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

Wir möchten BK August von der University of Wisconsin-Madison Electron Microscope Facility für die Elektronenmikroskopie danken. Diese Arbeit wurde vom NIH gewährt R01-DA026067 und P30-HD03352 (bis JSM) unterstützt.

1. Die Vorbereitungen 1-4 <ol><li> Bereiten Sie 500 mL Gradienten-Medium (GM)-Puffer durch Mischen von 50 mL einer 2,5 M Saccharose Gülle, 2,5 mL einer 1M Tris-HCl, pH 7,5 Lager, und 0,1 ml einer 0,5 m EDTA, pH 8,0 Lager mit Milli- Q-Wasser, um die Lautstärke. Filter sterilisieren die Lösung, Aliquot und speichern eingefroren. </li><li> Bereiten Sie eine 1000x Lager von Tetrodotoxin (TTX), indem Sie eine 1 mM Lösung in Milli-Q-H 2 O. Aliquots der TTX kann bei -20 ° C gelagert werden </li><li...

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Die diskontinuierliche Percoll-Saccharose-Gradienten Vorbereitung hierin beschriebenen ist eine schnelle, zuverlässige Methode, um aktiv SNS, die in einer Vielzahl von synaptischen Übertragung Experimente verwendet werden können isolieren. Dieser Gradient-Methode, die auf der Methode von Dunkley et al., 3,4 entwickelten, ist eine subzelluläre Fraktionierung Gehirn Verfahren, das sowohl prä-und postsynaptischen Membran-derived Bläschen, die miteinander verbunden sind, isoliert. Ohne weitere Reinigung die...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> Micro BCA Protein Assay Kit </td><td> Durchstechen </td><td> 23235 </td><td></td></tr><tr><td> CaCl 2 </td><td> Fischer </td><td> C79-500 </td><td></td></tr><tr><td> CO 2-Gas </td><td> Airgas (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> EtOH </td><td> Fischer </td><td> A407SK-4 </td><td></td></tr><tr><td> HCl </td><td> Fischer </td><td> A142-212 </td><td></td></tr><tr><td> Percoll </td><td> GE Healthcare </td><td> 17-0891-01 </td><td></td></tr><tr><td> KH 2 PO 4 </td><td> Fischer </td><td> P285-500 </td><td></td></tr><tr><td> PierceSDS-PAGE Sample Prep Kit </td><td> Durchstechen </td><td> 89888 </td><td></td></tr><tr><td> NaCl </td><td> RPI </td><td> S23020 </td><td></td></tr><tr><td> NaHCO 3 </td><td> Fischer </td><td> BP328-500 </td><td></td></tr><tr><td> Na 2 PHO 4 </td><td> Fischer </td><td> S381-500 </td><td></td></tr><tr><td> Sucrose </td><td> RPI </td><td> S24060 </td><td></td></tr><tr><td> Tris Base </td><td> RPI </td><td> T60040 </td><td></td></tr><tr><td> Tetrodotoxin </td><td> Sigma </td><td> T5651 </td><td></td></tr><tr><td> Express Pro-Label-Mix S35 Einfache Tag </td><td> Perkin Elmer </td><td> NEG772 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><th> Ausstattung </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> Dissection-Tools </td><td></td><td></td><td></td></tr><tr><td> Dounce Homogenisator, 7 mL (kommt mit zwei Glas-Stößel mit der Aufschrift &quot;A&quot; und &quot;B&quot;) </td><td> Wheaton </td><td></td><td></td></tr><tr><td> P1000 Gilson Pipetman </td><td> Gilson </td><td> F123602 </td><td></td></tr><tr><td> Allegra 6KR Centrifuge </td><td> Beckman Coulter </td><td> 366830 </td><td></td></tr><tr><td> GH 3,8 Rotor, Ausschwingrotor </td><td> Beckman Coulter </td><td> 360581 </td><td></td></tr><tr><td> Beckman J2-21 Zentrifuge </td><td> Beckman </td><td></td><td></td></tr><tr><td> Beckman Röhrchen mit Kappen </td><td> Beckman </td><td> 355672 </td><td></td></tr><tr><td> Weiß ummauerten Adapter </td><td> Beckman </td><td> 342327 </td><td></td></tr><tr><td> Blau ummauerten Adapter </td><td> Beckman </td><td></td><td></td></tr><tr><td> JA-17 Rotor, Festwinkelrotor </td><td> Beckman </td><td> 369691 </td><td></td></tr></tbody></table> Tabelle der Antikörper für Western Blots verwendet: <table border="1"><tbody><tr><th> Name der Antikörper </th><th> Firma </th><th> Katalog-Nummer </th><th> Wirtsarten </th><th> Verdünnungsfaktor </th></tr><tr><td> β-Aktin </td><td> Sigma </td><td> A5441 </td><td> Maus </td><td> 1:2000 </td></tr><tr><td> GFAP </td><td> Santa Cruz </td><td> sc-65343 </td><td> Maus </td><td> 1:200 </td></tr><tr><td> GP73 </td><td> Santa Cruz </td><td> Sc-134509 </td><td> Kaninchen </td><td> 1:200 </td></tr><tr><td> HSC70 </td><td> Santa Cruz </td><td> sc-7298 </td><td> Maus </td><td> 1:200 </td></tr><tr><td> Laminβ </td><td> Santa Cruz </td><td> Sc-6261 </td><td> Ziege </td><td> 1:200 </td></tr><tr><td> Prohibitin </td><td> Santa Cruz </td><td> sc-28259 </td><td> Kaninchen </td><td> 1:200 </td></tr><tr><td> PSD95 </td><td> Millipore </td><td> MAB1596 </td><td> Maus </td><td> 5 ug / ul </td></tr><tr><td> SNAP25 </td><td> AbCam </td><td> ab5666-100 </td><td> Kaninchen </td><td> 1:2000 </td></tr><tr><td> Synaptophysin </td><td> Millipore </td><td> MAB368 </td><td> Maus </td><td> 1:500 </td></tr><tr><td> β-3-Tubulin </td><td> Santa Cruz </td><td> sc-80016 </td><td> Maus </td><td> 1:200 </td></tr></tbody></table>

