この作品は、国家自然科学基金、中国の (U1503223、81673407、21475131)、浙江省の非営利技術 (2016 C 37110)、寧波国際科学技術に関する応用研究プロジェクトからの補助金によって支えられました。協力プロジェクト (2014 D 10019)、生命科学と健康 (2015 C 110026)、寧波科学技術プロジェクト共通の富 (2017 C 50042) オフイオチューカンあご・ ケネス ・李海洋バイオ医薬品開発基金李 Dak 合計イップ ・寧波市革新チーム・寧波大学 k. c. ウォン マグナ基金。

1. ソリューションの準備
注: は、試薬ソースの材料表を参照してください。
<ol><li>5% ウシ血清アルブミン (BSA) ソリューションを準備するには、二重蒸留水 100 mL に BSA の 5 g を追加します。ボルテックスによってそれらを完全にミックスします。1 ヶ月の 4 ° C でソリューションを保存します。</li>
	<li>5 %bsa 溶液 10 mL に抗体原液の 10 μ L の追加によって反オリゴマー抗体 A11 溶液 (1:1, 000) を準備します。ボルテックスによってそれらを完全にミックスします。1 ヶ月の 4 ° C でソリューションを保存します。</li>
	<li>抗 a β 抗体 6E10 溶液 (1:1, 000) を準備するには、5 %bsa 溶液 10 mL に抗体原液の 10 μ L を追加します。ボルテックスでは、完全にそれらをミックスします。1 ヶ月の 4 ° C でソリューションを保存します。</li>
	<li>アンチ維オリゴマー抗体 OC 溶液 (1:1, 000) を準備するには、5 %bsa 溶液 10 mL に抗体原液の 10 μ L を追加します。ボルテックスでは、完全にそれらをミックスします。1 ヶ月の 4 ° C でソリューションを保存します。</li>
	<li>二重蒸留水 1,000 mL に 24 g のトリス基地と NaCl の 88 g を追加することによって、含有トリス緩衝生理食塩水 (TBS) 原液を準備します。7.4 に pH を調整します。3 ヵ月 4 ° C でソリューションを保存します。</li>
	<li>準備トリス緩衝生理食塩水とトゥイーン 20 (TBST) ソリューション Tween 20 1 mL を 100 mL の TBS に追加することによって在庫ソリューションと 900 mL ダブル蒸留水。</li>
	<li>わさびの 10 μ L 追加ペルオキシダーゼ (HRP) ヤギ抗うさぎ IgG (H + L) TBST 溶液 10 mL に二次抗体溶液を準備します。ボルテックスによってそれらを完全にミックスします。1 ヶ月の 4 ° C でソリューションを保存します。</li>
	<li>ジメチルスルホキシド (DMSO) 10 mM クルクミン在庫ソリューションを形成するための 1 つの mL でクルクミンの 3.68 g を溶解します。</li>
	<li>2 mL の 1,1,1,3,3,3-ヘキサフルオロ-2-プロパノール (HFIP) 2.5 Mg/ml と a β の単量体ソリューションを形成するのに合成 a β1-42の 5 mg を溶解します。室温 (25 ° C) で 20 分、100 μ 因数の配置し、-20 ° C で 6 ヶ月保存します。 注: この手順をできるだけ速く実行する必要があります。ピペット チップは、正確なピペッティングを確実に切断する必要があります。</li>
	<li>1:1 の容積比で ECL 流体 A と B を混ぜて電気化学発光 (ECL) 液を準備します。このソリューションは、使用直前準備する必要があります。</li>
