일레인 Pranski 우수한 기술 지원 캐롤린 Suwyn와 해리 레빈 III, M. 폴 머피, 중요한 대화를 게어링 말라 많은 감사드립니다. 기금은 RR - 00165, PO1AG026423, P50AG025688, AG030539, 우드러프 재단과 에모리 대학 조사위원회에 의해 제공되었다.

1 부 : 명확히 조직 homogenates의 작성 <ol><li> 약 30도 유봉 스트로크로 (즉, 추가 400μl 버퍼 - 얼음처럼 차가운 버퍼 (플러스 2X 프로 테아제 억제제 칵테일 [산타크루즈] 0.1M 인산 버퍼 호수 [와 MG + + 무료 칼슘 + +])의 4 번 볼륨에서 수정되지 않은 대뇌 피질의 조직을 균질 다운스 100mg 조직). </li><li> 3000 5 분 스핀 homogenates g (4 ° C). 조심스럽게 -80에서 명확히 supernatants 이들 추출물의 저장 aliquots를 제거 ° C를 사용까지. </li><li> 제조 업체의 지침 (ThermoFisher)에 의하면, bicinchoninic 산성 (BCA) 분석을 사용하여 명확히 homogenates에 대한 총 단백질 농도 (μg / μl)를 결정합니다. </li></ol> 2 부 : SDS - PAGE 샘플 준비 <ol><li> 물론 10으로 10~20% Tricine 젤 (Invitrogen), 잘마다 로드할 수있는 최대 볼륨 ~ 25μl이다. 총 볼륨 2X SDS 샘플 버퍼와 10X 감소 에이전트를 포함, 잘마다 로드할 수 명확히 homogenate의 최대 볼륨이 10μl가되도록. 20% (W / V) 명확히 피질 homogenates는 당 잘 50μg 총 단백질에 대한 허용, 5 μg / μl 이상의 총 단백질 농도가 있어야합니다. 이하 5mg/ml 단백질과 샘플 당 자 이하 총 단백질을 로딩 할거 것입니다. 그러나, 높은 단백질 수준은 monomeric 및 집계 Aβ의 검색 (50 - 60μg 총 단백질이 최적입니다)에 최적입니다. 항상마다 잘 총 단백질의 동일한 금액을로드합니다. </li><li> 각 젤 들면 SeeBlue Plus2 (Invitrogen)로 분자량 마커의 10μl와 적어도 하나를로드합니다. 긍정적인 제어로, 합성 Aβ40 또는 다른 우물에 Aβ42 펩타이드 (1xPBS에 희석) 10 - 100ng 실행합니다. </li><li> 갓 해동, 명확히 homogenates을 사용하여 얼음에서 반응 혼합물을 준비합니다. 5-10초에 대한 소용돌이 튜브, 100에서 건조 욕조에 열을 ° C 5 분 후, 신속하게 모자의 결로를 제거하는 모든 샘플을 스핀. </li></ol> 파트 3 : SDS - PAGE 젤 전기 영동 <ol><li> , 제조 업체의 지침 (Invitrogen)에 따라, Tricine SDS 실행 버퍼를 사용하여 XCell 물론 잠금 미니 셀 Gelbox에 10~20%의 Tricine 젤로 vortexed 샘플을로드 </li><li> 약 90 분 동안 125V의 일정한 전압에서 젤을 실행합니다. 4KDa 마커 밴드가 젤의 바닥에서 1cm에 대한 때까지 샘플을 실행하자. </li></ol> 4 부 : 젤에서 세포막으로 전송 단백질 <ol><li> 조심스럽게 제조 업체의 지침 (Invitrogen)에 따라, 플라스틱 케이스에서 젤류를 제거하고 XCell II 얼룩 모듈 내부 전송 샌드위치를​​ 조립. 사전 서부 유럽 표준시 트리스 - 글리신 전송 버퍼 (20 % 메탄올)과 패드와 0.2μm nitrocellulose 막을 모래 바닥, 그리고 모래 바닥 패드 또는 여과지 / 멤브레인 샌드위치에서 모든 거품을 제거하십시오. </li><li> XCell Gelbox에서 전송 버퍼와 내부 챔버를 기입 dIH 2 O (메탄올의 노출 시간이 지남에 따라 플라스틱 gelbox을 입을 수)와 외부 챔버를 입력합니다. </li><li> 25mA의 일정한 암페어에서 2~3시간에 대한 전송을 실행합니다. </li><li> 전송이 완료되면, 샌드위치를 ​​재조 두 사각형 플라스틱 배양 접시에 1xPBS (또는 다른 적절한 컨테이너)에 dIH 2 O와 점막에 젤 넣습니다. </li><li> 단백질 전송의 효율성을 시각화하기 위해, 젤 단순히 블루 SafeStain (Invitrogen) 물들일 수 있으며, 개양귀비빛의 S 붉은 얼룩 (시그마 알드리치)와 안정, 두 제조 업체의 지시에 따라. 