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

우리는 전자 현미경에 대한 위스콘신 매디슨 전자 현미경 시설의 대학에서 BK 8 월 감사드립니다. 이 작품은 NIH 보조금 R01 - DA026067 및 P30 - HD03352 (JSM)을 지원했다.

1. 준비 1-4 <ol><li> 2.5 M의 자당 슬러리의 50 ML을 혼합하여 500 그라디언트 매체의 ML (GM) 버퍼를 준비, 1M 트리스 - HCL, pH7.5 주식 및 0.5 m EDTA (에틸렌 다이아 민 테트라 초산) 0.1 ML 2.5 ML, 산도와 8.0 주식 밀리 볼륨 Q 물. 솔루션 나누어지는 및 저장 냉동을 소독 필터. </li><li> 밀리 Q H 2 O.에서 1 ㎜ 솔루션을 만들어 테트로도톡신 (TTX)의 1000x 재고를 준비 TTX의 Aliquots는 -20 ° C.에 저장할 수 있...

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여기에 설명된 불연속 Percoll - 자당 기울기 준비는 시냅스 전송 실험의 다양한 사용할 수 있습니다 적극적인 SNS를 분리하기 위해 빠르고 안정적​​인 방법입니다. Dunkley 외., 3,4가 개발한 방법을 기반으로 그라데이션이 방법은 서로 관련된 두 - 사전 및 postsynaptic 멤브레인 - 파생 vesicles를 분리 subcellular 두뇌 분획화 절차입니다. 더 정화 않고 이러한 SNS가 immunoprecipitation, 서부 blotting, 유동?...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> 마이크로 BCA 단백질 분석 키트 </td><td> 찌르다 </td><td> 23,235 </td><td></td></tr><tr><td> CaCl 2 </td><td> 어부 </td><td> C79 - 500 </td><td></td></tr><tr><td> CO 2 가스 </td><td> Airgas (UW - MDS) </td><td> CD 50 </td><td></td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) </td><td> RPI </td><td> E57020 </td><td></td></tr><tr><td> EtOH </td><td> 어부 </td><td> A407SK - 4 </td><td></td></tr><tr><td> HCL </td><td> 어부 </td><td> A142 - 212 </td><td></td></tr><tr><td> Percoll </td><td> GE 헬스케어 </td><td> 17-0891-01 </td><td></td></tr><tr><td> KH 2 PO 4 </td><td> 어부 </td><td> P285 - 500 </td><td></td></tr><tr><td> PierceSDS 페이지 샘플 준비 키트 </td><td> 찌르다 </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> 어부 </td><td> BP328 - 500 </td><td></td></tr><tr><td> 나이 검사하고 사진을 사 </td><td> 어부 </td><td> S381 - 500 </td><td></td></tr><tr><td> 자당 </td><td> RPI </td><td> S24060 </td><td></td></tr><tr><td> 트리스베이스 </td><td> RPI </td><td> T60040 </td><td></td></tr><tr><td> 테트로도톡신 </td><td> 시그마 </td><td> T5651 </td><td></td></tr><tr><td> 익스프레스 프로 라벨 S35 쉬운 태그 믹스 </td><td> Perkin 엘머 </td><td> NEG772 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><th> 장비 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> 해부 도구 </td><td></td><td></td><td></td></tr><tr><td> 다운스 호모 지 나이저, 7 ML (두 유리와 함께 제공 &quot;A&quot;와 &quot;B&quot;표시 pestles) </td><td> Wheaton의 </td><td></td><td></td></tr><tr><td> P1000 Gilson Pipetman </td><td> Gilson </td><td> F123602 </td><td></td></tr><tr><td> Allegra 6KR의 원심 분리기 </td><td> 베크 만 보습 바로 앞에 달린 풀베는 날 </td><td> 366830 </td><td></td></tr><tr><td> GH 3.8 로터, 섹시한 버킷 로터 </td><td> 베크 만 보습 바로 앞에 달린 풀베는 날 </td><td> 360581 </td><td></td></tr><tr><td> 베크 만 J2 - 21 원심 분리기 </td><td> 베크 만 </td><td></td><td></td></tr><tr><td> 모자와 베크 만 튜브 </td><td> 베크 만 </td><td> 355672 </td><td></td></tr><tr><td> 흰색 벽으로 둘러싸인 어댑터 </td><td> 베크 만 </td><td> 342327 </td><td></td></tr><tr><td> 블루 벽으로 둘러싸인 어댑터 </td><td> 베크 만 </td><td></td><td></td></tr><tr><td> JA - 17 로터, 고정 앵글 로터 </td><td> 베크 만 </td><td> 369691 </td><td></td></tr></tbody></table> 서부 blots에 사용되는 항체의 테이블 : <table border="1"><tbody><tr><th> 항체의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 호스트 종 </th><th> 희석 팩터 </th></tr><tr><td> β - 굴지 </td><td> 시그마 </td><td> A5441 </td><td> 마우스 </td><td> 1:2000 </td></tr><tr><td> GFAP </td><td> 산타 크루즈 </td><td> SC - 65343 </td><td> 마우스 </td><td> 1:200 </td></tr><tr><td> GP73 </td><td> 산타 크루즈 </td><td> SC - 134509 </td><td> 토끼 </td><td> 1:200 </td></tr><tr><td> HSC70 </td><td> 산타 크루즈 </td><td> SC - 7298 </td><td> 마우스 </td><td> 1:200 </td></tr><tr><td> Laminβ </td><td> 산타 크루즈 </td><td> SC - 6261 </td><td> 염소 </td><td> 1:200 </td></tr><tr><td> Prohibitin </td><td> 산타 크루즈 </td><td> SC - 28259 </td><td> 토끼 </td><td> 1:200 </td></tr><tr><td> PSD95 </td><td> Millipore </td><td> MAB1596 </td><td> 마우스 </td><td> 5 μg / μL </td></tr><tr><td> SNAP25 </td><td> AbCam </td><td> ab5666 - 100 </td><td> 토끼 </td><td> 1:2000 </td></tr><tr><td> Synaptophysin </td><td> Millipore </td><td> MAB368 </td><td> 마우스 </td><td> 1:500 </td></tr><tr><td> β - 3 - Tubulin </td><td> 산타 크루즈 </td><td> SC - 80016 </td><td> 마우스 </td><td> 1:200 </td></tr></tbody></table>