</ol>2. サンプル準備
注: は、ブロッティング解析ドットの前に 2 日間サンプル準備を実行します。
<ol><li>A β1-42モノマー溶液のチューブに二重蒸留水の 900 μ L を追加します。a β1-42の濃度は 0.25 mg/mL になります。20 分間室温で混合物を配置します。</li>
	<li>その量は約 850 μ L まで高純度の窒素ガスでソリューションを蒸発させます。A β1-42溶液の濃度は、0.29 mg/mL になります。 注: ソリューションを随時で動揺して、HFIP が完全に蒸発することを確認する必要があります。通常、蒸発の 30 分後に残液量は約 850 μ L です。</li>
	<li>二重蒸留水でクルクミン作業ソリューション (0.2 2 μ M) にクルクミン原液を希釈します。A β1-42のソリューションとクルクミン作業ソリューション 1:1 の容積比で混ぜます。クルクミンの最終濃度は、0.1 と 1 μ M になります。 注: 必要な場合任意の割合で a β1-42のソリューションの潜在的なオリゴマー阻害剤に混在できます。</li>
	<li>磁気攪拌機でソリューションを継続的に振る (材料の表を参照してください)。<ol>
<li>磁気攪拌機にプラスティック ボックスを修正します。場所 2 磁気は 2 ボックスのコーナーでバーをかき混ぜるし、ボックスの中央にサンプル チューブを置きます。</li>
		<li>48 時間室温 (25 ° C) でボックスを振る。 注: 磁気攪拌機の速度は約 60 rpm です。</li>
	</ol>
</li>
	<li>4 ° C で 15 分間チューブを遠心し、18,000 x g. 収集上清。</li>
</ol>3. ドットのしみが付く分析
注: すべての孵化は水平シェーカーで行われます。
<ol><li>硝酸セルロースの膜は、1 cm 幅の短冊状にカットします。 注: 硝酸セルロースの膜の幅は、実験のニーズに応じて調整できます。</li>
	<li>2 ストリップ (ストリップ 1 と 2) 各ポイント間隔 0.5 cm に 2 μ L のサンプルを均等に配置します。</li>
	<li>バンドに液滴が乾くまで、5 分間室温でストリップを配置します。</li>
	<li>室温で 30 分間 5 %bsa 溶液によるストリップを孵化させなさい。</li>
	<li>室温で 5 分間 TBST ソリューションとストリップをすすいでください。</li>
	<li>TBST ソリューションを吸い出しなさい。室温で 1 時間インキュベート ストリップ 1 反オリゴマー抗体 A11 ソリューションとストリップ抗 a β 抗体 6E10 ソリューションと 2。</li>
	<li>3 回部屋の温度で 5 分間ずつ TBST ソリューションとストリップをすすいでください。</li>
	<li>TBST ソリューションを吸い出しなさい。室温で 40 分間二次抗体溶液でストリップを孵化させなさい。</li>
	<li>3 回部屋の温度で 5 分間ずつ TBST ソリューションとストリップをすすいでください。</li>
	<li>ストリップの表面に混合の ECL 流体を均等に適用されます。イメージング システム自動発光でストリップを公開 (材料の表を参照してください)。 注: 通常、300 μ L の ECL 流体膜 1 つのための十分です。膜は、露光中に湿った保たれなければなりません。露出時間は、イメージング システムによって自動的に計算されます。</li>
	<li>半定量的な結果を取得する ImageJ (健康の国民の協会) または別の画像処理ソフトウェアを使用してグレースケール分析を行います。</li>
</ol>

