工 Aβ寡聚的形貌和结构, 并利用体外模型2,3对 Aβ寡聚物的抑制剂进行研究。,4. 然而, 不同的体外形成协议的 Aβ寡聚物可能导致不同形态特征的聚类, 这可能导致不同研究组之间的不可比拟的结果。因此, 迫切需要一种标准的 Aβ齐聚物生成协议。
到目前为止, 还没有多少方法可以直接检测 Aβ寡聚物。透射电镜 (TEM)、非变性凝胶电泳、酶联免疫吸附试验 (ELISA) 和斑点印迹分析可用于检测 Aβ寡聚物的数量和/或形态学体外5,6. 例如, Aβ齐聚物的形态和结构可以在 TEM 中观察到。用非变性凝胶电泳法测定 Aβ聚合的相对含量和分子量。ELISA 可用于测定血清、血浆和脑组织提取物中的 Aβ寡聚体。最后, 斑点印迹分析, 一种用于检测、分析和识别蛋白质的技术, 可用于评价不同样品中 Aβ齐聚物的相对浓度, 并借助于寡聚特异性和 Aβ特异抗体。此外, 斑点印迹检测提供了大量的时间节省, 因为凝胶电泳和印迹程序的凝胶是不需要的。因此, 这种检测通常用于筛查潜在的 Aβ寡聚物抑制剂。本协议的总体目标是描述一种相对简单、可靠且可重现的方法, 用于准备 Aβ1-42富聚物的样本, 通过斑点印迹分析来分析 Aβ1-42低聚物的数量, 并筛选 Aβ寡聚使用半定量实验结果的抑制剂。

<table><tbody><tr><td>A11 (ab126892 Rb pAb to Amyloid Oligomers)</td><td>Abcam</td><td>GR91739-58</td><td></td></tr><tr><td>6E10 (beta-Amyloid Rabbit Ab)</td><td>Cell&amp;nbsp;Signalling Technology</td><td>2454S</td><td></td></tr><tr><td>OC (Anti-Amyloid Fibrils OC Antibody)</td><td>Millipore</td><td>AB2286</td><td></td></tr><tr><td>Horseradish Peroxidase (HRP) Marker Goat anti rabbit IgG (H+L)&amp;nbsp;</td><td>Beyotime</td><td>A0208</td><td></td></tr><tr><td>A&amp;beta;&lt;sub&gt;1-42&lt;/sub&gt;</td><td>GL Biochem</td><td>52487</td><td></td></tr><tr><td>1,1,1,3,3,3-Hexafiuoro-2-propanol</td><td>Aladdin</td><td>I1523078</td><td></td></tr><tr><td>Curcumin</td><td>Sigma</td><td>C1386</td><td></td></tr><tr><td>Albumin Bovine V</td><td>Solarbio</td><td>A8020</td><td></td></tr><tr><td>Sodium chloride</td><td>Sangon Biotech</td><td>D920BA0003</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>SCR</td><td>30166428</td><td></td></tr><tr><td>TRIS</td><td>Solarbio</td><td>T8060</td><td></td></tr><tr><td>Glycine</td><td>Solarbio</td><td>G8200</td><td></td></tr><tr><td>&amp;nbsp;Dimethyl sulfoxide</td><td>Solarbio</td><td>D8370</td><td></td></tr><tr><td>5&amp;times;nondenaturing gel PAGE Protein Marker</td><td>Beyotime</td><td>P0016</td><td></td></tr><tr><td>Genshare CFAS anyKD PAGE</td><td>Genshare</td><td>JC-PE022</td><td></td></tr><tr><td>Pure Nitrocellulose Blotting Membrane</td><td>Pall Corporation</td><td>T50189</td><td></td></tr><tr><td>Methanol</td><td>SCR</td><td>10014118</td><td></td></tr><tr><td>Ethanol</td><td>SCR</td><td>10009218</td><td></td></tr><tr><td>Super low range&amp;nbsp;protein Marker</td><td>Solarbio</td><td>PR1300</td><td></td></tr><tr><td>Transfer Membranes</td><td>Immobilon-PSQ</td><td>ISEQ00010</td><td></td></tr><tr><td>BeyoECL Star</td><td>Beyotime</td><td>P0018A</td><td></td></tr><tr><td>Commassie Blue Fast staining solution</td><td>Beyotime</td><td>P0017</td><td></td></tr><tr><td>All - automatic chemiluminescence imaging system</td><td>Tanon</td><td>Tanon 5200</td><td></td></tr><tr><td>Image J</td><td>National Institutes of Health</td><td></td><td></td></tr><tr><td>Image processing software</td><td>Adobe</td><td>Photoshop CS6</td><td></td></tr><tr><td>Magnetic agitator</td><td>Shanghai Huxi</td><td></td><td></td></tr></tbody></table>

a11-positive &#946;-amyloid oligomer preparation and assessment using dot blotting analysis

β淀粉样蛋白 (Aβ) 是一种疏水性肽, 具有自组装成骨料的内在倾向。在各种团聚体中, Aβ寡聚物作为阿尔茨海默病 (ad) 进展的主要神经毒素被广泛接受, 被认为是 ad 发病的关键事件。因此, Aβ寡聚物抑制剂可以防止变性, 并有潜力发展为疾病修改治疗的 AD。然而, Aβ寡聚物的不同形成方案可能会导致不同特征的寡聚。此外, 有效筛选 Aβ1-42寡聚物抑制剂的方法也不多。A11 抗体可以与含有反平行β片结构的毒性 Aβ1-42齐聚物的子集发生反应。在本协议中, 我们描述了如何从 Aβ的合成 Aβ1-42肽的体外准备一个 A11-positive 的1-42富齐聚的样本, 并通过斑点印迹来评估样本中 A11-positive Aβ1-42齐聚物的相对数量使用 A11 和 Aβ1-42特定6E10 抗体进行分析。使用该协议, A11-positive Aβ1-42齐聚物的抑制剂也可以从半定量实验结果中筛选出来。

为研究 Aβ1-42单体在制备后是否能形成 Aβ1-42齐聚物, 采用 TEM 分析。在 HFIP 溶解 Aβ1-42单体样本中未观察到可见骨料 (图 1A)。此外, 在48小时的震动后, 在 Aβ1-42样本中观察到了直径约 10-80 nm 的球状骨料, 这表明 Aβ1-42在准备后形成寡聚 (图 1B)。
<p class="jove_content...

