エレインPranskiと優秀な技術支援のためのキャロラインSuwynへとハリーレヴァインIII、M.ポールマーフィー、および重要な会話のためにギアリング​​マーラに感謝します。資金は、RR - 00165、PO1AG026423、P50AG025688、AG030539、ウッドラフ財団、エモリー大学研究委員会によって提供されていました。

パート1：明確に組織ホモジネートの調製<ol><li>約30にも乳棒ストローク（すなわち400μlのバッファを追加すると - （プラス2倍プロテアーゼ阻害剤カクテル[サンタクルス] [およびMg + + -遊離Ca + +] 0.1Mリン酸緩衝生理食塩水）の氷冷緩衝液の4倍のボリュームで固定されていない皮質組織をホモジナイズダウンス100mgの組織への）。 </li><li> 3000グラム（4℃）で5分間ホモジェネートを回転させる。慎重に-80明確上澄み液と、これらの抽出物のストアアリコートを削除° Cを使用するまで。 </li><li>メーカーの指示（ThermoFisher）によると、ビシンコニン酸（BCA）アッセイを用いて明らかにホモジネートの総タンパク質濃度（μg/μl）を決定します。 </li></ol>パート2：SDS - PAGEのサンプル調製<ol><li>よく10、10から20パーセントトリシンゲル（Invitrogen）を用いて、ウェル毎にロードできる最大量は、〜25μlのです。総体積が2倍のSDSサンプルバッファーと10倍還元剤を含む、ウェル当たり読み込むことができる明確ホモジネートの最大量は10μlのですので。明らかに20％（w / v）の皮質ホモジネートは、ウェルあたり50μgの総蛋白質を考慮し、5μg/μLのより大きいの総タンパク質濃度を持つ必要があります。 5mg/mlのタンパク質より少ないサンプルでは、​​ウェルあたり少なく総タンパク質をロードする必要となります。しかし、高タンパク質レベルは、単量体および凝集したAβ（50 -60μgの総タンパク質が最適である）の検出に最適です。 常にウェルあたりの総タンパク質の同量をロードする。 </li><li>それぞれのゲルのため、負荷などSeeBlue Plus2（Invitrogen社）などの分子量マーカーの10μlを持つだけでなく少なくとも一つ。ポジティブコントロールとして、合成Aβ40または別の同様のAβ42ペプチド（1xPBSで希釈）を10 - 100ngを実行します。 </li><li>たての融解、明らかホモジネートを用いて、氷上で反応混合物を準備します。 5〜10秒間ボルテックスチューブ、100℃、乾燥槽内の熱℃で5分間、その後すぐにキャップで結露を除去するためにすべてのサンプルをスピン。 </li></ol>パート3：SDS - PAGEゲル電気泳動<ol><li> 、メーカーの指示（Invitrogen社）によると、トリシンSDSランニングバッファーを用いて、X細胞確かにロックミニセルGelboxで10〜20％トリシンゲル上にボルテックスのサンプルをロードする</li><li>約90分間125Vの定電圧で泳動する。 4KDaマーカーのバンドがゲルの下端から約1cmになるまでのサンプルが実行してみましょう。 </li></ol>パート4：ゲルから膜への転送タンパク質<ol><li>慎重にメーカーの指示（Invitrogen社）によると、プラスチックケースからゲルを取り出して、X細胞IIブロットモジュール内部の転送サンドイッチを組み立てる。トリス - グリシントランスファーバッファー（20％メタノール）によるプリウェットブロッティングパッドおよび0.2μmのニトロセルロース膜、およびパッドまたはろ紙/メンブレンサンドイッチをブロッティングから気泡を取り除く。 </li><li> X細胞Gelboxでは、転送バッファで、内部チャンバーを記入し、DIH 2 O（メタノールへの暴露が時間をかけてプラスチックgelboxを身に着けることができる）と外室を埋める。 </li><li> 25mAの一定のアンペア数で2-3時間の転送を実行します。 </li><li>転送が完了したら、サンドイッチを分解すると、両方の正方形のプラスチックペトリ皿で1xPBS、（または他の適当な容器）にDIH 2 Oと膜にゲルを置く。 </li><li>タンパク質の転送の効率を可視化するために、ゲルは単にブルーSafeStain（インビトロジェン社）で染色することができ、ポンソーSレッド染色（シグマアルドリッチ）を有する膜、両方の製造元の指示に従って。この染色は、免疫ブロット法に干渉することはありません。染色は、非効率的なタンパク質の転送があることが判明した場合は、手順3で転送時間を変更します。 </li></ol>第5部。抗原エピトープの検索＆イムノ<ol><li>抗原賦活化は、免疫ブロット中に抗体の結合のための膜にAβエピトープを明らかにする重要なステップです。どんな蒸し器、電子レンジ、または100の温度を一定に維持° Cで十分です水浴。蒸し器での抗原の検索の場合は、室温で、1xPBSで満たされたヘビーデューティKapakのプラスチック袋の膜を熱融着させる。あらかじめ温めておいた蒸し器のフラットポーチを置く、ポーチは、さらに15分間インキュベート、拡大して始まると。膜が過度のしわを防ぐため、袋から取り出す前に、ゆっくりと冷却してください。 </li><li>室温で、慎重に蒸し袋からメンブレンを取り出し、5分（0.05％のTween - 20で生理食塩水、pH8.0のをTris緩衝TBS - Tで洗浄、続い1xPBSで5分間メンブレンを、すすぎ[シグマ] ）、オービタルシェーカー上で。揺れ、一時間ブロッキング溶液で膜（TBS - Tで2.5％脱脂乳）をインキュベートする。洗浄せずに、泡を避け、（1:1,000で6E10 [1μg/mlの] 5,000 [0.2μg/ml]希釈、Covance）ブロッキング溶液で希釈した一次抗体を含んでいる皿やプラスチック製の袋に膜を転送S.一時間してから、4℃で振盪24〜48時間室温でオービタルシェーカー上でインキュベートする（より長いインキュベーションがより良好な信号を与えるかもしれない）。 </li><li> TBS - Tで30分間メンブレンをリンス（ワンクイック3 × 10分のリンスに続いてリンス）。 </li><li>室温でシェーカー上で90分間ブロッキング溶液に1:10,000に希釈したHRP -標識二次抗体のメンブレン（Amersham ECLヒツジ抗マウス、GE Healthcare）を、、インキュベートする。 </li><li> TBS - Tで30分間メンブレンをリンス（ワンクイック3 × 10分のリンスに続いてリンス）。 </li><li>シェーカー上で、プラスチックシートプロテクターの間に、糸くずのろ紙上に余分な試薬をオフブロットおよびフィルムカセットにメンブレンを置き、5分間たてのSuperSignalウエストのピコの電気化学発光試薬（ThermoFisher）で膜をインキュベートする。ダストフリーキムワイプ、必要に応じて追加のSuperSignal試薬をオフに汚点。 </li><li>最大30分30秒の間隔でコダックバイオマックスMRフィルムに感光させるとフィルムの開発者で開発する。 5分後、Aβモノマーのバンドは、おそらく飽和状態になることがあります。 </li><li>膜は、室温で30分間の復元プラスストリッピングバッファーで振盪することによって（TBS - Tで洗浄後に）取り除かれ、必要に応じて追加の抗体で再プローブすることができます。 </li></ol>パート6：代表イムノブロット： <img src="/files/ftp_upload/1916/1916fig1a.jpg" alt="図1a" /> <img src="/files/ftp_upload/1916/1916fig1b.jpg" alt="図1b" /> <img src="/files/ftp_upload/1916/1916fig1c.jpg" alt="図1c" /> 図1a - C。7ヒト被験者から50μgの総タンパク質を含む清澄ホモジネートは、多量体AβとAPPの存在について分析されています。抗体6E10でイムノブロッティングするAβモノマー、ダイマー、トリマー、テトラマー、およびすべてのアルツハイマー病のケースでAPP（上部バンド）、同様に2段のAD症例の豊富な高い分子量のAβの多量体を明らかにする。合成Aβ40は、低分子量のバンドのアイデンティティを確認する。 AD：アルツハイマー病、DLB：レビー小体型、NDと認知症：ヒトNondemented（）30分のフィルム露光、（b）に 5分間のフィルム露光、（C）30秒のフィルム露光。