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このプロトコルでは a β1-42のオリゴマーを含む試料を準備する分析をしみが付くことのドットで A11 正 a β1-42のオリゴマーの量を分析する方法を報告しています。A β1-42オリゴマーの豊富なサンプルの準備のための私たちの方法はかなりシンプルで、信頼性と再現性のある、まだ気づかれるべきいくつかのポイントがあります。まず、HFIP、合成 a β1-42ペプチド...

アルツハイマー病 (AD) は、高齢者の世界的な1に影響を与える最も重要な神経変性疾患の一つです。Β アミロイド (a β) の異常凝集が広告の主要な病理要因かもしれないことが広く受け入れられます。A β の凝集体は、老人斑、AD 患者の脳の生物学的マーカーの 1 つの主要なコンポーネントです。さらに、特に、オリゴマーを含めて、a β の集計は、AD の進行性神経細胞死の原因かもしれない強力な毒性を作成します。したがって、a β オリゴマー形成の抑制、神経変性を妨げる可能性し、a β オリゴマー阻害剤は、疾患修飾として開発される可能性 AD の治療。多くの研究は、オリゴマー体外を形成、人工の a β オリゴマーの構造と形態を探検し、調査を使用して生体外モデル2,3 a β オリゴマーの阻害剤を合成 a β ペプチドを使用しています。,4します。 しかし、a β オリゴマー形成プロトコル別の in vitroの異なる研究グループ間で比類のない結果が生じる別の形態学的特性を持つオリゴマーに 。したがって、a β オリゴマーの形成の標準的なプロトコルが急務します。
今までは、a β オリゴマーが直接検出することがない多くの方法を報告されています。透過電子顕微鏡 (TEM)、非変性ゲル電気泳動法、酵素免疫測定法 (ELISA) と分析にしみが付く点は、量および/または a β オリゴマー体外5,の形態を検討する使用ことができます。6。 形態と a β オリゴマー構造を TEM で観察するたとえば、します。相対的な量、a β の集計の分子サイズは、非変性ゲル電気泳動法によって測定できます。Elisa 法は、血清、血漿、脳組織からの抽出物で a β オリゴマーを決定する使用できます。最後に、ドット解析技術で検出すると、しみが付くことを分析して、タンパク質の同定に使えるオリゴマーおよび a β に固有の抗体の助けを借りて、異なるサンプル a β オリゴマーの相対濃度を評価します。また、試金を吸い取りドット ゲル電気泳動およびゲルのあぶらとりのプロシージャは必要ないため大幅な時間節約を提供しています。したがって、この試金は通常、画面 a β オリゴマー阻害剤を使用します。このプロトコルの全体的な目標は、ブロッティング解析、ドット、画面 a β オリゴマー a β1-42のオリゴマーの量を分析するため、a β1-42オリゴマーの豊富なサンプルを準備する比較的単純な信頼性と再現性のあるメソッドを記述するのには半定量的実験結果を用いた阻害剤。

<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 アミロイド (a β) は集計に自己組み立てるに本質的な傾向と疎水性ペプチドです。各種骨材の中で a β オリゴマーはアルツハイマー病 (AD) の進行中の主要な神経毒として広く受け入れられている、AD の発症に重要なイベントであると見なされます。したがって、a β オリゴマー阻害剤が神経変性を防ぐため、疾患修飾として開発される可能性を秘めている AD の治療。しかし、a β オリゴマーの異なる形成プロトコルは、異なる特性を持つオリゴマーに可能性があります。さらに、効果的に画面 a β1-42オリゴマー阻害する多くの方法がないです。A11 抗体は逆平行 β シート構造を持つ有毒な a β1-42のオリゴマーのサブセットと反応できます。このプロトコルでは、合成 a β1-42ペプチド体外から A11 正 a β1-42オリゴマーの豊富なサンプルを準備し、ドットのしみが付くことによってサンプルの A11 正 a β1-42のオリゴマーの相対量を評価する方法について述べるA11 と a β1-42を用いた解析-特定 6E10 抗体。このプロトコルを使用して、A11 正 a β1-42のオリゴマーの阻害剤は、半定量的な実験結果から上映されることができます。

A β1-42の単量体が準備の後 a β1-42オリゴマーを形成できるかどうかを調べるには、TEM 分析を使用しました。HFIP 溶解 a β1-42モノマー サンプル (図 1A) で表示される集計は認められなかった。また、周りの直径を持つ主に球状骨材 10 80 nm で観察された a β1-42サンプルの振動、48 時間後 a β1-42の?...

