Name: quantifying synapses: an immunocytochemistry-based assay to quantify synapse number
Uploaded: 2010-11-16T17:30:00
Description: Watch this Scientific Journal Video about Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number at JoVE.com

Puncta Analyzer Plug-in för Image J är skriven av Barry Wark (nuvarande adress: Physion Consulting) i labbet av Ben A. Barres (Stanford University). Finansiering; <ul><li> Alfred P. Sloan Foundation </li><li> Den Ester A. och Joseph Klingenstein fonden, Inc. </li><li> Bred Biomedical Research Foundation </li><li> Cure för Huntingtons s Disease Initiative </li></ul>

Lösningar förbereda: <ol><li> Antibody buffert: <ul><li> 150 mM NaCl </li><li> 50 mM Tris-base (Fisher, Cat nr:. BP152-5, 50 mm) - 1,21 g </li><li> 1% BSA (Sigma, Cat nr:. A2153, 1%) - 2,0 g </li><li> 100 mm L-lysin (Sigma, Cat nr:. L-1137, 100 mm) - 3,65 g </li><li> Justera pH till 7,4 </li><li> 0,04% Azid </li><li> Justera volymen till 200 ml med destillerat H 2 O. </li><li> Filtrera genom 0.22μm filter (Millipore, Cat nr:. SCGPU02RE). </li></ul></li><li> PFA Diluant: <ul><li> 168 ml 0,5 M N...

<ol>
	<li>Barres, B. A., Silverstein, B. E., Corey, D. P., Chun, L. L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological,+morphological,+and+electrophysiological+variation+among+retinal+ganglion+cells+purified+by+panning.">Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning.</a> Neuron. 1, 791-803 (1988).</li><li>Meyer-Franke, A., Kaplan, M. R., Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+signaling+interactions+that+promote+the+survival+and+growth+of+developing+retinal+ganglion+cells+in+culture.">Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture.</a> Neuron. 15, 805-819 (1995).</li><li>Eroglu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gabapentin+receptor+alpha2delta-1+is+a+neuronal+thrombospondin+receptor+responsible+for+excitatory+CNS+synaptogenesis.">Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis.</a> Cell. 139, 380-392 (2009).</li><li>Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+efficacy+enhanced+by+glial+cells+in+vitro.">Synaptic efficacy enhanced by glial cells in vitro.</a> Science. 277, 1684-1687 .</li><li>Ullian, E. M., Sapperstein, S. K., Christopherson, K. S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+synapse+number+by+glia.">Control of synapse number by glia.</a> Science. 291, 657-661 (2001).</li><li>Christopherson, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombospondins+are+astrocyte-secreted+proteins+that+promote+CNS+synaptogenesis.">Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis.</a> Cell. 120, 421-433 (2005).</li><li>Godement, P., Salaun, J., Imbert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+and+postnatal+development+of+retinocollicular+projections+in+the+mouse.">Prenatal and postnatal development of retinocollicular projections in the mouse.</a> J. Comp. Neurol. 230, 552-575 (1985).</li><li>Schmidt, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+retinotopic+connections:+selective+stabilization+by+an+activity-dependent+mechanism.">Formation of retinotopic connections: selective stabilization by an activity-dependent mechanism.</a> Cell. Mol. Neurobiol. 5, 65-84 (1985).</li><li>Sachs, G. M., Jacobson, M., Caviness, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+changes+in+arborization+patterns+of+murine+retinocollicular+axons.">Postnatal changes in arborization patterns of murine retinocollicular axons.</a> J. Comp. Neurol. 246, 395-408 (1986).</li><li>Elmariah, S. B., Oh, E. J., Hughes, E. G., Balice-Gordon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+regulate+inhibitory+synapse+formation+via+Trk-mediated+modulation+of+postsynaptic+GABAA+receptors.">Astrocytes regulate inhibitory synapse formation via Trk-mediated modulation of postsynaptic GABAA receptors.</a> J. Neurosci. 25, 3638-3650 (2005).</li><li>Hughes, E. G., Elmariah, S. B., Balice-Gordon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+secreted+proteins+selectively+increase+hippocampal+GABAergic+length,+branching+and+synaptogenesis.">Astrocyte secreted proteins selectively increase hippocampal GABAergic length, branching and synaptogenesis.</a> Moll. Cell. Neurosci. 43, 136-145 (2010).</li></ol>