<ol>
	<li>Hardy, J., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+hypothesis+of+Alzheimer's+disease:+progress+and+problems+on+the+road+to+therapeutics.">The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.</a> Science. 297, 353-356 (2002).</li><li>Haass, C., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+protein+oligomers+in+neurodegeneration:+lessons+from+the+Alzheimer's+amyloid+beta-peptide.">Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide.</a> Nat. Rev. Mol. Cell. Biol. 8, 101-112 (2007).</li><li>Walker, L. C., LeVine, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cerebral+proteopathies.">The cerebral proteopathies.</a> Neurobiol. Aging. 21, 559-561 (2000).</li><li>Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging,+amyloid,+and+Alzheimer's+disease:+a+perspective+in+honor.">Aging, amyloid, and Alzheimer's disease: a perspective in honor.</a> of Carl Cotman. Neurochem. Res. 28, 1705-1713 (2003).</li><li>Walsh, D. M., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abeta+oligomers+-+a+decade+of+discovery.">Abeta oligomers - a decade of discovery.</a> J. Neurochem. 101, 1172-1184 (2007).</li><li>Swerdlow, P. S., Finley, D., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+immunoblot+sensitivity+by+heating+of+hydrated+filters.">Enhancement of immunoblot sensitivity by heating of hydrated filters.</a> Anal. Biochem. 156, 147-153 (1986).</li><li>Ida, N., Hartmann, T., Pantel, J., Schr&#246;der, J., Zerfass, R., F&#246;rstl, H., Sandbrink, R., Masters, C. L., Beyreuther, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+hetergeneous+A4+peptides+in+human+cerebrospinal+fuid+and+blood+by+a+newly+developed+sensitive+Western+blot+assay.">Analysis of hetergeneous A4 peptides in human cerebrospinal fuid and blood by a newly developed sensitive Western blot assay.</a> J. Biol. Chem. 271, 22908-22914 (1996).</li></ol>

アルツハイマー病1,4,5の病因におけるAβ凝集の重要性にもかかわらず、いくつかの研究ではヒト皮質組織試料2に、多様なAβ多量体の分布を説明したり、定量化している。一般的に使用される免疫組織化学法は、固定皮質組織内で個別の多量Aβの種の識別のために許可されていません。固定されていない皮質組織ホモジネートでは、Aβ多量体を分離し、生化学的にゲル電気泳動...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Complete Protease Inhibitor Cocktail Tablets</td>
<td>&nbsp;</td>
<td>Santa Cruz Biotechnology</td>
<td>Sc-29130</td>
<td>1 tablet in 25ml buffer</td>
</tr>
<tr>
<td>BCA Protein Assay kit</td>
<td>&nbsp;</td>
<td>ThermoFisher Scientific</td>
<td>23225</td>
<td>&nbsp;</td>
<tr>
<td>XCell SureLock Mini-Cell and XCell II Blot Module Kit CE Mark</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EI0002</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Sample Buffer (2X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1676</td>
<td>&nbsp;</td>
<tr>
<td>NuPAGE Sample Reducing Agent (10X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>NP0004</td>
<td>&nbsp;</td>
<tr>
<td>SeeBlue Plus2 Pre-Stained Standard</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC5925</td>
<td>&nbsp;</td>
<tr>
<td>Novex 10-20% Tricine Gel 1.0 mm, 10 well</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EC6625BOX</td>
<td>&nbsp;</td>
<tr>
<td>Nitrocellulose membranes, 0.2 &mu;m pore size</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC2000</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Running Buffer (10X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1675</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tris-Glycine Transfer Buffer (25X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC3675</td>
<td>&nbsp;</td>
<tr>
<td>SimplyBlue SafeStain</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6060</td>
<td>Will not interfere with immunostaining</td>
</tr>
<tr>
<td>ATX Ponceau S Red staining solution</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>09276</td>
<td>Will not interfere with immunostaining</td>
</tr>
<tr>
<td>Kapak heat sealable plastic sample pouches</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>0181225AA</td>
<td>&nbsp;</td>
<tr>
<td>6E10 mouse monoclonal antibody to A&beta;(1-16)</td>
<td>&nbsp;</td>
<td>Covance</td>
<td>SIG-39320</td>
<td>Dilute 1:1,000 up to 1:5,000 for WB</td>
</tr>
<tr>
<td>Tween 20</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P2287</td>
<td>&nbsp;</td>
<tr>
<td>ECL Mouse IgG, HRP-Linked Whole A&beta; (from sheep)</td>
<td>&nbsp;</td>
<td>GE Healthcare Life Sciences</td>
<td>NA931-1ML</td>
<td>Dilute at 1:10,000</td>
</tr>
<tr>
<td>SuperSignal West Pico Chemiluminescent Substrate</td>
<td>&nbsp;</td>
<td>ThermoFisher</td>
<td>34077</td>
<td>&nbsp;</td>
<tr>
<td>Kodak Biomax MR Film</td>
<td>&nbsp;</td>
<td>Carestream Health</td>
<td>870 1302</td>
<td>&nbsp;</td>		</tbody>
		</table>
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sds-page/immunoblot detection of a&beta; multimers in human cortical tissue homogenates using antigen-epitope retrieval

