Biz, elektron mikroskobu için, Wisconsin-Madison Elektron Mikroskobu Tesisi Üniversitesi&#39;nden BK Ağustos teşekkür etmek istiyorum. Bu çalışma NIH hibe R01-DA026067 ve P30-HD03352 (JSM) tarafından desteklenmiştir.

1. Hazırlıklar 1-4 <ol><li> 50 ml 2.5 M sakkaroz bulamaç karıştırılarak 500 mL (GM), degrade orta tampon hazırlayın, 1M Tris-HCl, pH7.5 hisse senedi ve 0,5 m EDTA 0.1 ml 2.5 ml, pH 8.0, stok Mili S su hacmi. Çözümü, kısım ve mağaza dondurulmuş sterilize edin Filtre. </li><li> Mili-Q H 2 O. 1 mM çözüm yaparak tetrodotoxin (TTX) 1000x stok hazırlayın TTX alikotları -20 ° C&#39;de saklanabilir </li><li> Mili Q suyu 100 mM Tris (pH 7.5), 5 mM Na 2 HPO 4, 4 mM KH 2 PO 4, 40 mM NaHCO 3 ve 800 mM NaCl çözeltisi yaparak 10x Uyarım Tampon hazırlayın. Stok 4 saklanabilir ° C </li><li> Pre-cool santrifüjler için 4 ° C </li><li> Mili Q suyu 2.5 M sakkaroz (2 ml) 1 hacim Percoll (18 ml) 9 hacim ekleme Isosmotic Percoll (SIP) stok solüsyonu hazırlayın ve iyice karıştırın. </li><li> GM tampon SIP ilgili tutarların eklenmesi ve her karıştırma 3, 10, 15, ve% 23 degrade katmanları hazırlayın: </li></ol><img alt="Tablo 0" src="/files/ftp_upload/3196/3196table0.jpg" /><ol start="7"><li> P1000 Gilson Pipetman kullanarak kapaklar Beckman santrifüj tüplerine 2 ml 23, 15, 10, ve% 3 isosmotic Percoll çözümleri pipetleme degrade katmanları dökün. Katmanlar arasında arayüz katmanları hiçbir karıştırma ile açıkça ayırt edilebilir olmalıdır. Gradyanlar kullanmadan önce en az 20 dakika süreyle 4 ° C&#39;de saklayın. Gradyanlar aynı gün kullanılması tavsiye edilir rağmen 24 saate kadar saklanabilir. [Deneyimsiz laboratuvar personeli mavi boya katmanı arayüzleri görünüm için isomotic Percoll çözümler ekleyerek hazırlanıyor gradyanlar pratik.] </li></ol> 2. Fare diseksiyonu 1 <ol><li> Tüm fare hayvancılık ve ötenazi prosedürleri NIH ve Üniversite kurallarına uygun olarak yapılır emin olun. Karbondioksit boğulma ya da hayvan protokolü olarak onaylanmış bir yöntem ile yavrular (yaş P13 P21) Euthanize. </li><li> Diseksiyonu kuruluna fare Pin ve etanol ile boyun ve baş arkasında sprey. Kafatası tabanında omurilik kesti keskin bir makas kullanarak, kafatasının üst deri kaldırmak parietal kemik ve interparietal kemik arasında veya beyin ve korteks bölgelerinde arasındaki yanal kafatası kesmek. Sonra burun tabanı (sagital sütür boyunca) kafatasının kesilmiş, dikkatle yumuşak parietal kemik tarafında her yarımkürede çekerek çıkarın ve bir spatula ile korteks kaldırmak. Spatula beyincik Yukarıdaki beyin içine takın ve daha sonra korteks kepçe ve buz GM tampon yerleştirmek. Homojenizasyon adıma hemen geçin. Korteks bu SNS oluşumunu olumsuz etkileyecek, çok uzun süre buz tampon oturmak izin vermeyin. </li></ol> 3. Homojenat ve SNS 1-4 hazırlanması <ol><li> Buz GM tampon korteks yıkayın ve soğuk GM tampon 5 ml içeren bir cam