Synapsen analys som beskrivs ovan är baserad i samband med våra experimentella mål, där vi fokuserar till stor del på excitatoriska projektioner av RGC: er, antingen renas kultur eller i hjärnans avsnitt. Vi har lämnat en hänvisning antikroppar tabell över som fungerar bra för märkning retande synapser (tabell 1). Denna synaps analys kan anpassas för att kvantifiera synaps på alla neuronala befolkning eller någon annan synaptiska subtyp som det är en selektiv pre-och postsynap...

<table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> Antigen </td><td> Arter </td><td> Monoklonala / Polyklonala </td><td> Leverantör </td><td> Katalognummer </td><td> Utspädning </td><td> Fungerar i Kultur </td><td> Fungerar i avsnitten </td></tr><tr><td rowspan="10"> Presynaptiska </td><td> Synapsin </td><td> Kanin </td><td> Polyklonala </td><td> Synaptic Systems </td><td> 106.004 </td><td> 1:750 </td><td> Y </td><td> ND </td></tr><tr><td> Synapsin </td><td> Mus </td><td> Monoklonala </td><td> Synaptic Systems </td><td> 106.001 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> Fagott </td><td> Mus </td><td> Monoklonala </td><td> Analys Designs </td><td> VAM-PS003F </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> Fagott </td><td> Guinea Pig </td><td> Polyklonala </td><td> Synaptic Systems </td><td> 141.004 </td><td> 1:1000 </td><td> Y </td><td> ND </td></tr><tr><td> Synaptotagmin 1 </td><td> Kanin </td><td> Polyklonala </td><td> Synaptic Systems </td><td> 105.002 </td><td> 1:750 </td><td> Y </td><td> N </td></tr><tr><td> Synaptobrevin 2 (C1.69.1) </td><td> Mus </td><td> Monoklonala </td><td> Synaptic Systems </td><td> 104.211 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> Synaptophysin (C1.7.2) </td><td> Mus </td><td> Monoklonala </td><td> Synaptic Systems </td><td> 101.011 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> VGlut1 </td><td> Mus </td><td> Monoklonala </td><td> Millipore </td><td> MAB5502 </td><td> 1:2500 </td><td> N </td><td> Y </td></tr><tr><td> VGlut1 </td><td> Marsvin </td><td> Polyklonala </td><td> Millipore </td><td> AB5905 </td><td> 1:2500 </td><td> N </td><td> Y </td></tr><tr><td> VGlut2 </td><td> Marsvin </td><td> Polyklonala </td><td> Millipore </td><td> AB2251 </td><td> 1:2500 </td><td> N </td><td> Y </td></tr><tr><td rowspan="6"> Postsynaptiska </td><td> PSD-95 (6G6-1C9 klon) </td><td> Mus </td><td> Monoklonala </td><td> Affinity Bio Reagenser </td><td> MA1-045 </td><td> 1:750 </td><td> Y </td><td> N </td></tr><tr><td> PSD-95 </td><td> Kanin </td><td> Polyklonala </td><td> Zymed </td><td> 51-6900 </td><td> 1:500 </td><td> N </td><td> Y </td></tr><tr><td> Homer </td><td> Mus </td><td> Monoklonala </td><td> Synaptic Systems </td><td> 160.011 </td><td> 1:500 </td><td> Y </td><td> ND </td></tr><tr><td> Homer </td><td> Råtta </td><td> Polyklonala </td><td> Millipore </td><td> AB5875 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> Gephyrin </td><td> Kanin </td><td> Polyklonala </td><td> Synaptic Systems </td><td> 147.003 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> Gephyrin </td><td> Mus </td><td> Monoklonala </td><td> Synaptic Systems </td><td> 147 </td><td> 1:200 </td><td> Y </td><td> Y </td></tr></tbody></table> Tabell 1: Listor exempel på goda pre-och postsynaptiska markörer som vi har med framgång använts i vår synaps analys. Var medveten om att detta inte är en uttömmande lista över alla tillgängliga markörer. Y = Ja, N = Nej, ND = Ej fastställd. <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> Reagens </td><td> Företag </td><td> Kat. Nej. </td></tr><tr><td> PBS </td><td> Invitrogen </td><td> 20012-027 </td></tr><tr><td> poly-D-lysin </td><td> Sigma </td><td> P6407 </td></tr><tr><td> Laminin </td><td> Cultrex </td><td> 3400-010-01 </td></tr><tr><td> Triton X-100 </td><td> Roche Diagnostics GmbH </td><td> 9002-93-1 </td></tr><tr><td> Normal Goat Serum </td><td> Gibco </td><td> 16.210 </td></tr><tr><td> VectaShield med DAPI </td><td> Vector Laboratories </td><td> H-1200 </td></tr><tr><td> Oktober </td><td> Tissue-Tek </td><td> 4583 </td></tr><tr><td> Tris-Base (50 mm) </td><td> Fisher </td><td> BP152-5 </td></tr><tr><td> Bovint serumalbumin </td><td> Sigma </td><td> A2153 </td></tr><tr><td> L-lysin </td><td> Sigma </td><td> L-1137 </td></tr><tr><td> 16% PFA-lösning </td><td> Elektronmikroskopi Sciences </td><td> 15.711 </td></tr><tr><td> Granular PFA </td><td> Elektronmikroskopi Sciences </td><td> 19.210 </td></tr><tr><td> 24-bra odlingsplatta </td><td> Falcon </td><td> 35-3047 </td></tr><tr><td> Get-anti-mus Alexa konjugerade antikroppar </td><td> Invitrogen </td><td> --- </td></tr><tr><td> Supply </td><td> Företag </td><td> Kat. Nej. </td></tr><tr><td> 12mm, nr 0 glas täckglas </td><td> Karl Hecht Gmbh </td><td> 1105209 </td></tr><tr><td> Nej 1,5 glas täckglas (för skivor) </td><td> VWR Vetenskapliga </td><td> 48393241 </td></tr><tr><td> Glas diabilder </td><td> VWR Vetenskapliga </td><td> 48311-703 </td></tr></tbody></table>

quantifying synapses: an immunocytochemistry-based assay to quantify synapse number

En av de viktigaste målen inom neurovetenskap är att förstå de molekylära signaler som instruerar tidiga stadier av synaps bildas. Som sådant har det blivit nödvändigt att utveckla objektiva metoder för att kvantifiera förändringar i synaptisk anslutning. Från prov fixering, detta protokoll beskriver hur att kvantifiera synaps antal både i dissocierade neuronala kultur och i hjärnan sektioner med hjälp av immuncytokemi. Använda fack-specifika antikroppar, etikett vi presynaptiska terminaler samt platser av postsynaptiska specialisering. Vi definierar synapser som punkter av colocalization mellan de signaler som genereras av dessa markörer. Antalet sådana colocalizations är kvantifieras med hjälp av en plug in Puncta Analyzer (skriven av Bary Wark, på begäran, c.eroglu @ cellbio.duke.edu) under ImageJ analysen mjukvaruplattform. Synapsen analys som beskrivs i detta protokoll kan tillämpas på alla nervvävnad eller kultur förberedelse för vilka du har selektiv pre-och postsynaptiska markörer. Denna synaps-analysen är ett värdefullt verktyg som kan vara allmänt används i studiet av synaptiska utveckling.

Watch this Scientific Journal Video about Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number at JoVE.com

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

Detta protokoll beskriver hur att kvantifiera synaps antal både i dissocierade neuronala kultur och i hjärnan sektioner med hjälp av immuncytokemi. Använda fack-specifika antikroppar, etikett vi presynaptiska terminaler samt platser av postsynaptiska specialisering. Vi definierar synapser som punkter av colocalization mellan de signaler som genereras av dessa markörer.

Kvantifiera synapser: en immuncytokemi-baserad analys att kvantifiera Synapse Antal

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

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