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

Synaptoneurosomen (SNS) sind nach der Homogenisierung und Fraktionierung von Maus Hirnrinde erhalten. Sie sind Vesikel oder isolierte Terminals, die ausbrechen aus Axonterminalen wenn die kortikale Gewebe wird homogenisiert und verschließen. Die SNS behält prä-und postsynaptischen Eigenschaften, wodurch sie sinnvoll in das Studium der synaptischen Übertragung. Sie behalten die molekulare Maschinerie in neuronalen Signalübertragung verwendet und sind in der Lage die Aufnahme, Speicherung und Freisetzung von Neurotransmittern. Die Herstellung und Isolierung von aktiven SNs kann problematisch mit Medien wie Ficoll, die zytotoxisch können und müssen verlängert Zentrifugation aufgrund der hohen Dichte und Filtration und Zentrifugation Methoden, die in geringer Aktivität durch eine mechanische Beschädigung der SNS können die Folge sein. Allerdings ist die Verwendung von diskontinuierlichen Percoll-Saccharose-Dichtegradienten an SNS isolieren eine schnelle Methode, um gute Ausbeuten an translationsaktiven SNs produzieren. Die Percoll-Saccharose-Gradienten-Methode ist schnell und sanft wie es isotonischen Bedingungen beschäftigt, hat weniger und kürzere Zentrifugation Spins und vermeidet Zentrifugationsschritte, dass Pellet SNs und mechanische Schäden verursachen.

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

Eine Methode, um translationsaktiven, intakte Synaptoneurosomen (SNS) von der Maus Hirnrinde vorbereiten beschrieben. Das Verfahren nutzt eine diskontinuierliche Percoll-Saccharose-Dichtegradienten Dies ermöglicht die schnelle Erstellung von aktiven SNS.

Vorbereitung der Synaptoneurosomen von Maus Cortex mit einem diskontinuierlichen Percoll-Saccharose-Dichtegradienten

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

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Chronische Imaging of Mouse Visual Cortex Mit einem ausgedünnten Schädel Vorbereitung

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Visualisierung und genetische Manipulation von Dendriten und Dornen in der Maus Hirnrinde und im Hippocampus mit<em&gt; In utero</em&gt; Die Elektroporation

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

Funktionelle Bildgebung Auditory Cortex bei erwachsenen Katzen mit Hochfeld-fMRI

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

Live-Imaging der Mitose in der Entwicklung von embryonalen Maus-Cortex

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

Zwei-Photonen-<em&gt; In vivo</em&gt; Imaging von dendritischen Dornen in der Maus Cortex Mit einem ausgedünnten Schädel Vorbereitung

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

Vorbereitung der synaptischen Plasmamembran und Postsynaptische Dichte Proteinen mit einer diskontinuierlichen Saccharosegradienten

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

Die Untersuchung der Funktion von Deep kortikalen und subkortikalen Strukturen Mit Stereotaktische Elektroenzephalographie: Lehren aus der anterioren cingulären Cortex

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