Dounce homojenizatör iki korteks transferi. </li><li> Yavaşça gevşek havaneli 5-10 vuruş ile korteks homojenize (havaneli &quot;A&quot;), 5-10 vuruş sıkı havaneli (havaneli &quot;B&quot;) izledi. Darbeleri hızlı ama hava kabarcıkları neden yeterince yavaş olmalıdır. Programı 1 homojenizasyon sonra Homojenat bir resim. Vuruş sayısı, bireysel Homojenizatör ve beraberindeki havan bağlı olarak değişecektir. Gradyanlar vuruş sayısı değilse, güçlü bir SN band verdiği ayarlamanız gerekir bitirdi. </li><li> 4 15 ml konik ve 1000xg az 10 dakika süreyle santrifüj ° C sallanan bir kova rotor (Allegra 6KR santrifüj) Homojenat pelet hücresel enkaz ve çekirdekler aktarın. Bükülmüş Homojenat Resim Programı 1. </li><li> 4 5 dakika 32.500 xg&#39;de Percoll-sukroz gradient, kap tüpleri, santrifüj başına süpernatant Layer 2 mL ° C (Beckman J2-21 santrifüj, JA-17 rotor) sabit açılı rotor (uygun adaptörler kullanarak Reaktifler ve Ekipmanları Masa bakınız. ) </li><li> Pipet ve cam Pasteur pipeti kullanarak SN bant çözümü yukarıda atın. 15/23% arayüzü SN bant kapalı Pipet, buz üzerinde konik ve mağaza aktarın. Her degrade veya bir korteks 0,9-1,1 ml SN üretecektir. </li><li> 12 nM bir final konsantrasyon 1000x CaCl 2 ve nonspesifik uyarma bastırmak için 1 mcM bir son konsantrasyonu 1 mM TTX stok eklemek SNS tuz konsantrasyonu, 10x stimülasyon tampon onda biri hacmi ekleyerek ayarlayın. (Downstream SN uygulamaları ile engel olacak CaCl 2 ilavesi isteğe bağlıdır). </li><li> Micro BCA Protein Testi Kiti kullanarak SNS protein konsantrasyonu belirleyin. (Bradford protein testi Percoll çıkarımlar nedeniyle tavsiye edilmez.) </li><li> SNS protein transla doğrudan ve hızlı bir şekilde kullanılabilirTION çalışmalar. Diğer uygulamalar için SN lizat temizlenmeli veya Pierce SDS-PAGE örnek Hazırlık Seti kullanarak konsantre olabilir. </li></ol> 4. Temsilcisi sonuçları: Fare korteks süreksiz Percoll-sakaroz gradyanlar (Şekil 1), 6 bant veya kesirler homojenize ve ayrıldı (Şekil 1) görüldü. Daha önce, yüksek molekül ağırlıklı gruplarından bileşenlerinin membran ve miyelin kırık ve pelet malzeme (Tablo 1) mitokondri veya organelleri yer alan olduğunu serebral korteks 3,5 sıçan gösterildi . % 23 /% 15 arayüzü (Band 5) zenginleştirilmiş SNS içeriyordu. Sağlam SNS içeren% 23 /% 15 arayüzü, bant kaldırılır ve daha önce açıklandığı gibi, elektron mikroskobu (EM) tarafından incelenmiştir. 2,6 EM sağlam sinaptik veziküllerin varlığı ve presinaptik ve postsinaptik elemanlarının korunması (Şekil gösterdi 2). Pre-ve postsinaptik bölmeleri izolasyonu, sinyalizasyon ve her iki bölümlerinde meydana protein sentezi, incelenmesi için önemlidir. 