β-アミロイドの異常折り畳み及び重合（Aβ）ペプチドは、アルツハイマー病の発症の神経変性カスケードを開始すると考えられている<sup&gt; 1</sup&gt;。アルツハイマー病の脳老人斑のコアに見られるβ-シートが豊富なアミロイド線維への自己凝集する傾向があるまたは42アミノ酸のペプチド - Aβは、主に40です。証拠を増やすと、その低分子量は、可溶性Aβの多量の線維Aβのアミロイドよりも毒性が示唆<sup&gt; 2</sup&gt;。脳組織における低および高分子量の多量のAβ種の同定と定量は、アルツハイマー病の研究に不可欠な目標である、とも用いられる方法は他のproteopathiesに有毒な多量体の同定と特性評価に適用することができます<sup&gt; 3</sup&gt;。天然由来のAβ多量体は、Aβ特異的抗体を用いたイムノブロッティングに続いてSDS -ポリアクリルアミドゲル電気泳動によって検出することができます。しかし、多量Aβの分離と検出には小さいポアサイズのニトロセルロース膜に高濃度の皮質ホモジネートと抗原の検索を使用する必要があります。ここでは、明らかに人間の大脳皮質のホモジネート、SDS - PAGEによるタンパク質の分離、および抗原エピトープの検索/AβペプチドのN末端領域に対する抗体6E10とウェスタンブロッティングの準備のためのテクニックを説明します。このプロトコルを使用して、我々は一貫してアルツハイマー病の病理と人間からの皮質組織でAβモノマー、ダイマー、トリマー、テトラマー、およびより高い分子量のマルチマーを検出する。

Watch this Scientific Journal Video about SDS-PAGE/Immunoblot Detection of AÎ² Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval at JoVE.com

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

我々は明らかに人間の皮質ホモジネート、SDS - PAGEによるタンパク質の分離、抗原の検索とAβペプチドに対する抗体でイムノブロットを調製するためのテクニックを説明します。このプロトコルを使用して、我々は一貫してアルツハイマー病の病理と人間からの皮質組織で単量体と多量体Aβを検出する。

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 ...

sds-pageimmunoblot-detection-multimers-human-cortical-tissue

Research

JoVE Journal

Neuroscience

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

24.0K ビュー.  Emory University. 我々は明らかに人間の皮質ホモジネート、SDS - PAGEによるタンパク質の分離、抗原の検索とAβペプチドに対する抗体でイムノブロットを調製するためのテクニックを説明します。このプロトコルを使用して、我々は一貫してアルツハイマー病の病理と人間からの皮質組織で単量体と多量体Aβを検出する。

