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

Vorremmo ringraziare BK agosto presso la University of Wisconsin-Madison strumento microscopio elettronico per la microscopia elettronica. Questo lavoro è stato sostenuto da NIH R01-DA026067 e P30-HD03352 (a JSM).

1. Preparativi 1-4 <ol><li> Preparare 500 mL di tampone di media pendenza (GM) mescolando 50 mL di una sospensione saccarosio 2,5 M, 2,5 mL di una 1M Tris-HCl, pH7.5 stock, e 0,1 mL di una m 0,5 EDTA, pH 8,0 stock con millisecondi Q acqua al volume. Filtro sterilizzare la soluzione, aliquota e conservare congelati. </li><li> Preparare uno stock 1000x di tetrodotossina (TTX) facendo una soluzione 1 mM in milli-Q H 2 O. Aliquote di TTX possono essere conservati a -20 ° C. </li><li> Preparare Buffe...

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	<li>Westmark, C. J., Malter, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FMRP+mediates+mGluR5-dependent+translation+of+amyloid+precursor+protein.">FMRP mediates mGluR5-dependent translation of amyloid precursor protein.</a> PLoS Biol. 5, e52-e52 (2007).</li><li>Westmark, P. R., Westmark, C. J., Wang, S., Levenson, J., O'Riordan, K. J., Burger, C., Malter, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pin1+and+PKMzeta+sequentially+control+dendritic+protein+synthesis.">Pin1 and PKMzeta sequentially control dendritic protein synthesis.</a> Sci Signal. 3, ra18-ra18 (2010).</li><li>Dunkley, P. R., Heath, J. W., Harrison, S. M., Jarvie, P. E., Glenfield, P. J., Rostas, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+Percoll+gradient+procedure+for+isolation+of+synaptosomes+directly+from+an+S1+fraction:+Homogeneity+and+morphology+of+subcellular+fractions.">A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: Homogeneity and morphology of subcellular fractions.</a> Brain Res. 441, 59-71 (1988).</li><li>Dunkley, P. R., Jarvie, P. E., Robinson, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+Percoll+gradient+procedure+for+preparation+of+synaptosomes.">A rapid Percoll gradient procedure for preparation of synaptosomes.</a> Nat. Protoc. 3, 1718-1728 (2008).</li><li>Hollingsworth, E. B., McNeal, E. T., Burton, J. L., Williams, R. J., Daly, J. W., Creveling, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+a+filtered+synaptoneurosome+preparation+from+guinea+pig+cerebral+cortex:+cyclic+adenosine+3':5'-monophosphate-generating+systems,+receptors,+and+enzymes.">Biochemical characterization of a filtered synaptoneurosome preparation from guinea pig cerebral cortex: cyclic adenosine 3':5'-monophosphate-generating systems, receptors, and enzymes.</a> J. Neurosci. 5, 2240-2253 (1985).</li><li>Yi, H., Leunissen, J., Shi, G., Gutekunst, C., Hersch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+procedure+for+pre-embedding+double+immunogold-silver+labeling+at+the+ultrastructural+level.">A novel procedure for pre-embedding double immunogold-silver labeling at the ultrastructural level.</a> J. Histochem. Cytochem. 49, 279-284 (2001).</li><li>Akins, M. R., Berk-Rauch, H. E., Fallon, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+translation:+stepping+out+of+the+postsynaptic+shadow.">Presynaptic translation: stepping out of the postsynaptic shadow.</a> Front. Neural Circuits. 3, (2009).</li><li>Jin, I., Kandel, E. R., Hawkins, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whereas+short-term+facilitation+is+presynaptic,+intermediate-term+facilitation+involves+both+presynaptic+and+postsynaptic+protein+kinases+and+protein+synthesis.">Whereas short-term facilitation is presynaptic, intermediate-term facilitation involves both presynaptic and postsynaptic protein kinases and protein synthesis.</a> Learn. Mem. 18, 96-102 (2011).</li><li>Lyles, V., Zhao, Y., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse+formation+and+mRNA+localization+in+cultured+Aplysia+neurons.">Synapse formation and mRNA localization in cultured Aplysia neurons.</a> Neuron. 49, 349-356 (2006).</li><li>Wagatsuma, A., Azami, S., Sakura, M., Hatakeyama, D., Aonuma, H., Ito, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+Novo+synthesis+of+CREB+in+a+presynaptic+neuron+is+required+for+synaptic+enhancement+involved+in+memory+consolidation.">De Novo synthesis of CREB in a presynaptic neuron is required for synaptic enhancement involved in memory consolidation.</a> J. Neurosci. Res. 84, 954-9560 (2006).</li><li>Sutton, M. A., Schuman, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+translational+control+in+dendrites+and+its+role+in+long-term+synaptic+plasticity.">Local translational control in dendrites and its role in long-term synaptic plasticity.</a> J. Neurobiol. 64, 116-131 (2005).</li><li>Li, K. -. W., Hornshaw, M. P., Van der Schors, R. C., Watson, R., Tate, S., Casetta, B., Jimenez, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+analysis+of+rat+brain+postsynaptic+density:+implications+of+the+diverse+protein+functional+groups+for+the+integration+of+synaptic+physiology.">Proteomics analysis of rat brain postsynaptic density: implications of the diverse protein functional groups for the integration of synaptic physiology.</a> J. Biol. Chem. 279, 987-1002 (2004).</li><li>Peng, J., Kim, M. J., Cheng, D., Duong, D. M., Gygi, S. P., Sheng, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semi-quantitative+proteomic+analysis+of+rat+forebrain+postsynaptic+density+fractions+by+mass+spectrometry.">Semi-quantitative proteomic analysis of rat forebrain postsynaptic density fractions by mass spectrometry.</a> J. Biol. Chem. 279, 21003-21011 (2004).</li><li>Kim, S. H., Fraser, P. E., Westaway, D., St. George-Hyslop, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Group+II+metabotropic+glutamate+receptor+stimulation+triggers+production+and+release+of+Alzheimer's+amyloid+&#x03B2;42+from+isolated+intact+nerve+terminals.">Group II metabotropic glutamate receptor stimulation triggers production and release of Alzheimer's amyloid &#x03B2;42 from isolated intact nerve terminals.