7-13 Percoll sukroz gradient SN fraksiyonu küçük hücresel, nükleer ve organel kirlenme gördüm, böylece ham arıtma ama genel ayrılması iyi oldu. Western Blot bakıldığında, Percoll kesirler SN bant (Şekil 3, Tablo 2) sinaptik saflık bir artış bekleniyor protein damgalı içeriyordu. Synaptic ve nöronal belirteçler zenginleştirilmiş SN bant (Band 5) muhafaza edildi ama diğer belirteçlerin bolluk önemli ölçüde azaldı. Özellikle, yapısal ve sinaptik belirteçler muhafaza veya gelişmiş iken GFAP, glial kökenli astrosit ve neoplastik hücreler için bir marker ve Lamin-B1, nükleer bir marker, varlığı, azdı. Buna ek olarak, protein sentezinde önemli bir rol oynayan mitokondri ve golgi gibi organelleri, belirteçleri saklama vardı. BCA protein testi ile belirlenen SN grup için tipik verim mcL başına protein 250-500 ng. SNS 50 mcM glutamate/10 mcM glisin ile uyarılmış olduğu SNS faaliyet de novo protein sentezi miktarı tespit edildi [35S]-Met dahil edilmesi (Şekil 4) tarafından izlenir gibi mevcut. 2 Bazal translasyonel aktivite 1.56 kat arttı (Glu) ya da aktif kat 5.12 Pin1 inhibitörü juglone&#39;un. Pin1 inhibisyonu [35S]-Met eklenmesi de novo protein sentezinin artması nedeniyle olduğunu onaylamak için artan protein sentezini 2 bilinir, biz 40 mcM anisomycin, protein sentezi inhibitörü eklenen ve belirgin bir azalma dahil gördüm bazal düzeylere göre Glu Mobile varlığı ve yokluğu. Ayrıca,% 2 SNS bütünlüğünü bozan Triton-X 100, ek üzerine, bazal çeviri% 75 oranında azalmıştır. 2 Buna ek olarak, en sinaptik işaretleyici zenginleştirme gösterdi bantları (B4-B6) incelerken, SNS veya Band 5 de novo protein sentezi (Şekil 5) en büyük faaliyet vardı. Böylece, süreksiz Percoll sakaroz gradyanlar hızlı protein çeviri çalışma için kullanılabilecek bir aktif ve yüksek derecede zenginleştirilmiş SN bant üretir. Çünkü sert koşulları tarafından mekanik hasarlara neden süreksiz Percoll sukroz gradient işlemi diğer yöntemlere göre daha avantajlıdır. Kortikal Homojenat temizlenir filtrasyon yöntemi ile karşılaştırıldığı zaman boyutunda azalma, 3,14-15 Percoll yöntemi, daha fazla aktivite ile daha fazla SNS formları filtreleri bir dizi geçirilir. Şekil 6, 15/23% arayüzü çok daha az kırık membranların SNS ve büyük miktarda vardır Percoll sukroz gradient üzerinde filtrasyon yöntemi ile hazırlanan SNS bir kısım geçti görüldüğü gibi. De novo protein sentezi S35-met birleşme yoluyla ölçüldü, filtrasyon yöntemi ile hazırlanan SNS çok daha az aktif olan (Şekil 7). Translasyonel aktivite en üst düzeye çıkarmak için P14 ve P18 yaş arasında olan fareler kullanın. SN yetişkin farelerin hazırlanan, ancak juvenil fareler (Şekil 8) hazırlanan SN translasyonel aktivite önemli ölçüde artış görülmektedir olabilir. <img alt="Tablo 1" src="/files/ftp_upload/3196/3196table1.jpg" /> Tablo 1. Homojenize fare korteksin süreksiz Percoll degrade since gruplarından bileşenleri 3,5 <img alt="Tablo 2" src="/files/ftp_upload/3196/3196table2.jpg" /> Tablo 2. Süreksiz Percoll sukroz gradient bant bileşenlerinin Batı cıvata analizinin özeti her grubun