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

Vi vill tacka BK augusti från University of Wisconsin-Madison Electron Microscope mikrokreditinstrumentet för elektronmikroskop. Detta arbete stöddes av NIH bidrag R01-DA026067 och P30-HD03352 (till JSM).

1. Förberedelser 1-4 <ol><li> Förbered 500 ml gradient medium (GM) buffert genom att blanda 50 ml av en 2,5 M sackaros flytgödsel, 2,5 ml av en 1M Tris-HCl, pH7.5 lager, och 0,1 ml av en 0,5 m EDTA, pH 8,0 lager med milli- Q-vatten till volym. Filtrera sterilisera lösningen, alikvot och förvara fryst. </li><li> Förbered en 1000x lager av tetrodotoxin (TTX) genom att göra en 1 mM lösning i Milli-Q H 2 O. Alikvoter av TTX kan lagras vid -20 ° C. </li><li> Förbered 10x Stimulering buffert ...

<ol>
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Det diskontinuerliga Percoll-sackaros gradient förberedelser som beskrivs häri är en snabb, tillförlitlig metod att isolera aktiva SNS, som kan användas i en mängd olika synaptisk transmission experiment. Denna gradient metod, som bygger på den metod som utvecklats av Dunkley et al., 3,4 är en subcellulära hjärna fraktionering förfarande som isolerar både pre-och postsynaptiska membran som härrör blåsor som är kopplade till varandra. Utan ytterligare rening dessa SNS är kompatibla med en män...

<table border="1"><tbody><tr><th> Reagensets namn </th><th> Företag </th><th> Katalognummer </th><th> Kommentarer (frivilligt) </th></tr><tr><td> Micro BCA Protein Assay Kit </td><td> Pierce </td><td> 23.235 </td><td></td></tr><tr><td> CaCl 2 </td><td> Fisher </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> 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 Sample Prep Kit </td><td> Pierce </td><td> 89.888 </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 FO 4 </td><td> Fisher </td><td> S381-500 </td><td></td></tr><tr><td> Sackaros </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 Blanda S35 Lätt Tagg </td><td> Perkin Elmer </td><td> NEG772 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><th> Utrustning </th><th> Företag </th><th> Katalognummer </th><th> Kommentarer (frivilligt) </th></tr><tr><td> Dissection verktyg </td><td></td><td></td><td></td></tr><tr><td> Dounce homogeniseringsblandare, 7 mL (levereras med två glas pestles märkt &quot;A&quot; och &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 Centrifugera </td><td> Beckman Coulter </td><td> 366.830 </td><td></td></tr><tr><td> GH 3,8 Rotor, Swinging hink rotor </td><td> Beckman Coulter </td><td> 360.581 </td><td></td></tr><tr><td> Beckman J2-21 Centrifug </td><td> Beckman </td><td></td><td></td></tr><tr><td> Beckman rör med lock </td><td> Beckman </td><td> 355.672 </td><td></td></tr><tr><td> Vita väggar adaptrar </td><td> Beckman </td><td> 342.327 </td><td></td></tr><tr><td> Blå väggar adaptrar </td><td> Beckman </td><td></td><td></td></tr><tr><td> JA-17 Rotor, fast-vinkel rotor </td><td> Beckman </td><td> 369.691 </td><td></td></tr></tbody></table> Tabell av antikroppar används för Western blotting: <table border="1"><tbody><tr><th> Namn på antikroppar </th><th> Företag </th><th> Katalognummer </th><th> Värdarter </th><th> Spädningsfaktor </th></tr><tr><td> β-Actin </td><td> Sigma </td><td> A5441 </td><td> mus </td><td> 1:2000 </td></tr><tr><td> GFAP </td><td> Santa Cruz </td><td> sc-65.343 </td><td> mus </td><td> 1:200 </td></tr><tr><td> GP73 </td><td> Santa Cruz </td><td> Sc-134.509 </td><td> kanin </td><td> 1:200 </td></tr><tr><td> HSC70 </td><td> Santa Cruz </td><td> sc-7298 </td><td> mus </td><td> 1:200 </td></tr><tr><td> Laminβ </td><td> Santa Cruz </td><td> Sc-6261 </td><td> get </td><td> 1:200 </td></tr><tr><td> Prohibitin </td><td> Santa Cruz </td><td> sc-28.259 </td><td> kanin </td><td> 1:200 </td></tr><tr><td> PSD95 </td><td> Millipore </td><td> MAB1596 </td><td> mus </td><td> 5 mikrogram / mikroliter </td></tr><tr><td> SNAP25 </td><td> AbCam </td><td> ab5666-100 </td><td> kanin </td><td> 1:2000 </td></tr><tr><td> Synaptophysin </td><td> Millipore </td><td> MAB368 </td><td> mus </td><td> 1:500 </td></tr><tr><td> β-3-tubulin </td><td> Santa Cruz </td><td> sc-80.016 </td><td> mus </td><td> 1:200 </td></tr></tbody></table>

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

Synaptoneurosomes (SNS) erhålls efter homogenisering och fraktionering av musen hjärnbarken. De är återförslutas blåsor eller isolerade terminaler bryta sig loss från axon terminaler när kortikala vävnad homogeniserad. SNS behålla pre-och postsynaptiska egenskaper, vilket gör dem användbara i studiet av synaptisk transmission. De behåller de molekylära maskiner som används i neuronala signalering och kan upptag, lagring och frisättning av signalsubstanser. Produktionen och isolering av aktiva SNS kan vara problematiskt att använda medier som Ficoll, som kan vara cytotoxiska och kräva förlängd centrifugering på grund av hög densitet, och filtrering och metoder centrifugering, vilket kan resultera i låg aktivitet på grund av mekaniska skador på SNS. Ger dock användning av diskontinuerliga Percoll-sackaros densitetsgradienter att isolera SNS en snabb metod för att producera bra skördar av translationally aktiva SNS. Den Percoll-sackaros gradientmetoden är snabb och skonsam som sysselsätter isoton förhållanden, har färre och kortare centrifugering snurrar och undviker centrifugering steg som pellets SNS och orsaka mekaniska skador.

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

En metod för att förbereda translationally aktiv, intakt synaptoneurosomes (SNS) från mus hjärnbarken beskrivs. Metoden använder en diskontinuerlig Percoll-sackaros densitetsgradient möjliggör en snabb beredning av aktiva SNS.

Beredning av Synaptoneurosomes från mus Cortex med en kontinuerliga Percoll-Sackaros densitetsgradient

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

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

Funktionell avbildning av hörselbarken hos vuxna katter med hjälp av hög-fält 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 avbildning av Mitos i utvecklings Mouse embryonala 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

Two-Photon<em&gt; In vivo</em&gt; Imaging av Dendritutskotten i mus Cortex Använda en förtunnad-skalle Förberedelse

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

Beredning av Synaptic plasmamembranet och Postsynaptiska Density Proteiner hjälp av en kontinuerliga sukrosgradient

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

Undersöka Funktion av djupa kortikala och subkortikala strukturer Använda Stereotactic Elektroencefalografi: Lärdomar från främre cingulum Cortex

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