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

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, ...

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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 ...

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Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using 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

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

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 ...

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

Yüksek alan fMRI kullanılarak Yetişkin kedilerde işitme korteksi Fonksiyonel Görüntüleme

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

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

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

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

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

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 ...