</a> J. Neurosci. 30, 3870-3875 (2010).</li><li>Weiler, I. J., Greenough, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+ion+stimulation+triggers+protein+translation+in+synaptoneurosomal+poiyribosomes.">Potassium ion stimulation triggers protein translation in synaptoneurosomal poiyribosomes.</a> Mol. Cell. Neurosci. 2, 305-314 (1993).</li><li>Billard, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ageing,+hippocampal+synaptic+activity+and+magnesium.">Ageing, hippocampal synaptic activity and magnesium.</a> Magnes Res. 19, 199-215 (2006).</li><li>Murphy, G. G., Fedorov, N. B., Giese, K. P., Ohno, M., Friedman, E., Chen, R., Silva, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+neuronal+excitability,+synaptic+plasticity,+and+learning+in+aged+Kvbeta1.1+knockout+mice.">Increased neuronal excitability, synaptic plasticity, and learning in aged Kvbeta1.1 knockout mice.</a> Curr. Biol. 14, 1907-1915 (2004).</li><li>Norris, C. M., Halpain, S., Foster, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+Age-Related+Alterations+in+Synaptic+Plasticity+by+Blockade+of+L-Type+Ca21+Channels.">Reversal of Age-Related Alterations in Synaptic Plasticity by Blockade of L-Type Ca21 Channels.</a> J. Neurosci. 18, 3171-3179 (1998).</li><li>Sallert, M., Malkki, H., Segerstr&#228;lea, M., Tairaa, T., Lauri, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+kainate+receptor+agonist+ATPA+on+glutamatergic+synaptic+transmission+and+plasticity+during+early+postnatal+development.">Effects of the kainate receptor agonist ATPA on glutamatergic synaptic transmission and plasticity during early postnatal development.</a> Neuropharm. 52, 1354-1365 (2007).</li><li>Quinlan, E. M., Philpot, B. D., Huganir, R. L., Bear, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+experience-dependent+expression+of+synaptic+NMDA+receptors+in+visual+cortex+in+vivo.">Rapid, experience-dependent expression of synaptic NMDA receptors in visual cortex in vivo.</a> Nat. Neurosci. 2, 357-35 (1999).</li><li>Scheetz, A. J., Nairn, A. C., Constantine-Patton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+receptor-mediated+control+of+proteins+synthesis+at+developing+synapses.">NMDA receptor-mediated control of proteins synthesis at developing synapses.</a> Nat. Neurosci. 3, 211-216 (2000).</li><li>Villasana, L. E., Klann, E., Tejada-Simon, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+synaptoneurosomes+and+postsynaptic+densities+from+adult+mouse+hippocampus.">Rapid isolation of synaptoneurosomes and postsynaptic densities from adult mouse hippocampus.</a> J. Neurosci. Methods. 158, 30-36 (2006).</li><li>Weiler, I. J., Greenough, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabotropic+glutamate+receptors+trigger+postsynaptic+protein+synthesis.">Metabotropic glutamate receptors trigger postsynaptic protein synthesis.</a> Proc. Natl. Acad. Sci. U.S.A. 90, 7168-7171 (1993).</li><li>Gray, E. G., Whittaker, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+of+nerve+endings+from+brain:+An+electron+microscopic+study+of+cell+fragments+derived+by+homogenization+and+centrifugation.">The isolation of nerve endings from brain: An electron microscopic study of cell fragments derived by homogenization and centrifugation.</a> J. Anat. 96, 79-88 (1962).</li><li>Gylys, K. H., Fein, J. A., Yang, F., Cole, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+Presynaptic+and+Postsynaptic+Markers+by+Size-Based+Gating+Analysis+of+Synaptosome+Preparations+From+Rat+and+Human+Cortex.">Enrichment of Presynaptic and Postsynaptic Markers by Size-Based Gating Analysis of Synaptosome Preparations From Rat and Human Cortex.</a> Cytometry A. 60, 90-96 (2004).</li><li>Haj&#243;s, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+the+preparation+of+synaptosomal+fractions+in+high+purity.">An improved method for the preparation of synaptosomal fractions in high purity.</a> Brain Res. 93, 485-489 (1975).</li><li>Bai, F., Witzmann, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptosome+Proteomics.">Synaptosome Proteomics.</a> Subcell. Biochem. 43, 77-98 (2007).</li><li>Rao, A., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+protein+constituents+of+postsynaptic+membrane+specializations+are+locally+synthesized:+analysis+of+proteins+synthesized+within+synaptosomes.">Evidence that protein constituents of postsynaptic membrane specializations are locally synthesized: analysis of proteins synthesized within synaptosomes.</a> J Neurosci. 11, 2881-2895 (1991).</li><li>Pertoft, H., Laurent, T. C., Catsimpoolas, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isopycnic+separation+of+cells+and+cell+organelles+by+centrifugation+and+modified+colloidal+silica+gradients.">Isopycnic separation of cells and cell organelles by centrifugation and modified colloidal silica gradients.</a> Methods of Cell Separation. , 25-65 (1977).</li><li>Ramarao, C. S., Acharya, S. R., Krishnan, K. S., Kenkare, U. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+affinity+uptake+of+L-glutamate+and+&amp;gamma;-aminobutyric+acid+in+Drosophila+melanogaster.">High affinity uptake of L-glutamate and &amp;gamma;-aminobutyric acid in Drosophila melanogaster.</a> J. Biosci. 11, 119-135 (1987).</li><li>Munteanu, L. S., Dinu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractionation+of+granulocytes+from+whole+human+blood+by+centrifugation.+Practical+hints.">Fractionation of granulocytes from whole human blood by centrifugation. Practical hints.</a> Romanian J. Biophys. 14, 53-58 (2004).</li><li>Vlasselaer, P., Van Palathumpat, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation+composition,+kit+and+method.">Cell separation composition, kit and method.</a> US patent. , (1997).</li></ol>