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

このプロトコルでは、体外で合成ペプチド a β オリゴマーを準備しドット解析をしみが付くことによって a β オリゴマーの相対量を評価する方法について説明します。

A11 肯定的なアミロイド β オリゴマーの合成と評価を使用してドットのしみが付く分析

&#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

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JoVE Journal

Biochemistry

11.7K ビュー.  Ningbo University. このプロトコルでは、体外で合成ペプチド a β オリゴマーを準備しドット解析をしみが付くことによって a β オリゴマーの相対量を評価する方法について説明します。

ビデオ: 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.

β-アミロイドオリゴマーの調製 1から42まで</sub海馬スライスでのシナプス可塑性の減損の>と誘導

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

神経科学

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.

ヒト皮質組織におけるAβ多量体のSDS-PAGE/Immunoblotの検出は、抗原エピトープの検索を使用してホモジネート

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.

オリゴマー形成可能な高純度のアミロイドベータ42とアミロイドベータ40ペプチドを、利回りテーラードHPLC精製プロトコル

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

生化学

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.

アミロイド組織イメージング ステンド グラス ハイパー スペクトルで発光共役オリゴチオフェンと共焦点顕微鏡と蛍光寿命イメージング

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.

アルツハイマー病のマウスモデルにおける神経突起斑の検出

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.

合成アミロイド β 蛋白の脳室内投与によるラットの動物モデルにおけるアルツハイマー病の貴重な模倣の確立

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

行動学

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.

βアミロイド斑とミクログリアの相互作用を研究するために相関光と電子顕微鏡

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.

定量的3D<em&gt;インシリコ</emアルツハイマー病の齧歯類モデルにおける脳アミロイドベータ食作用の&gt;モデリング（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.

脳からのアミロイド線維コアの生化学的浄化とプロテオミクス特性評価

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

A11 肯定的なアミロイド β オリゴマーの合成と評価を使用してドットのしみが付く分析

要約

さらに動画を探す

この動画の章

β-アミロイドオリゴマーの調製_{1から42までと誘導}

ヒト皮質組織におけるAβ多量体のSDS-PAGE/Immunoblotの検出は、抗原エピトープの検索を使用してホモジネート

オリゴマー形成可能な高純度のアミロイドベータ42とアミロイドベータ40ペプチドを、利回りテーラードHPLC精製プロトコル

アミロイド組織イメージングステンドグラスハイパースペクトルで発光共役オリゴチオフェンと共焦点顕微鏡と蛍光寿命イメージング

アルツハイマー病のマウスモデルにおける神経突起斑の検出

合成アミロイド β 蛋白の脳室内投与によるラットの動物モデルにおけるアルツハイマー病の貴重な模倣の確立

βアミロイド斑とミクログリアの相互作用を研究するために相関光と電子顕微鏡

定量的3D

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

脳からのアミロイド線維コアの生化学的浄化とプロテオミクス特性評価

A11 肯定的なアミロイド β オリゴマーの合成と評価を使用してドットのしみが付く分析

要約

さらに動画を探す

この動画の章

β-アミロイドオリゴマーの調製 1から42までと誘導

ヒト皮質組織におけるAβ多量体のSDS-PAGE/Immunoblotの検出は、抗原エピトープの検索を使用してホモジネート

オリゴマー形成可能な高純度のアミロイドベータ42とアミロイドベータ40ペプチドを、利回りテーラードHPLC精製プロトコル

アミロイド組織イメージング ステンド グラス ハイパー スペクトルで発光共役オリゴチオフェンと共焦点顕微鏡と蛍光寿命イメージング

アルツハイマー病のマウスモデルにおける神経突起斑の検出

合成アミロイド β 蛋白の脳室内投与によるラットの動物モデルにおけるアルツハイマー病の貴重な模倣の確立

βアミロイド斑とミクログリアの相互作用を研究するために相関光と電子顕微鏡

定量的3D

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

脳からのアミロイド線維コアの生化学的浄化とプロテオミクス特性評価

β-アミロイドオリゴマーの調製_{1から42までと誘導}

アミロイド組織イメージングステンドグラスハイパースペクトルで発光共役オリゴチオフェンと共焦点顕微鏡と蛍光寿命イメージング