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

Nous tenons à remercier BK août de l&#39;Université de Wisconsin-Madison Facilité microscope électronique pour la microscopie électronique. Ce travail a été soutenu par des subventions du NIH R01-DA026067 et P30-HD03352 (pour la JSM).

1. Les préparatifs 1-4 <ol><li> Préparer tampon de 500 mL de milieu de gradient (GM) en mélangeant 50 ml d&#39;une suspension de 2,5 M de saccharose, 2,5 mL d&#39;un 1M Tris-HCl, pH 7,5 stock, et 0,1 mL d&#39;une EDTA 0,5 M, pH 8,0 milli-actions avec Q eau au volume. Filtre stériliser la solution, et de stocker aliquot congelé. </li><li> Préparer un stock de 1000x de tétrodotoxine (TTX) en faisant une solution à 1 mM dans milli-Q H 2 O. Des aliquotes de TTX peuvent être stockés à -20 ?...

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La préparation gradient discontinu de Percoll-sucrose décrite ici est une méthode rapide et fiable d&#39;isoler actifs SN, qui peut être utilisé dans une variété d&#39;expériences de la transmission synaptique. Cette méthode de gradient, qui est basé sur la méthode développée par Dunkley et al., 3,4 est une procédure cerveau subcellulaire fractionnement qui isole à la fois pré-et post-synaptiques à la membrane des vésicules dérivées qui sont associés à un autre. Sans autre purification d...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> Kit Micro BCA Protein Assay </td><td> Pierce </td><td> 23235 </td><td></td></tr><tr><td> CaCl 2 </td><td> Fisher </td><td> C79-500 </td><td></td></tr><tr><td> Gaz CO 2 </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> Fisher </td><td> A407SK-4 </td><td></td></tr><tr><td> HCl </td><td> Fisher </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> Fisher </td><td> P285-500 </td><td></td></tr><tr><td> PierceSDS-PAGE de préparation d&#39;échantillons Kit </td><td> Pierce </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> Fisher </td><td> BP328-500 </td><td></td></tr><tr><td> Na 2 PHO 4 </td><td> Fisher </td><td> S381-500 </td><td></td></tr><tr><td> Saccharose </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> Tetrodotoxine </td><td> Sigma </td><td> T5651 </td><td></td></tr><tr><td> Express Pro label Mix S35 Tag Facile </td><td> Perkin Elmer </td><td> NEG772 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><th> Équipement </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> Des outils de dissection </td><td></td><td></td><td></td></tr><tr><td> Dounce homogénéisateur, 7 ml (livré avec deux pilons en verre étiqueté «A» et «B») </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 Centrifugeuse </td><td> Beckman Coulter </td><td> 366830 </td><td></td></tr><tr><td> GH 3.8 Rotor, rotor swing </td><td> Beckman Coulter </td><td> 360581 </td><td></td></tr><tr><td> Beckman J2-21 Centrifugeuse </td><td> Beckman </td><td></td><td></td></tr><tr><td> Beckman tubes avec des bouchons </td><td> Beckman </td><td> 355672 </td><td></td></tr><tr><td> Blanc adaptateurs fortifiée </td><td> Beckman </td><td> 342327 </td><td></td></tr><tr><td> Bleu adaptateurs fortifiée </td><td> Beckman </td><td></td><td></td></tr><tr><td> JA-17 Rotor, Rotor angulaire </td><td> Beckman </td><td> 369691 </td><td></td></tr></tbody></table> Table des anticorps utilisés pour Western blot: <table border="1"><tbody><tr><th> Nom de l&#39;anticorps </th><th> Société </th><th> Numéro de catalogue </th><th> Espèce Hôte </th><th> Facteur de dilution </th></tr><tr><td> β-actine </td><td> Sigma </td><td> A5441 </td><td> la souris </td><td> 1:2000 </td></tr><tr><td> GFAP </td><td> Santa Cruz </td><td> sc-65343 </td><td> la souris </td><td> 1:200 </td></tr><tr><td> GP73 </td><td> Santa Cruz </td><td> Sc-134509 </td><td> lapin </td><td> 1:200 </td></tr><tr><td> HSC70 </td><td> Santa Cruz </td><td> SC-7298 </td><td> la souris </td><td> 1:200 </td></tr><tr><td> Laminβ </td><td> Santa Cruz </td><td> Sc-6261 </td><td> de chèvre </td><td> 1:200 </td></tr><tr><td> Prohibitin </td><td> Santa Cruz </td><td> sc-28259 </td><td> lapin </td><td> 1:200 </td></tr><tr><td> PSD95 </td><td> Millipore </td><td> MAB1596 </td><td> la souris </td><td> 5 ug / uL </td></tr><tr><td> SNAP25 </td><td> Abcam </td><td> ab5666-100 </td><td> lapin </td><td> 1:2000 </td></tr><tr><td> Synaptophysine </td><td> Millipore </td><td> MAB368 </td><td> la souris </td><td> 1:500 </td></tr><tr><td> β-3-tubuline </td><td> Santa Cruz </td><td> sc-80016 </td><td> la souris </td><td> 1:200 </td></tr></tbody></table>

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

Synaptoneurosomes (SNS) sont obtenus après homogénéisation et fractionnement de cortex cérébral de la souris. Ils sont refermés vésicules ou des terminaux isolés que rompre avec terminaisons axonales lorsque le tissu cortical est homogénéisé. Le SNS conserver des caractéristiques pré-et post-synaptiques, ce qui les rend utiles dans l&#39;étude de la transmission synaptique. Ils conservent la machinerie moléculaire utilisée dans la signalisation neuronale et sont capables d&#39;absorption, le stockage et la libération de neurotransmetteurs. La production et l&#39;isolement de SNS active peut être problématique médias utilisant comme Ficoll, qui peuvent être cytotoxiques et nécessitent une centrifugation prolongée due à haute densité, et la filtration et des méthodes de centrifugation, ce qui peut entraîner une faible activité en raison de dommages mécaniques de la SNS. Cependant, l&#39;utilisation des gradients de densité discontinu de Percoll-sucrose pour isoler SN fournit une méthode rapide pour produire de bons rendements de SNS translation active. La méthode du gradient Percoll-sucrose est rapide et en douceur car il emploie des conditions isotoniques, a fait tourner la centrifugation moins nombreux et plus courts et évite les étapes de centrifugation que les SRS granulés et causer des dommages mécaniques.

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

Une méthode pour préparer translation active, synaptoneurosomes intacte (SNS) de la souris cortex cérébral est décrite. La méthode utilise un gradient de densité discontinu de Percoll-sucrose permettant la préparation rapide de SNS active.

Préparation de Synaptoneurosomes de Mouse Cortex en utilisant un gradient de densité discontinu de Percoll-sucrose

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

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Imagerie fonctionnelle du cortex auditif chez chats adultes à l&#39;aide de haut-champ IRMf

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

Imagerie en direct de la mitose dans les développement Cortex souris embryonnaires

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

Deux-Photon<em&gt; In vivo</em&gt; Imagerie des épines dendritiques dans le cortex de souris utilisant un crâne-Diluée Préparation

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

Préparation de Synaptic membrane plasmique et de protéines de densité post-synaptique en utilisant un gradient discontinu de saccharose

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

Étude de la fonction de Deep corticale et structures sous-corticales Utilisation stéréotaxique électroencéphalographie: Leçons du cortex cingulaire antérieur

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