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

Nos gustaría agradecer a BK agosto en la Universidad de Wisconsin-Madison Fondo microscopio electrónico para la microscopía electrónica. Este trabajo fue apoyado por subvenciones del NIH R01-DA026067 y P30 HD03352 (a JSM).

1. Preparativos 4.1 <ol><li> Preparar 500 mL de tampón de gradiente medio (GM) al mezclar 50 ml de una suspensión de sacarosa 2,5 M, 2,5 ml de 1M Tris-HCl, pH 7,5 acciones, y 0,1 mL de EDTA 0,5 M, pH 8,0 con valores de mili- Q agua a volumen. Filtro de esterilización de la solución, alícuota y almacenar congelado. </li><li> Preparar un stock de 1.000 veces la tetrodotoxina (TTX), haciendo una solución 1 mM en mili-Q H 2 O. Alícuotas de TTX se pueden almacenar a -20 ° C. </li><li> Prepare ...

<ol>
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Lo discontinuo de Percoll-sacarosa gradiente de preparación descrito en este documento es un método rápido y fiable para aislar activos del SNS, que se puede utilizar en una variedad de experimentos de transmisión sináptica. Este método del gradiente, que se basa en el método desarrollado por Dunkley et al. 3,4, es un procedimiento de fraccionamiento subcelular cerebro que aísla tanto de pre-y post-sinápticas derivados vesículas de membrana que se asocian unas con otras. Sin más purificación estos...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> Micro BCA Protein Kit de ensayo </td><td> Atravesar </td><td> 23235 </td><td></td></tr><tr><td> CaCl2 </td><td> Pescador </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> Pescador </td><td> A407SK-4 </td><td></td></tr><tr><td> HCl </td><td> Pescador </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> Pescador </td><td> P285-500 </td><td></td></tr><tr><td> PierceSDS-PAGE preparación de muestras Kit </td><td> Atravesar </td><td> 89888 </td><td></td></tr><tr><td> NaCl </td><td> RPI </td><td> S23020 </td><td></td></tr><tr><td> NaHCO3 </td><td> Pescador </td><td> BP328-500 </td><td></td></tr><tr><td> Na 2 PHO 4 </td><td> Pescador </td><td> S381-500 </td><td></td></tr><tr><td> Sacarosa </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> Tetrodotoxina </td><td> Sigma </td><td> T5651 </td><td></td></tr><tr><td> Expresa Pro Label Mix S35 etiquetas de uso sencillo </td><td> Perkin Elmer </td><td> NEG772 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><th> Equipo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> Herramientas de disección </td><td></td><td></td><td></td></tr><tr><td> Dounce homogeneizadora, 7 ml (viene con dos morteros de vidrio tipo &quot;A&quot; y &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 centrífuga </td><td> Beckman Coulter </td><td> 366830 </td><td></td></tr><tr><td> GH 3.8 del rotor, el rotor basculante </td><td> Beckman Coulter </td><td> 360581 </td><td></td></tr><tr><td> Beckman J2-21 Centrífuga </td><td> Beckman </td><td></td><td></td></tr><tr><td> Beckman tubos con tapas </td><td> Beckman </td><td> 355672 </td><td></td></tr><tr><td> Blancas paredes de adaptadores </td><td> Beckman </td><td> 342327 </td><td></td></tr><tr><td> Azul adaptadores de pared </td><td> Beckman </td><td></td><td></td></tr><tr><td> JA-17 Rotor, Rotor de ángulo fijo </td><td> Beckman </td><td> 369691 </td><td></td></tr></tbody></table> Tabla de los anticuerpos utilizados para western blots: <table border="1"><tbody><tr><th> Nombre de anticuerpos </th><th> Empresa </th><th> Número de catálogo </th><th> Especie huésped </th><th> Factor de dilución </th></tr><tr><td> β-actina </td><td> Sigma </td><td> A5441 </td><td> ratón </td><td> 1:2000 </td></tr><tr><td> GFAP </td><td> Santa Cruz </td><td> sc-65343 </td><td> ratón </td><td> 1:200 </td></tr><tr><td> GP73 </td><td> Santa Cruz </td><td> Sc-134509 </td><td> conejo </td><td> 1:200 </td></tr><tr><td> HSC70 </td><td> Santa Cruz </td><td> sc-7298 </td><td> ratón </td><td> 1:200 </td></tr><tr><td> Laminβ </td><td> Santa Cruz </td><td> SC-6261 </td><td> cabra </td><td> 1:200 </td></tr><tr><td> Prohibitina </td><td> Santa Cruz </td><td> sc-28259 </td><td> conejo </td><td> 1:200 </td></tr><tr><td> PSD95 </td><td> Millipore </td><td> MAB1596 </td><td> ratón </td><td> 5 g / l </td></tr><tr><td> SNAP25 </td><td> AbCam </td><td> ab5666-100 </td><td> conejo </td><td> 1:2000 </td></tr><tr><td> Sinaptofisina </td><td> Millipore </td><td> MAB368 </td><td> ratón </td><td> 1:500 </td></tr><tr><td> β-3-tubulina </td><td> Santa Cruz </td><td> sc-80016 </td><td> ratón </td><td> 1:200 </td></tr></tbody></table>

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

Synaptoneurosomes (SN) se obtienen después de la homogeneización y fraccionamiento de la corteza cerebral del ratón. Que se hara vesículas o terminales aislados que rompen con terminales de los axones en el tejido cortical se homogeneiza. El SNS conservar las características pre-y post-sináptica, lo que hace que sean útiles en el estudio de la transmisión sináptica. Conservan la maquinaria molecular utilizados en la señalización neuronal y que son capaces de captación, almacenamiento y liberación de neurotransmisores. La producción y el aislamiento de ganglios centinela activa puede ser problemático, utilizando como medios de Ficoll, que pueden ser citotóxicos y requieren centrifugación extendido debido a la alta densidad, y la filtración y el método de centrifugado, que puede resultar en una baja actividad debido a los daños mecánicos del SNS. Sin embargo, el uso de gradientes de densidad de sacarosa discontinua de Percoll para aislar SN proporciona un método rápido para producir un buen rendimiento del SNS translationally activo. El método del gradiente de Percoll-sacarosa es rápida y suave, ya que emplea a condiciones isotónicas, tiene menos y más cortas tiradas centrifugación y evita pasos de centrifugación que la SNS pellet y causar daños mecánicos.

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

Un método para preparar translationally activa, synaptoneurosomes intacta (SN) de la corteza cerebral del ratón se describe. El método utiliza una discontinua de Percoll-sacarosa gradiente de densidad que permite la rápida preparación de los ganglios centinela activa.

Preparación de Synaptoneurosomes de ratón con una corteza discontinua de Percoll-sacarosa gradiente de densidad

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

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La visualización y la manipulación genética de las dendritas y espinas en la corteza cerebral y el hipocampo del ratón usando<em&gt; En el útero</em&gt; La electroporación

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

Imágenes funcionales de la corteza auditiva en los gatos adultos el uso de alto campo 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

Imágenes en vivo de la mitosis en el desarrollo de la corteza de embriones de ratón

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

De dos fotones<em&gt; In vivo</em&gt; Imágenes de las espinas dendríticas en la corteza del ratón Usando un Diluido-cráneo Preparación

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

Preparación de sináptica Membrana plasmática y proteínas postsináptica densidad utilizando un gradiente discontinuo de sacarosa

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

La investigación de la función de profunda cortical y estructuras subcorticales Usando estereotáctica Electroencefalografía: Lecciones de la corteza cingulada anterior

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