이 얼룩은 immunoblotting 영향을주지 않습니다. 얼룩은 비효율적인 단백질 전송을 공개하는 경우 3 단계에서 전송 시간을 수정합니다. </li></ol> 제 5 부. 항원 - 에피토프 복구 및 Immunoblotting <ol><li> 항원 검색은 immunoblotting 동안 항체 바인딩을위한 멤브레인에 Aβ epitopes을 드러내는에서 중요한 단계입니다. 100 일정한 온도를 유지 ° 모든 증기선, 전자렌지, 또는 물이 욕조가 C는 충분합니다. 기선의 항원 검색을 위해, 상온에서, 1xPBS 가득한 강력 Kapak 플라스틱 주머니에서 막을 열 - 인감. 미리 온수 기선의 평면 주머니 라이, 파우치는 추가로 15 분 동안 품어, 확장하기 시작 한때. 멤브레인가 과도하게 주름을 방지하기 위해, 주머니에서 제거하기 전에 천천히 냉각 수 있습니다. </li><li> 상온에서, 신중하게 피어오르는 주머니에서 멤브레인를 제거하고 5 분 (0.05 % 트윈 - 20과 함께 살린, 산도 8.0을 트리스 버퍼 TBS - T에 린스 다음 1xPBS 5 분 동안 멤브레인을 린스 [시그마] ), 궤도 흔드는에. 떨고 한 시간 동안 차단 솔루션에 멤브레인 (TBS - T에서 2.5 %의 무지방 우유)를 품어. rinsing없이, 피하 풍선 (1:1,000 [1μg/ml] 1:5,000 [0.2μg/ml] 희석, Covance에서 6E10) 차단 솔루션에 희석 기본 항체를 포함하는 음식 또는 플라스틱 주머니에 막를 전송S. 4 떨고 ° C (더 이상 부화 더 나은 신호를 줄 수 있습니다.) 1 시간 후 24~48시간에 대한 실온에서 궤도 흔드는에 품어 </li><li> TBS - T 30 분 동안 막을 린스 (한 빠른 3 X 10 분 rinses 다음 린스). </li><li> 상온에서 흔드는에서 90 분간 차단 솔루션 1:10,000에서 희석 HRP - 복합 이차 항체에 멤브레인 (애머스햄 ECL 양 안티 - 마우스, GE 헬스케어)을, 알을 품다. </li><li> TBS - T 30 분 동안 막을 린스 (한 빠른 3 X 10 분 rinses 다음 린스). </li><li> 흔드는에서 플라스틱 시트 보호 장치 사이에 보푸라기가없는 필터 종이에 여분의 시약을 얼룩 및 필름 카세트에 막을 장소, 5 분 갓 만든 SuperSignal 웨스트 피코 Electrochemiluminescence 시약 (ThermoFisher)에서 막을 품어. 먼지없는 Kimwipes 필요한 경우와 추가 SuperSignal 시약을 얼룩. </li><li> 30 분정도 30 초 간격에 대한 코닥 Biomax MR 필름에 노출 및 필름 개발에 개발할 수 있습니다. 오분 후, Aβ 모노머 밴드는 아마 포화됩니다. </li><li> 세포막은 복원에 떨고 플러스 실온에서 30 분 버퍼가 벗겨진하고 원하는 경우 추가 항체와 reprobed하여 (TBS - T에 rinsing 이후) 송두리째 수 있습니다. </li></ol> 6 부 : 대표 Immunoblot : <img src="/files/ftp_upload/1916/1916fig1a.jpg" alt="그림 1A" /> <img src="/files/ftp_upload/1916/1916fig1b.jpg" alt="그림 1B" /> <img src="/files/ftp_upload/1916/1916fig1c.jpg" alt="그림 1C" /> 7 인간의 과목에서 50μg 총 단백질을 포함하는 수치 1A - C. 명확히 homogenates는 multimeric Aβ와 APP의 존재에 대해 분석하고 있습니다. 항체 6E10와 Immunoblotting 것은 Aβ 단량체, dimers, trimers, tetramers, 그리고 알츠하이머의 경우 APP (상단 밴드)뿐만 아니라,이 광고의 경우에 풍부 고분자량 Aβ의 multimers를 보여줍니다. 합성 Aβ40은 낮은 분자량 밴드의 신원을 확인합니다. AD : 알츠하이머 질환, DLB : Nondemented 인간 (A) 30 분 필름 노출, (B) 5 분 필름 노출, (C) 30초 필름 노출 :. Lewy 기관, ND와 치매.

