Name: preparation of synaptoneurosomes from mouse cortex using a discontinuous percoll-sucrose density gradient
Uploaded: 2011-09-17T14:00:00
Description: Watch this Scientific Journal Video about Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient at JoVE.com

我々は電子顕微鏡のためのウィスコンシン大学マディソン電子顕微鏡施設の大学からBKアウグスト感謝したいと思います。この作品は、NIHの助成金R01 - DA026067とP30 - HD03352（JSMへ）によってサポートされていました。

1。準備1月4日 <ol><li>で、2.5 Mのショ糖のスラリー50mLを、1Mトリス - 塩酸2.5mLを、pH7.5株式、および0.5 M EDTA 0.1 mLを混合してpH8.0のストックを勾配培地（GM）バッファーを500mLを準備ミリボリュームへのQ水。凍結溶液、分注して店を滅菌フィルタします。 </li><li>ミリ- Q H 2 Oで1 mM溶液を作ることによってテトロドトキシン（TTX）の1000倍の株式を準備TTXのアリコートを-20℃で保存す...

<ol>
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ここに記載さ不連続パーコール-ショ糖勾配の準備は、シナプス伝達の実験の様々な使用することができるアクティブなSNを、分離するために迅速、信頼性の高い方法です。 Dunkleyら、3,4によって開発された方法に基づいてこの勾配法は、互いに関連付けられている両方の前およびシナプス後膜由来の小胞を分離した細胞内脳分画の手順です。さらに精製することなくこれらのSNは免疫?...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td>マイクロBCAタンパク質アッセイキット</td><td>ピアース</td><td> 23235 </td><td></td></tr><tr><td> CaCl 2で </td><td>フィッシャー</td><td> C79 - 500 </td><td></td></tr><tr><td> CO 2ガス</td><td> Airgasの（UW - MDS） </td><td> CD 50 </td><td></td></tr><tr><td> EDTA </td><td> RPI </td><td> E57020 </td><td></td></tr><tr><td>エタノール</td><td>フィッシャー</td><td> A407SK - 4 </td><td></td></tr><tr><td>塩酸</td><td>フィッシャー</td><td> A142 - 212 </td><td></td></tr><tr><td>パーコール</td><td> GEヘルスケア</td><td> 17-0891-01 </td><td></td></tr><tr><td> KH 2 PO 4 </td><td>フィッシャー</td><td> P285 - 500 </td><td></td></tr><tr><td> PierceSDS - PAGEサンプル前処理キッ​​ト</td><td>ピアース</td><td> 89888 </td><td></td></tr><tr><td> NaClの</td><td> RPI </td><td> S23020 </td><td></td></tr><tr><td> NaHCO 3の </td><td>フィッシャー</td><td> BP328 - 500 </td><td></td></tr><tr><td>のNa 2 PHO 4 </td><td>フィッシャー</td><td> S381 - 500 </td><td></td></tr><tr><td>スクロース</td><td> RPI </td><td> S24060 </td><td></td></tr><tr><td>トリスベース</td><td> RPI </td><td> T60040 </td><td></td></tr><tr><td>テトロドトキシン</td><td>シグマ</td><td> T5651 </td><td></td></tr><tr><td>エクスプレスProのラベルには、S35簡単タグをミックス</td><td>パーキンエルマー</td><td> NEG772 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><th>機器の</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td>解剖ツール</td><td></td><td></td><td></td></tr><tr><td>ダウンスホモジナイザー、7 mLの（二枚のガラスに付属する&quot;A&quot;と&quot;B&quot;とラベルされた乳棒） </td><td>ホイートン</td><td></td><td></td></tr><tr><td> P1000ギルソンPipetman </td><td>ギルソン</td><td> F123602 </td><td></td></tr><tr><td>アレグラ6KR遠心機</td><td>ベックマンコールター</td><td> 366830 </td><td></td></tr><tr><td> GH 3.8ロータ、スイングバケットローター</td><td>ベックマンコールター</td><td> 360581 </td><td></td></tr><tr><td>ベックマンJ2 - 21遠心</td><td>ベックマン</td><td></td><td></td></tr><tr><td>キャップ付きベックマンチューブ</td><td>ベックマン</td><td> 355672 </td><td></td></tr><tr><td>白壁のアダプタ</td><td>ベックマン</td><td> 342327 </td><td></td></tr><tr><td>青い壁のアダプター</td><td>ベックマン</td><td></td><td></td></tr><tr><td> JA - 17ローター、固定アングルローター</td><td>ベックマン</td><td> 369691 </td><td></td></tr></tbody></table>ウエスタンブロットのために使用される抗体の表： <table border="1"><tbody><tr><th>抗体の名前</th><th>会社</th><th>カタログ番号</th><th>宿主動物種</th><th>希釈係数</th></tr><tr><td> β-アクチン</td><td>シグマ</td><td> A5441 </td><td>マウスを</td><td> 1:2000 </td></tr><tr><td> GFAP </td><td>サンタクルス</td><td> SC - 65343 </td><td>マウスを</td><td> 1:200 </td></tr><tr><td> GP73 </td><td>サンタクルス</td><td> SC - 134509 </td><td>ウサギ</td><td> 1:200 </td></tr><tr><td> HSC70 </td><td>サンタクルス</td><td> SC - 7298 </td><td>マウスを</td><td> 1:200 </td></tr><tr><td> Laminβ </td><td>サンタクルス</td><td> SC - 6261 </td><td>ヤギ</td><td> 1:200 </td></tr><tr><td> Prohibitin </td><td>サンタクルス</td><td> SC - 28259 </td><td>ウサギ</td><td> 1:200 </td></tr><tr><td> PSD95 </td><td>ミリポア</td><td> MAB1596 </td><td>マウスを</td><td> 5μgの/μL </td></tr><tr><td> SNAP25 </td><td>アブカム</td><td> ab5666 - 100 </td><td>ウサギ</td><td> 1:2000 </td></tr><tr><td>シナプトフィジン</td><td>ミリポア</td><td> MAB368 </td><td>マウスを</td><td> 1:500 </td></tr><tr><td> β- 3 -チューブリン</td><td>サンタクルス</td><td> SC - 80016 </td><td>マウスを</td><td> 1:200 </td></tr></tbody></table>