kesirler Bağıl protein konsantrasyonları süreksiz Percol kullanarak izolel-sukroz gradient Western Blot tarafından belirlenir. Proteinini yok (-) 0 ≥ 0.2 (+), 0.2 ≥ 0.8 (++), veya&gt; 0.8 (+++) kat daha fazla n süpernatant (S, santrifüj korteks Homojenat), temsilci protein mevcut = 3. <img alt="Programı 1" src="/files/ftp_upload/3196/3196scheme1.jpg" /> Programı 1: süreksiz Percoll sakaroz yoğunluk gradiyenti kullanarak SN hazırlanması şematik Fare beynin korteks disseke homojenize ve homojenize olmayan doku, hücre çekirdekleri ve membranlar gibi tüm organelleri kaldırmak için düşük hızda santrifüj edildi. Süreksiz degrade katmanları açıkça tanımlanmış olması gerekir. Inset resim sadece katmanları görselleştirmek için amaçlıdır mavi boyalı olan katmanları, bir örnek gösterir. Süpernatant süreksiz Percoll sakaroz degrade üst katmanlı ve orta hızda santrifüj edilir. SN grup, daha sonra degrade alınan ve kullanıma hazır. <img alt="Şekil 1" src="/files/ftp_upload/3196/3196fig1.jpg" /> Şekil 1. Süreksiz Percoll sakaroz degrade Homojenat fraksiyonasyon. Fare korteks homojenize ve 6 bantlar veya kesirler sebebiyet veren bir süreksiz Percoll-sukroz gradient ayrıldı. Saflaştırılmış, aktif SNS (*) Band 5 bulundu. <img alt="Şekil 2" src="/files/ftp_upload/3196/3196fig2.jpg" /> Şekil 2. SN hazırlıkları presinaptik ve postsinaptik unsurlarını muhafaza SNS heterojen bir karışım verir. SN hazırlık C57BL / 6 farelerinde (yaş P13 ile P21) hazırlanan bir elektron mikrografı korunmuş presinaptik ve postsinaptik elemanları ile daha önce açıklanan ve gösterir SNS olarak yapıldı. 2 6 Kırmızı oklar noktası post-sinaptik yoğunlukları. Ölçek çubuğu 1 mikron. <img alt="Şekil 3" src="/files/ftp_upload/3196/3196fig3.jpg" /> Şekil 3. SN hazırlıkları Western Blot analizleri sinaptik ve nöronal işaretleri saflaştırılmış SNS muhafaza olduğunu gösterdi (Band 5), ancak diğer belirteçlerin bolluğu önemli ölçüde azaldı. Süpernatant Immunoblots (S, kortikal Homojenat santrifüjle) ve bir süreksiz Gruplar 1-6 Percoll-sukroz gradient (postsinaptik yoğunluğu β-Aktin (yapısal, yükleme kontrolü), GFAP (astrositik), GP73 (Golgi), HSC70 (sitoplazmik ve nükleer), Lamin-B1 (nükleer), Prohibitin (mitokondrial) PSD95 için probed ), SNAP25 (sinaptik), sinaptofizin (sinaptik) ve β3-tubulin (nöron-spesifik). Bantları temsilcisi n = 3, Storm 860 Phosphorimager kullanılarak görüntülendi. <img alt="Şekil 4" src="/files/ftp_upload/3196/3196fig4.jpg" /> Şekil 4. SNS aktif ve [35S]-Met eklenmesi ile de novo protein sentezi gösterirler. SNS 37 ° C&#39;de 10 dakika süreyle ön-dengelenmiş. Sonra SNS, 37, 40 mcM anisomycin veya 1 mcM juglone&#39;un ile 15 dakika süreyle tedavi edilmezse veya ön tedavi ° C-Met, 20 ul Express Pro Etiket artı veya eksi Glu 37 S35 Kolay Etiket Mix [35S] ilave edilerek takip ° C 30 dk. Örnekleri, Pierce SDS-PAGE örnek Hazırlık Seti kullanılarak hazırlanmış% 15 poliakrilamid jel üzerinde çalıştırmak, kuru ve Moleküler Dinamik Fırtına 860 phosphorimager, n = 3 temsilcisi, ± SEM kullanarak quantitated edildi. <img alt="Şekil 5" src="/files/ftp_upload/3196/3196fig5.jpg" /> Şekil 5. Band süreksiz Percoll sukroz gradient 5 de novo protein sentezinin en yüksek seviyede yer alıyor . 