Il discontinuo di Percoll-saccarosio preparazione pendenza qui descritto è un processo rapido, metodo affidabile per isolare attivo SN, che può essere utilizzato in una varietà di esperimenti di trasmissione sinaptica. Questo metodo del gradiente, che si basa sul metodo sviluppato da Dunkley et al. 3,4 è una procedura di frazionamento subcellulare del cervello che isola pre e post-sinaptici di membrana derivata vescicole che sono associati tra loro. Senza ulteriore purificazione queste SN sono compatibili...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> Micro BCA Protein Assay Kit </td><td> Perforare </td><td> 23235 </td><td></td></tr><tr><td> CaCl 2 </td><td> Pescatore </td><td> C79-500 </td><td></td></tr><tr><td> Gas 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> Pescatore </td><td> A407SK-4 </td><td></td></tr><tr><td> HCl </td><td> Pescatore </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> Pescatore </td><td> P285-500 </td><td></td></tr><tr><td> PierceSDS-PAGE kit di preparazione del campione </td><td> Perforare </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> Pescatore </td><td> BP328-500 </td><td></td></tr><tr><td> Na 2 PHO 4 </td><td> Pescatore </td><td> S381-500 </td><td></td></tr><tr><td> Saccarosio </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> Tetrodotossina </td><td> Sigma </td><td> T5651 </td><td></td></tr><tr><td> Esprimere Label Pro 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> Attrezzatura </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> Strumenti di dissezione </td><td></td><td></td><td></td></tr><tr><td> Dounce omogeneizzatore, 7 ml (vetro viene fornito con due pestelli etichetta &quot;A&quot; e &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 Centrifuga </td><td> Beckman Coulter </td><td> 366830 </td><td></td></tr><tr><td> GH 3,8 rotore, rotore oscillante </td><td> Beckman Coulter </td><td> 360581 </td><td></td></tr><tr><td> Beckman J2-21 Centrifuga </td><td> Beckman </td><td></td><td></td></tr><tr><td> Beckman tubi con tappi </td><td> Beckman </td><td> 355672 </td><td></td></tr><tr><td> Bianco adattatori murata </td><td> Beckman </td><td> 342327 </td><td></td></tr><tr><td> Blu adattatori murata </td><td> Beckman </td><td></td><td></td></tr><tr><td> JA-17 Rotor, Rotore ad angolo fisso </td><td> Beckman </td><td> 369691 </td><td></td></tr></tbody></table> Tabella di anticorpi utilizzati per Western blot: <table border="1"><tbody><tr><th> Nome di anticorpi </th><th> Azienda </th><th> Numero di catalogo </th><th> Specie Host </th><th> Fattore di diluizione </th></tr><tr><td> β-Actina </td><td> Sigma </td><td> A5441 </td><td> mouse </td><td> 1:2000 </td></tr><tr><td> GFAP </td><td> Santa Cruz </td><td> sc-65343 </td><td> mouse </td><td> 1:200 </td></tr><tr><td> GP73 </td><td> Santa Cruz </td><td> Sc-134509 </td><td> coniglio </td><td> 1:200 </td></tr><tr><td> HSC70 </td><td> Santa Cruz </td><td> sc-7298 </td><td> mouse </td><td> 1:200 </td></tr><tr><td> Laminβ </td><td> Santa Cruz </td><td> Sc-6261 </td><td> capra </td><td> 1:200 </td></tr><tr><td> Prohibitin </td><td> Santa Cruz </td><td> sc-28259 </td><td> coniglio </td><td> 1:200 </td></tr><tr><td> PSD95 </td><td> Millipore </td><td> MAB1596 </td><td> mouse </td><td> 5 mcg / mL </td></tr><tr><td> SNAP25 </td><td> AbCam </td><td> ab5666-100 </td><td> coniglio </td><td> 1:2000 </td></tr><tr><td> Sinaptofisina </td><td> Millipore </td><td> MAB368 </td><td> mouse </td><td> 1:500 </td></tr><tr><td> β-3-tubulina </td><td> Santa Cruz </td><td> sc-80016 </td><td> mouse </td><td> 1:200 </td></tr></tbody></table>