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

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

高濃縮ニューロン培養の世代のための皮質組織ブロックの凍結保存

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

神経科学

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

開発ゼブラフィッシュ前脳におけるクローン関連の神経前駆細胞のタイムラプスライブイメージング

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

のBalb / Cの脳組織の高感度5 hydroxymethylcytosine検出

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and focused on psychological aspects. Until now, objective observation of neurophysiological parameters has not been carried out - undoubtedly because the technical and methodological means have not been available -, investigations into the neurophysiological effects of weightlessness are in their infancy (Schneider et al. 2008).
While imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) would be hardly applicable in space, the non-invasive near-infrared spectroscopy (NIRS) technique represents a method of mapping hemodynamic processes in the brain in real time that is both relatively inexpensive and that can be employed even under extreme conditions. The combination with electroencephalography (EEG) opens up the possibility of following the electrocortical processes under changing gravity conditions with a finer temporal resolution as well as with deeper localization, for instance with electrotomography (LORETA).
Previous studies showed an increase of beta frequency activity under normal gravity conditions and a decrease under weightlessness conditions during a parabolic flight (Schneider et al. 2008a+b). Tilt studies revealed different changes in brain function, which let suggest, that changes in parabolic flight might reflect emotional processes rather than hemodynamic changes. However, it is still unclear whether these are effects of changed gravity or hemodynamic changes within the brain. Combining EEG/LORETA and NIRS should for the first time make it possible to map the effect of weightlessness and reduced gravity on both hemodynamic and electrophysiological processes in the brain. Initially, this is to be done as part of a feasibility study during a parabolic flight. Afterwards, it is also planned to use both techniques during medium- and long-term space flight.
It can be assumed that the long-term redistribution of the blood volume and the associated increase in the supply of oxygen to the brain will lead to changes in the central nervous system that are also responsible for anaemic processes, and which can in turn reduce performance (De Santo et al. 2005), which means that they could be crucial for the success and safety of a mission (Genik et al. 2005, Ellis 2000).
Depending on these results, it will be necessary to develop and employ extensive countermeasures. Initial results for the MARS500 study suggest that, in addition to their significance in the context of the cardiovascular and locomotor systems, sport and physical activity can play a part in improving neurocognitive parameters. Before this can be fully established, however, it seems necessary to learn more about the influence of changing gravity conditions on neurophysiological processes and associated neurocognitive impairment.

The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. 
A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.

脳皮質機能および変更された重力状態の間、神経認知パフォーマンスとの間のコヒーレンス

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and ...

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

廃棄されたヒト胎児脳組織から神経幹細胞の生成

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

皮質ネットワークの開発で機能的カルシウムイメージング

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

ラット皮質ニューロンにおける繊維状アクチン（F-アクチン）涙点の定量化

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

蛍光プローブを用いた培養ニューロンにおけるエンドソームおよびリソソームの動力学の定量

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

平坦化された哺乳類の皮質皮質モジュールの可視化

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...

Statistical Modelling of Cortical Connectivity Using Non-invasive Electroencephalograms

Non-invasive electrophysiological recordings are useful for the evaluation of nervous system function. These techniques are inexpensive, fast, replicable, and less resource-intensive than imaging. Further, the functional data produced have excellent temporal resolution, which is not achievable with structural imaging.
Current applications of electroencephalograms (EEG) are limited by data processing methods. Standard analysis techniques using raw time series data at individual channels are very limited methods of interrogating nervous system activity. More detailed information about cortical function can be achieved by examining relationships between channels and deriving statistical models of how areas are interacting, allowing visualization of connectivity between networks.
This manuscript describes a method for deriving statistical models of cortical network activity by recording EEG in a standard manner, then examining the interelectrode coherence measures to assess relationships between the recorded areas. Higher order interactions can be further examined by assessing the covariance between the coherence pairs, producing high-dimensional &#34;maps&#34; of network interactions. These data constructs can be examined to assess cortical network function and its relationship to pathology in ways not achievable with traditional techniques.
This approach offers greater sensitivity to network level interactions than is achievable with raw time series analysis. It is, however, limited by the complexity of drawing specific mechanistic conclusions about the underlying neural populations and the high volumes of data generated, requiring more advanced statistical techniques for evaluation, including dimensionality reduction and classifier-based approaches.

Standard EEG analysis techniques offer limited insight into nervous system function. Deriving statistical models of cortical connectivity offers far greater ability to investigate underlying network dynamics. Improved functional assessment opens new possibilities for diagnosis, prognostication, and outcome prediction in nervous system diseases.

非侵襲的脳波を用いた皮質接続の統計的モデリング

Non-invasive electrophysiological recordings are useful for the evaluation of nervous system function. These techniques are inexpensive, fast, replicable, ...