4, 5 ve 6 Gruplar ve supernatant (S), kortikal Homojenat santrifüjle 37 ön-dengelenmiş ° C 10 dak. Sonra, [35S]-Met, Express Pro Etiket Mix S35 Kolay Etiket eklendi, artı ya da eksi 37 ° Glu ° C de 30 dakika oldu. Örnekleri, Pierce SDS-PAGE örnek Hazırlık Seti kullanılarak hazırlanmış% 15 poliakrilamid jel üzerinde çalıştırmak, kuru ve Moleküler Dinamik Fırtına 860 phosphorimager, n = 3 temsilcisi, ± SEM kullanarak quantitated edildi. <img alt="Şekil 6" src="/files/ftp_upload/3196/3196fig6.jpg" /> Şekil 6. Filtrasyon yöntemi hazırlanan SNS süreksiz Percoll sukroz gradient yöntemi hazırlanan SNS daha kırık membranlar ve daha az bütün SNS içerir . SNS, daha önce açıklandığı gibi gözenek boyutu azalan filtreler bir dizi homojenize korteks geçerek hazırlanmıştır. 3,14-15 filtrasyon yöntemi ile hazırlanan SNS bir kısım süreksiz Percoll sukroz gradient üzerinde santrifüj ve eşdeğer bir miktara göre süreksiz Percoll-sukroz gradient yöntemi kullanılarak malzeme. <img alt="Şekil 7" src="/files/ftp_upload/3196/3196fig7.jpg" /> Şekil 7. Filtrasyon yöntemi hazırlanan SNS SNS fro hazırlanan daha az de novo protein sentezi aktivitesi içerenm süreksiz Percoll sukroz gradient yöntemi. SNS azalan gözenek boyutu ile daha önce açıklandığı gibi filtreler, 3,14-15 bir dizi ile homojenize korteks geçirerek ve süreksiz Percoll-sukroz gradient metodlar tarafından hazırlanmıştır. SNS hem hazırlıkları 37 ön-dengelenmiş ° C 10 dak. Sonra, [35S]-Met, Express Pro Etiket Mix S35 Kolay Etiket eklendi, artı ya da eksi 37 ° Glu ° C de 30 dakika oldu. Örnekleri, Pierce SDS-PAGE örnek Hazırlık Seti kullanılarak hazırlanmış% 15 poliakrilamid jel üzerinde çalıştırmak, kuru ve Moleküler Dinamik Fırtına 860 phosphorimager, n = 3 temsilcisi, ± SEM kullanarak quantitated edildi. <img alt="Şekil 8" src="/files/ftp_upload/3196/3196fig8.jpg" /> Şekil 8. Genç farelerde (P14) SNS yaşlı farelerin (P85) daha büyük translasyonel bir faaliyet var. SNS Glu Mobile ile 10 dakika süreyle 37 ° preequilibrated süreksiz Percoll-sukroz gradient yöntemi, ° C kullanarak farklı yaşlı farelerde hazırlanan tedavi veya tedavi edilmemiş edildi varlığı [35S]-Met, çıtçıt dondurulmuş, 30 dakika (Express Pro Etiket S35 Kolay Etiket Mix) ve daha sonra Pierce, SDS-PAGE Örnek Hazırlık Seti ile temizlenir. SNS, sonra% 12 SDS-poliakrilamid jel üzerinde çalıştırmak kurutulmuş ve n = 3, ± SEM Moleküler Dinamik Fırtına 860 phosphorimager, temsilci görüntülü edildi. P14 fareler SNS P85 farelerin en az 3 kat daha aktif.