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

Synaptoneurosomes (SN) si ottengono dopo omogeneizzazione e frazionamento della corteccia cerebrale del mouse. Sono risigillati vescicole o terminali isolati che rompere con terminali dell&#39;assone quando il tessuto corticale è omogeneizzato. La SN mantengono caratteristiche di pre-e post-sinaptici, che li rende utili per lo studio della trasmissione sinaptica. Essi conservano le macchine molecolari utilizzati nella segnalazione neuronale e sono capaci di captazione, stoccaggio, e il rilascio di neurotrasmettitori. La produzione e l&#39;isolamento di SN attivo può essere problematico media usando come Ficoll, che possono essere citotossici e richiedono estese a causa di centrifugazione ad alta densità, e la filtrazione e metodi di centrifugazione, che può portare a bassa attività a causa di danni meccanici del SN. Tuttavia, l&#39;uso discontinuo di Percoll-saccarosio gradienti di densità per isolare SN offre un metodo rapido per produrre buone rese di SN traduzionali attivo. Il Percoll-saccarosio metodo del gradiente è veloce e gentile come impiega condizioni isotoniche, ha un minor numero di giri centrifugazione e più brevi ed evita le fasi di centrifugazione che SN pellet e provocare danni meccanici.

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 metodo per preparare traduzionali attivo, synaptoneurosomes intatto (SN) da topo corteccia cerebrale è descritto. Il metodo utilizza un discontinuo di Percoll-saccarosio gradiente di densità che permette per la preparazione veloce di SN attivo.

Preparazione di Synaptoneurosomes da mouse Cortex con un discontinuo di Percoll-Saccarosio gradiente di densità

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

Imaging funzionale di Auditory Cortex in gatti adulti utilizzando ad 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

Immagini dal vivo della mitosi in via di sviluppo embrionali di topo 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 di spine dendritiche nella corteccia mouse Uso un teschio Diluito-Preparazione

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

Preparazione di Synaptic membrana plasmatica e Proteine ​​densità postsinaptica Utilizzo di un gradiente di saccarosio discontinuo

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

Indagare la funzione di Deep corticale e strutture sottocorticali Usando stereotassica Elettroencefalografia: gli insegnamenti della corteccia cingolata anteriore

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