Watch this Scientific Journal Video about A11-positive Î²-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis at JoVE.com

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

本协议描述如何从合成肽体外制备 Aβ寡聚物, 并通过斑点印迹分析来评价 Aβ的相对含量。

用斑点印迹分析 A11-positive β淀粉样寡聚物的制备与评价

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer ...

a11-positive-amyloid-oligomer-preparation-assessment-using-dot

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

11.7K 次观看.  Ningbo University. 本协议描述如何从合成肽体外制备 Aβ寡聚物, 并通过斑点印迹分析来评价 Aβ的相对含量。

视频: A11-positive β-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

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

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

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

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

低聚β-淀粉样蛋白的制备 1-42和突触可塑性损伤诱导海马脑片

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

科研

神经科学

SDS-PAGE/Immunoblot Detection of A&beta; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis1. A&beta; is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into &beta;-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble A&beta; multimers are more toxic than fibrillar A&beta; amyloid2. The identification and quantification of low- and high-molecular weight multimeric A&beta; species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies3. Naturally occurring A&beta; multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with A&beta;-specific antibodies. However, the separation and detection of multimeric A&beta; requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the A&beta; peptide. Using this protocol, we consistently detect A&beta; monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.

SDS-PAGE/Immunoblot Detection of A&#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the A&beta; peptide. Using this protocol, we consistently detect monomeric and multimeric A&beta; in cortical tissue from humans with Alzheimer's pathology.

SDS-PAGE/Immunoblot检测人脑皮层组织中的Aβ多聚匀浆使用抗原表位检索

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's ...

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

一个定制HPLC纯化协议，产量高纯度淀粉样蛋白β42和贝塔淀粉样蛋白肽40，有能力寡聚物形成的

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

生物化学

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

用高光谱共聚焦显微镜和荧光寿命成像对发光共轭吩染色的淀粉样组织的成像

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

在阿尔茨海默氏症小鼠模型神经炎斑检测

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

Establishment of a Valuable Mimic of Alzheimer's Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure change. With the increase of AD patients worldwide, the pathology and treatment of the disease has become a focus in the International Pharmaceutical Industry. Thus, the establishment of the animal model to mimic AD in the laboratory is of great importance.
Here, we describe a detailed protocol for establishing a mimic of AD in a rat animal model though intracerebroventricular injection of amyloid beta protein 25-35 (A&#946;25-35) combined with aluminum trichloride (AlCl3) and anterodorsal thalamic nucleus injection of recombinant human transforming growth factor-&#946;1 (RHTGF-&#946;1) to rats. The related markers of AD were measured, including: spatial memory, neuronal structure and substructure, neuronal A&#946;, and neurofibrillary tangles (NFT) production. This rat model demonstrates spatial memory impairment, neuronal structure and substructure pathological changes, neuron intracellular A&#946; burden, and NFT aggregation, and provides a close mimic of the neuronal structure and function disorder to that of clinical AD patients. Thus, the presented AD rat modelprovides a valuable in vivo tool for exploring neuronal function, neuronal pathology, and drug screening of AD.

This is a protocol to mimic Alzheimer&#39;s Disease in rats by evaluation of spatial memory impairment, neuronal pathological changes, neuronal amyloid beta protein (A&#946;) burden, and neurofibrillary tangles aggregation, induced by the injection of A&#946;25-35 combined with aluminum trichloride and recombinant human transforming growth factor-&#946;1.

脑室注射复合淀粉样蛋白β模型建立大鼠阿尔茨海默病的价值模拟

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure ...

行为学

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label &#946;-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for A&#946; (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing A&#946; plaques at the right location. Importantly, A&#946; plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

This article describes a protocol for visualizing amyloid A&#946; plaques in Alzheimer&#39;s disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

定量3D<em&gt;在硅片</em&gt;建模阿尔茨海默病的啮齿动物模型脑淀粉样蛋白-β吞噬（q3DISM）

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

医学

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

来自大脑的淀粉样原纤维核心的生化纯化和蛋白质组学表征

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

用斑点印迹分析 A11-positive β淀粉样寡聚物的制备与评价

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低聚β-淀粉样蛋白的制备_1-42和突触可塑性损伤诱导海马脑片

SDS-PAGE/Immunoblot检测人脑皮层组织中的Aβ多聚匀浆使用抗原表位检索

一个定制HPLC纯化协议，产量高纯度淀粉样蛋白β42和贝塔淀粉样蛋白肽40，有能力寡聚物形成的

用高光谱共聚焦显微镜和荧光寿命成像对发光共轭吩染色的淀粉样组织的成像

在阿尔茨海默氏症小鼠模型神经炎斑检测

脑室注射复合淀粉样蛋白β模型建立大鼠阿尔茨海默病的价值模拟

相关光学和电子显微镜，研究与β淀粉样蛋白斑块的小神经胶质互动

定量3D

Detecting Amyloid-β Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

来自大脑的淀粉样原纤维核心的生化纯化和蛋白质组学表征