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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. 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Çıkar çatışması ilan etti.

Burada açıklanan süreksiz Percoll sukroz gradient hazırlık sinaptik iletim deneyler çeşitli kullanılabilir aktif SNS, izole etmek için hızlı ve güvenilir bir yöntemdir. Dunkley ve ark. 3,4 tarafından geliştirilen yöntem dayanmaktadır Bu gradyan yöntemi, birbiri ile ilişkili hem de pre-ve postsinaptik membran kaynaklı veziküller izole bir hücre içi beyin fraksiyonasyon işlemdir. Ileri arıtma olmadan bu SNS immunoprecipitation, batı blot, flow sitometri, [35S] üzerinden Met dahil, ele...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> Micro BCA Protein Testi Kiti </td><td> Delmek </td><td> 23235 </td><td></td></tr><tr><td> CaCl 2 </td><td> Balıkçı </td><td> C79-500 </td><td></td></tr><tr><td> CO 2 gaz </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> Balıkçı </td><td> A407SK-4 </td><td></td></tr><tr><td> HCl </td><td> Balıkçı </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> Balıkçı </td><td> P285-500 </td><td></td></tr><tr><td> PierceSDS-PAGE Örnek Hazırlık Seti </td><td> Delmek </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> Balıkçı </td><td> BP328-500 </td><td></td></tr><tr><td> Na 2 PHO 4 </td><td> Balıkçı </td><td> S381-500 </td><td></td></tr><tr><td> Sakaroz </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 Etiket Mix S35 Kolay Etiket </td><td> Perkin Elmer </td><td> NEG772 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><th> Ekipman </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> Diseksiyon araçları </td><td></td><td></td><td></td></tr><tr><td> Dounce homojenizatör, 7 ml (iki cam ile birlikte &quot;A&quot; ve &quot;B&quot; etiketli havan) </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 Santrifüj </td><td> Beckman Coulter </td><td> 366830 </td><td></td></tr><tr><td> GH 3.8 Rotor, Sallanır kova rotor </td><td> Beckman Coulter </td><td> 360581 </td><td></td></tr><tr><td> Beckman J2-21 Santrifüj </td><td> Beckman </td><td></td><td></td></tr><tr><td> Beckman tüpleri ile kapaklar </td><td> Beckman </td><td> 355672 </td><td></td></tr><tr><td> Beyaz duvarlı adaptörleri </td><td> Beckman </td><td> 342327 </td><td></td></tr><tr><td> Mavi duvarlı adaptörleri </td><td> Beckman </td><td></td><td></td></tr><tr><td> JA-17 Rotor, Sabit açılı rotor </td><td> Beckman </td><td> 369691 </td><td></td></tr></tbody></table> Batı lekeler için kullanılan antikorların Tablo: <table border="1"><tbody><tr><th> Antikor Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Ev Sahibi Türler </th><th> Seyreltme Faktörü </th></tr><tr><td> beta-aktin </td><td> Sigma </td><td> A5441 </td><td> fare </td><td> 1:2000 </td></tr><tr><td> GFAP </td><td> Santa Cruz </td><td> sc-65.343 </td><td> fare </td><td> 1:200 </td></tr><tr><td> GP73 </td><td> Santa Cruz </td><td> SC-134.509 </td><td> tavşan </td><td> 1:200 </td></tr><tr><td> HSC70 </td><td> Santa Cruz </td><td> sc-7298 </td><td> fare </td><td> 1:200 </td></tr><tr><td> Laminβ </td><td> Santa Cruz </td><td> SC-6261 </td><td> keçi </td><td> 1:200 </td></tr><tr><td> Prohibitin </td><td> Santa Cruz </td><td> sc-28.259 </td><td> tavşan </td><td> 1:200 </td></tr><tr><td> PSD95 </td><td> Millipore </td><td> MAB1596 </td><td> fare </td><td> 5 mg / ml </td></tr><tr><td> SNAP25 </td><td> AbCam </td><td> ab5666-100 </td><td> tavşan </td><td> 1:2000 </td></tr><tr><td> Sinaptofizin </td><td> Millipore </td><td> MAB368 </td><td> fare </td><td> 1:500 </td></tr><tr><td> β-3-tubulin </td><td> Santa Cruz </td><td> sc-80.016 </td><td> fare </td><td> 1:200 </td></tr></tbody></table>