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

Synaptoneurosomes (SNS)는 마우스 뇌 피질의 균질화 및 분별화 후 얻을 수 있습니다. 그들은 vesicles 또는 대뇌 피질의 조직이 균질 때 축삭 터미널에서 멀리 휴식 격리 터미널을 resealed 있습니다. SNS는 시냅스 전달의 연구에 유용하게하는, 사전과 postsynaptic 특성을 보유합니다. 그들의 연결 신호에 사용되는 분자 기계를 유지하고 이해, 스토리지 및 신경 전달 물질의 릴리스 할 수 있습니다. 적극적인 SNS의 생산과 격리 세포 독성하고 의한 SNS의 기계적 손상이 낮은 활동에 발생할 수 고밀도, 그리고 여과 및 원심 분리 방식에 의한 확장 원심 분리를 요구할 수 Ficoll 같은 문제를 사용하여 medias 수 있습니다. 그러나, SNS를 분리 불연속 Percoll - 자당 밀도 그라디언트의 사용 translationally 적극적인 SNS의 좋은 수율을 생산하는 빠른 방법을 제공합니다. 이 펠렛 SNS 그리고이 기계 고장의 원인이되는, isotonic 조건을 고용하고 적은 짧은 원심 분리를 돌면서을 보유하고 원심 분리 단계를 피할 수로 Percoll - 자당 기울기 방법은 신속하고 부드러운 있습니다.

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

마우스 뇌 피질에서 translationally 활성화, 손상 synaptoneurosomes (SNS)를 준비하는 방법을 설명합니다. 이 메서드는 활성화된 SNS의 빠른 준비를 위해 허용하는 불연속 Percoll - 자당 밀도 기울기를 사용합니다.

불연속 Percoll - 자당 밀도 구배를 사용하여 마우스를 접속에서 Synaptoneurosomes의 준비

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

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Neuroscience

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

Thinned - 해골 준비를 사용하여 마우스 시각 피질의 만성 이미징

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

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

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The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar ...

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분리 LacZ를 표현 유동세포계측법를 사용하여 마우스 내이 (内耳) 조직의 세포를

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 ...

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

사용하는 마우스 대뇌 피질과 해마의 시각화 및 Dendrites와 쪽이의 유전자 조작<em&gt; utero에서</em&gt; 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 ...

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하이 필드의 fMRI를 사용하여 성인 고양이의 청각 피질의 기능적 이미징

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

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개발 마우스 배아의 피질 유사 분열의 라이브 영상

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

두 광자<em&gt; 생체</em&gt; 시닝 두개골 준비를 사용하여 마우스 피질 돌기 쪽의 영상

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

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불연속 자당 구배를 사용 시냅틱 세포막과 시냅스 밀도 단백질의 제조

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

정위 뇌파를 사용하여 깊은 피질의 기능 및 피질 하 구조 조사 : 전방에 cingulate 피질 수업

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