<ol>
	<li>Hardy, J., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+hypothesis+of+Alzheimer's+disease:+progress+and+problems+on+the+road+to+therapeutics.">The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.</a> Science. 297, 353-356 (2002).</li><li>Haass, C., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+protein+oligomers+in+neurodegeneration:+lessons+from+the+Alzheimer's+amyloid+beta-peptide.">Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide.</a> Nat. Rev. Mol. Cell. Biol. 8, 101-112 (2007).</li><li>Walker, L. C., LeVine, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cerebral+proteopathies.">The cerebral proteopathies.</a> Neurobiol. Aging. 21, 559-561 (2000).</li><li>Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging,+amyloid,+and+Alzheimer's+disease:+a+perspective+in+honor.">Aging, amyloid, and Alzheimer's disease: a perspective in honor.</a> of Carl Cotman. Neurochem. Res. 28, 1705-1713 (2003).</li><li>Walsh, D. M., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abeta+oligomers+-+a+decade+of+discovery.">Abeta oligomers - a decade of discovery.</a> J. Neurochem. 101, 1172-1184 (2007).</li><li>Swerdlow, P. S., Finley, D., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+immunoblot+sensitivity+by+heating+of+hydrated+filters.">Enhancement of immunoblot sensitivity by heating of hydrated filters.</a> Anal. Biochem. 156, 147-153 (1986).</li><li>Ida, N., Hartmann, T., Pantel, J., Schr&#246;der, J., Zerfass, R., F&#246;rstl, H., Sandbrink, R., Masters, C. L., Beyreuther, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+hetergeneous+A4+peptides+in+human+cerebrospinal+fuid+and+blood+by+a+newly+developed+sensitive+Western+blot+assay.">Analysis of hetergeneous A4 peptides in human cerebrospinal fuid and blood by a newly developed sensitive Western blot assay.</a> J. Biol. Chem. 271, 22908-22914 (1996).</li></ol>

알츠하이머 병 1,4,5의 pathogenesis에서 Aβ 집합의 중요성에도 불구하고, 몇 가지 연구가 설명 또는 인간의 대뇌 피질의 조직 샘플 2 다양한 Aβ multimers의 분포를 계량 있습니다. 일반적으로 사용되는 immunohistochemical 기법은 고정 피질 조직에서 서로 다른 multimeric Aβ 종의 차별에 대한 허용하지 않습니다. 떼어낸 피질 조직 homogenates에서 Aβ multimers는 분리와 화학적 겔 전기 영동과 항체 ?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Complete Protease Inhibitor Cocktail Tablets</td>
<td>&nbsp;</td>
<td>Santa Cruz Biotechnology</td>
<td>Sc-29130</td>
<td>1 tablet in 25ml buffer</td>
</tr>
<tr>
<td>BCA Protein Assay kit</td>
<td>&nbsp;</td>
<td>ThermoFisher Scientific</td>
<td>23225</td>
<td>&nbsp;</td>
<tr>
<td>XCell SureLock Mini-Cell and XCell II Blot Module Kit CE Mark</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EI0002</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Sample Buffer (2X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1676</td>
<td>&nbsp;</td>
<tr>
<td>NuPAGE Sample Reducing Agent (10X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>NP0004</td>
<td>&nbsp;</td>
<tr>
<td>SeeBlue Plus2 Pre-Stained Standard</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC5925</td>
<td>&nbsp;</td>
<tr>
<td>Novex 10-20% Tricine Gel 1.0 mm, 10 well</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EC6625BOX</td>
<td>&nbsp;</td>
<tr>
<td>Nitrocellulose membranes, 0.2 &mu;m pore size</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC2000</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Running Buffer (10X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1675</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tris-Glycine Transfer Buffer (25X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC3675</td>
<td>&nbsp;</td>
<tr>
<td>SimplyBlue SafeStain</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6060</td>
<td>Will not interfere with immunostaining</td>
</tr>
<tr>
<td>ATX Ponceau S Red staining solution</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>09276</td>
<td>Will not interfere with immunostaining</td>
</tr>
<tr>
<td>Kapak heat sealable plastic sample pouches</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>0181225AA</td>
<td>&nbsp;</td>
<tr>
<td>6E10 mouse monoclonal antibody to A&beta;(1-16)</td>
<td>&nbsp;</td>
<td>Covance</td>
<td>SIG-39320</td>
<td>Dilute 1:1,000 up to 1:5,000 for WB</td>
</tr>
<tr>
<td>Tween 20</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P2287</td>
<td>&nbsp;</td>
<tr>
<td>ECL Mouse IgG, HRP-Linked Whole A&beta; (from sheep)</td>
<td>&nbsp;</td>
<td>GE Healthcare Life Sciences</td>
<td>NA931-1ML</td>
<td>Dilute at 1:10,000</td>
</tr>
<tr>
<td>SuperSignal West Pico Chemiluminescent Substrate</td>
<td>&nbsp;</td>
<td>ThermoFisher</td>
<td>34077</td>
<td>&nbsp;</td>
<tr>
<td>Kodak Biomax MR Film</td>
<td>&nbsp;</td>
<td>Carestream Health</td>
<td>870 1302</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

sds-page/immunoblot detection of a&beta; multimers in human cortical tissue homogenates using antigen-epitope retrieval

β - 아밀로이드의 변칙 접지와 중합도 (Aβ) 펩타이드는 알츠하이머 질병 pathogenesis에 neurodegenerative 층계를 시작 생각됩니다<sup&gt; 1</sup&gt;. 알츠하이머 질환에서 대뇌 치매 plaques의 코어에서 발견되는 β - 시트 풍부한 아밀로이드 원섬유로 자체 집계하는 경향이 있거나 42 아미노산 펩타이드 - Aβ는 주로 40. 증거를 증가하면 해당 낮은 분자량이 용해 Aβ의 multimers가 원섬유의의 Aβ의 아밀로이드보다 독성이 제안<sup&gt; 2</sup&gt;. 뇌 조직에서 낮은과 높은 분자량 multimeric Aβ 종의 식별 및 부량는 알츠하이머 질병 연구에 필수적인 목적이며, 또한 고용 방법은 다른 proteopathies에 독성 multimers의 식별 및 특성에 적용할 수있는<sup&gt; 3</sup&gt;. 자연스럽게 발생 Aβ multimers는 Aβ 특정 항체와 immunoblotting 다음 SDS - polyacrylamide 젤 전기 영동에 의해 감지하실 수 있습니다. 그러나, multimeric Aβ의 분리 및 검출은 고농도 대뇌 피질 homogenates 작은 기공 크기 nitrocellulose의 점막에서 항원 검색의 사용을 필요로합니다. 여기서 우리는 명확히 인간의 대뇌 피질 homogenates의 준비를 위해 기술, SDS - PAGE에 의해 단백질의 분리, 그리고 항원 에피토프 복구 / Aβ 펩타이드의 N - 터미널 지역에 항체 6E10과 모래 바닥 서양을 설명합니다. 이 프로토콜을 사용, 우리는 지속적으로 알츠하이머의 병리와 인간에서 Aβ 단량체, dimers, trimers, tetramers 및 대뇌 피질의 조직에서 높은 분자량의 multimers을 감지.

Watch this Scientific Journal Video about SDS-PAGE/Immunoblot Detection of AÎ² Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval at JoVE.com

SDS-PAGE/Immunoblot Detection of A&amp;#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

우리는 명확히 인간 대뇌 피질 homogenates, SDS - PAGE로 단백질 분리, 항원 검색 및 Aβ 펩타이드에 대한 항체와 immunoblotting의 준비를위한 기술을 설명합니다. 이 프로토콜을 사용하여, 우리는 지속적으로 알츠하이머의 병리 현상과 인간의 대뇌 피질 조직에서 monomeric 및 multimeric Aβ를 감지합니다.

인간의 두피 조직에서 Aβ Multimers의 SDS-PAGE/Immunoblot 감지는 항원 에피토프 검색을 사용 Homogenates

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's ...

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Neuroscience

SDS-PAGE/Immunoblot Detection of A&beta; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

24.0K 조회수.  Emory University. 우리는 명확히 인간 대뇌 피질 homogenates, SDS - PAGE로 단백질 분리, 항원 검색 및 Aβ 펩타이드에 대한 항체와 immunoblotting의 준비를위한 기술을 설명합니다. 이 프로토콜을 사용하여, 우리는 지속적으로 알츠하이머의 병리 현상과 인간의 대뇌 피질 조직에서 monomeric 및 multimeric Aβ를 감지합니다.

비디오: SDS-PAGE/Immunoblot Detection of A&#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

고도의 연결을 강화 문화의 생성을위한 두피 조직 블록의 Cryopreservation

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

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신경과학

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

개발 Zebrafish Forebrain에 Clonally 관련 신경 전구 세포의 시간 경과 라이브 영상

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

Balb / C 뇌 조직에 높은 민감도는 5 hydroxymethylcytosine 감지

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and focused on psychological aspects. Until now, objective observation of neurophysiological parameters has not been carried out - undoubtedly because the technical and methodological means have not been available -, investigations into the neurophysiological effects of weightlessness are in their infancy (Schneider et al. 2008).
While imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) would be hardly applicable in space, the non-invasive near-infrared spectroscopy (NIRS) technique represents a method of mapping hemodynamic processes in the brain in real time that is both relatively inexpensive and that can be employed even under extreme conditions. The combination with electroencephalography (EEG) opens up the possibility of following the electrocortical processes under changing gravity conditions with a finer temporal resolution as well as with deeper localization, for instance with electrotomography (LORETA).
Previous studies showed an increase of beta frequency activity under normal gravity conditions and a decrease under weightlessness conditions during a parabolic flight (Schneider et al. 2008a+b). Tilt studies revealed different changes in brain function, which let suggest, that changes in parabolic flight might reflect emotional processes rather than hemodynamic changes. However, it is still unclear whether these are effects of changed gravity or hemodynamic changes within the brain. Combining EEG/LORETA and NIRS should for the first time make it possible to map the effect of weightlessness and reduced gravity on both hemodynamic and electrophysiological processes in the brain. Initially, this is to be done as part of a feasibility study during a parabolic flight. Afterwards, it is also planned to use both techniques during medium- and long-term space flight.
It can be assumed that the long-term redistribution of the blood volume and the associated increase in the supply of oxygen to the brain will lead to changes in the central nervous system that are also responsible for anaemic processes, and which can in turn reduce performance (De Santo et al. 2005), which means that they could be crucial for the success and safety of a mission (Genik et al. 2005, Ellis 2000).
Depending on these results, it will be necessary to develop and employ extensive countermeasures. Initial results for the MARS500 study suggest that, in addition to their significance in the context of the cardiovascular and locomotor systems, sport and physical activity can play a part in improving neurocognitive parameters. Before this can be fully established, however, it seems necessary to learn more about the influence of changing gravity conditions on neurophysiological processes and associated neurocognitive impairment.

The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. 
A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.

브레인 두피 기능 및 변경 중력 조건 중 Neurocognitive 성능 사이의 일관성

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and ...

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

폐기 인간 태아 두피 조직에서 신경 줄기 세포의 생성

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

두피 네트워크 개발에 기능성 칼슘 이미징

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

쥐 두피 뉴런의 사상 액틴 (F - 굴지) puncta의 정량

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

형광 프로브를 이용한 배양 된 뉴런의 엔도 좀과 리소좀 기능의 정량화

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

병합 된 포유류 외피가 대뇌 피 질의 모듈의 시각화

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...

Statistical Modelling of Cortical Connectivity Using Non-invasive Electroencephalograms

Non-invasive electrophysiological recordings are useful for the evaluation of nervous system function. These techniques are inexpensive, fast, replicable, and less resource-intensive than imaging. Further, the functional data produced have excellent temporal resolution, which is not achievable with structural imaging.
Current applications of electroencephalograms (EEG) are limited by data processing methods. Standard analysis techniques using raw time series data at individual channels are very limited methods of interrogating nervous system activity. More detailed information about cortical function can be achieved by examining relationships between channels and deriving statistical models of how areas are interacting, allowing visualization of connectivity between networks.
This manuscript describes a method for deriving statistical models of cortical network activity by recording EEG in a standard manner, then examining the interelectrode coherence measures to assess relationships between the recorded areas. Higher order interactions can be further examined by assessing the covariance between the coherence pairs, producing high-dimensional &#34;maps&#34; of network interactions. These data constructs can be examined to assess cortical network function and its relationship to pathology in ways not achievable with traditional techniques.
This approach offers greater sensitivity to network level interactions than is achievable with raw time series analysis. It is, however, limited by the complexity of drawing specific mechanistic conclusions about the underlying neural populations and the high volumes of data generated, requiring more advanced statistical techniques for evaluation, including dimensionality reduction and classifier-based approaches.

Standard EEG analysis techniques offer limited insight into nervous system function. Deriving statistical models of cortical connectivity offers far greater ability to investigate underlying network dynamics. Improved functional assessment opens new possibilities for diagnosis, prognostication, and outcome prediction in nervous system diseases.

비침습적 뇌전도를 이용한 피질 연결의 통계적 모델링

Non-invasive electrophysiological recordings are useful for the evaluation of nervous system function. These techniques are inexpensive, fast, replicable, ...