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

Synaptoneurosomes (SNS) fare beyin korteksi homojenizasyon ve bölümleme sonra elde edilir. Bunlar kortikal doku homojenize akson terminalleri kurtulmak vezikül veya izole terminalleri kapatılmalı. SNS sinaptik iletim çalışmasında faydalı kılan, pre-ve postsinaptik özelliklerini korurlar. Nöronal sinyal kullanılan moleküler makine korumak ve alımı, depolama ve nörotransmitterlerin salınımı yeteneğine sahiptir. Aktif SNS üretim ve izolasyon sitotoksik ve SNS mekanik hasar nedeniyle düşük aktivite neden olabilir, yüksek yoğunluklu ve filtrasyon ve santrifüj yöntemleri nedeniyle genişletilmiş santrifüj gerektirebilir Ficoll gibi sorunlu kullanarak medias olabilir. Ancak, süreksiz Percoll sakaroz yoğunluğu gradyanlar SNS izole etmek için kullanmak translationally aktif SNS iyi verim üretmek için hızlı bir yöntem sağlar. Percoll sukroz gradient yöntemi, hızlı ve nazik bir pelet SNS ve mekanik hasara neden olduğu, izotonik koşullar istihdam ve daha az ve daha kısa santrifüj spin santrifüj adımları önler.

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

Fare beyin korteksi translationally etkin, sağlam synaptoneurosomes (SNS) hazırlamak için bir yöntem açıklanmıştır. Bu yöntem, aktif SNS hızlı hazırlanması için izin veren bir süreksiz Percoll sakaroz yoğunluk gradiyenti kullanır.

Süreksiz Percoll Sakkaroz Yoğunluk Gradient kullanarak Fare Cortex Synaptoneurosomes hazırlanması

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

preparation-synaptoneurosomes-from-mouse-cortex-using-discontinuous

Research

JoVE Journal

Neuroscience

31.2K Görüntüleme.  University of Wisconsin. Fare beyin korteksi translationally etkin, sağlam synaptoneurosomes (SNS) hazırlamak için bir yöntem açıklanmıştır. Bu yöntem, aktif SNS hızlı hazırlanması için izin veren bir süreksiz Percoll sakaroz yoğunluk gradiyenti kullanır.

Video: Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

Fare Visual Cortex İnceltilen kafatası Hazırlık Kronik Görüntüleme

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Nörobilim

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Patch Kelepçe bir Karanlık adapte Dilim Hazırlık Fare Retina Nöronlar Kayıtlar

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Yalıtımlı LacZ ifade Sitometrisi kullanarak Fare İç Kulak Dokular Hücreler

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

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 this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Kullanarak Fare Serebral Korteks ve Hippocampus Görselleştirme ve Dendritler ve Spine Genetik Manipülasyon<em&gt; Utero olarak</em&gt; Elektroporasyon

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 ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Gelişmekte Fare Embriyonik Cortex mitoz Canlı Görüntüleme

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

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

İki-Foton<em&gt; In vivo</em&gt; Bir inceltilmiş-kafatası Hazırlık kullanarak Fare Cortex dendritik dikenler Görüntüleme

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

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

Süreksiz Sukroz Degrade&#39;yi kullanma Sinaptik Plazma Membran ve postsinaptik Yoğunluk Proteinlerin Hazırlanması

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

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure involves the chronic placement of multiple depth electrodes into regions of the brain typically inaccessible via subdural grid electrode placement. SEEG thus provides a unique opportunity to investigate brain function. In this paper we demonstrate how SEEG can be used to investigate the role of the dorsal anterior cingulate cortex (dACC) in cognitive control. We include a description of the SEEG procedure, demonstrating the surgical placement of the electrodes. We describe the components and process required to record local field potential (LFP) data from consenting subjects while they are engaged in a behavioral task. In the example provided, subjects play a cognitive interference task, and we demonstrate how signals are recorded and analyzed from electrodes in the dorsal anterior cingulate cortex, an area intimately involved in decision-making. We conclude with further suggestions of ways in which this method can be used for investigating human cognitive processes.

Stereotactic Electroencephalography (SEEG) is an operative technique used in epilepsy surgery to help localize seizure foci. It also affords a unique opportunity to investigate brain function. Here we describe how SEEG can be used to investigate cognitive processes in human subjects.

Stereotaktik Elektroensefalografi kullanarak Derin KORTİKAL Fonksiyonu ve subkortikal yapıları incelenmesi: Ön Singulat Korteks Dersler

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

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

Primer serebral korteks hücre bir 2D kültür sistemi kullanarak görüntüleme yaşamak

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Karakterizasyonu ve yoğunluk Gradient Santrifüjü tarafından fare birincil Microglia izolasyonu

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...