preparation of synaptoneurosomes from mouse cortex using a discontinuous percoll-sucrose density gradient

Synaptoneurosomes（SNS）は、マウス脳皮質の均質化と分別の後に得られる。彼らは、小胞または皮質組織が均質化されたときに軸索末端からの脱却孤立端末が再シールされています。 SNはシナプス伝達の研究に有用になる前と後シナプスの特性を、保持する。彼らは神経シグナリングで使用されている分子機構を保持して取り込み、ストレージ、および神経伝達物質の放出が可能です。 アクティブなSNの生産と分離は、細胞毒性になるとSNの機械的損傷に起因する低活性につながる可能性の拡張、高密度のために遠心分離し、ろ過と遠心分離の方法を、必要とすることができるフィコール、のような問題に使用してマスコミのすることができます。しかし、SNを分離するために、不連続パーコール-ショ糖密度勾配の使用は、並進アクティブSNの良好な収率を生成するために迅速な方法を提供する。パーコール-ショ糖密度勾配法では、等張性の条件を採用して、迅速かつ穏やかであり少ないと短い遠心分離スピンを持っており、ペレット、SNSとは、機械的損傷を引き起こすことに遠心操作を避けることができます。

Watch this Scientific Journal Video about Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient at JoVE.com

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

マウス脳皮質から翻訳後のアクティブな、無傷のsynaptoneurosomes（SNS）を準備する方法について説明する。方法は、アクティブなSNの迅速な作成を可能にする不連続パーコール-ショ糖密度勾配を使用しています。

不連続パーコール-ショ糖密度勾配を用いてマウスの皮質からSynaptoneurosomesの調製

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

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Neuroscience

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

間伐-頭蓋骨の準備を使用してマウス視覚野の慢性的イメージング

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

暗順応スライス標本におけるマウス網膜神経細胞からパッチクランプ記録

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

人間の前頭前野皮質からfMRI信号への行動応答の相関：タスク分析を用いた認知プロセスを調べる

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

フローサイトメトリーを用いてマウスの内耳の組織からのLacZの発現細胞を単離する

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

使用して、マウス大脳皮質および海馬における可視化と樹状突起と棘の遺伝子操作<em&gt;子宮内で</em&gt;エレクトロポレーション

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

ハイフィールドのfMRIを使って大人の猫における聴覚皮質の機能イメージング

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

発生中のマウス胚性皮質における有糸分裂のライブイメージング

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

二光子<em&gt; in vivoで</em&gt;間引き頭蓋骨の準備を使用して、マウス皮質における樹状突起棘のイメージング

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

不連続ショ糖勾配を用いてシナプス形質膜とシナプス後肥厚タンパク質の調製

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

Investigating the Function of Deep Cortical and Subcortical Structures Using Stereotactic Electroencephalography: Lessons from the Anterior Cingulate Cortex

定位脳波を使用して、ディープ皮質の機能と下構造の調査：前帯状皮質からの教訓は、

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure ...