NIH / NIDCD 및 RP에 슈미트 재단의 보조금에 의해 지원 작동합니다.

이 프로토콜은 이전에 뇌 조직 1-7에 하나 또는 두 개의 사본 수종을 감지하는 우리와 다른 사람에 의해 개발된 표준 방사선이 아닌 방사선 원위치 하이브리드화 방법에 따라 개발 및 정제되었다. 아래에서 설명하는 프로토콜은 최종 사용자가 선택한 절차 중단의 수에 따라 2 ~ 3 일 총 길이있다. 아래의 자세한 모든 단계가 riboprobe의 하이브리드화 단계 및 사후 하이브 리다이 제이션 세척을 제외하고, 상온에서 실시하는 것입니다. 이 방법에 관련된 모든 단계에 필요한 솔루션 및 버퍼는이 프로토콜의 끝 부분에서 찾을 수 있습니다. 1. 조직 준비 및 Sectioning <ol><li> 참살 빠르게 주제의 두뇌를 추출하고, 적절한 크기의 플라스틱 금형에 넣습니다. </li><li> 조직 테크의 포함 매체와 두뇌를 커버하고 빠르게 빠른 냉동 dry-ice/alcohol 목욕탕에서 플라스틱 금형을 놓으십시오. 냉동 조직은 -80 ° C에서 사용까지 저장할 수 있습니다. </li><li> 그라 이오 스탯을 사용하여 청구 Superfrost 플러스 슬라이드에 슬라이드 당 2 ~ 3 두뇌 섹션을 수집합니다. 섹션의 두께는 10-12 μm의해야합니다. 슬라이드는 -80 ° C에서 사용까지 저장할 수 있습니다. </li></ol> 2. 프로브 정제에 대한 Sephadex G50 열 준비 &quot;Sephadex 컬럼이 상용 소스에서 구입할 수 있지만, 우리는 프로브 정화를 위해 필요합니다 컬럼의 생성을 위해 아래 저가의 대안을 제공합니다. <ol><li> 상온에서 RNase 무료, DEPC - 처리된 물 (예를 들어, DEPC - 처리된 물 100 ML에 분말 2g), 혼합 솔루션 간략하고 저장과 Sephadex G50 분말의 하이드 레이트 적절한 금액 초과 Sephadex G50을 촉진합니다. </li><li> Sephadex G50 강수량 다음 (물을 상위 계층) 뜨는 제거합니다. </li><li> 5 회 - 3 위의 과정을 반복합니다. </li><li> 최종 세척 후 사용까지 4 Sephadex G50 TE 버퍼 (1:1 비율)에 솔루션 및 저장 ° C를 다시 일시 중지합니다. </li><li> 이곳은 살균 한 ML 주사기로 글라스 울을 autoclaved하고 하단에 소형 레이어를 만들기 위해 플런저로 압축하고 15 ML 팔콘 튜브에 주사기를 놓습니다. </li><li> 잘 TE에 Sephadex G50의 솔루션을 섞는다. 이 솔루션과 함께 주사기 / 열을 채웁니다. </li><li> 1,000 rpm으로 30 초에 대한 컬럼을 원심 분리기. </li><li> 칼럼이 거의 Sephadex G50 비즈 가득 때까지이 절차를 반복합니다. </li><li> 그 결과 해당 컬럼에 대한 컬럼 세척 버퍼 200 μl를 적용하고 1000 rpm으로 2 분을 위해 원심 분리기. 흐름 -을 통해 폐기하십시오. </li><li> 그 결과 해당 컬럼에 대한 열의 차단 버퍼 200 μl를 적용하고 1000 rpm으로 2 분을 위해 스핀. 열 평형이 단계 4-5 번 반복합니다. ° C까지 사용 Parafilm와 봉인하면 열이 4 저장할 수 있습니다. </li></ol> 3. Riboprobes의 라벨링 및 정제 우리가 상세 한 riboprobe의 생성 및 정화를 아래. dFISH 각 프로브의 준비 프로브 중 하나가 비오틴 - 태그 UTP와 다른 반면 digoxigenin (발굴) 태그 UTP이 표시됩니다 것을 제외하고, 동일한 방법론을 포함합니다. <ol><li> 감각 (제어) 또는 안티 센스 riboprobes 중 하나를 생성하는 관심 집중 (&gt; 150 NG / μl)와 정화 선형 cDNA 솔루션을 준비합니다. </li><li> 1.5 ML의 microfuge 관에서 cDNA 템플릿, 배 프로브 라벨링 버퍼 2 μl, 10X 파다 1 μl (또는 비오틴) - 라벨 믹스, RNasin 0.5 μl하고 적절한 RNA 효소 1 μl를 정화의 0.5-1 μg을 추가 그리고 RNAse없는 (DEPC - 처리) 물 10 μl로 솔루션의 최종 볼륨을 가지고. </li><li> 37 물 목욕에있는 솔루션 ° 2 시간 C를 품어. </li><li> 솔루션 및 열 차단 버퍼 39 μl, tRNA 1 μl (20 μg / μl 주식)를 추가합니다. </li><li> 프로브 정화를위한 Sephadex G50 컬럼을 준비합니다. 이를 위해, 열 50 μl 차단 버퍼를 추가하고 1,000 rpm으로 10 초을 위해 스핀. 열 (즉, 그 결과 해당 컬럼에 적용되는 전체 50 μl가 원심 분리의 사이클 이후에 회복된다) 평형이 단계에게 2-3 번 반복합니다. </li><li> Sephadex G50 칼럼, 위치 칼럼의 하단에 새로운 microfuge 튜브에 프로브 솔루션을 적용하고, riboprobe 솔루션의 정화 50 μl를 얻기 위해 1,000 rpm으로 3 분을 위해 스핀. </li><li> 중 표준 포름 알데히드 - 아가로 오스 겔 RNA, 또는 분광 광도계를 사용하여 레이블 riboprobe의 품질 및 수율을 평가합니다. </li></ol> 4. 포스트 - 고정, 아세틸화 및 하이브리드화 <ol><li> -80 ° C의 냉장고에서 섹션을 제거하고 평형에 실내 온도에 대한 허용합니다. 그 후, 5 분 추위, 갓 만들어진 3 % paraformaldehyde 솔루션 섹션을 품어. </li><li> 간략 P의 부분을 씻어hosphate 두 번 호수 (PBS)를 버퍼. </li><li> 표준 알코올 시리즈 (70, 95, 및 100 %, 2 분마다)를 통해 섹션을 탈수하고, 그들이 공기 건조하게. </li><li> 10 분 아세틸화 솔루션 섹션을 품어. </li><li> 2X SSPE에 섹션 3 번 린스. </li><li> 표준 알코올 시리즈 위에서 설명한을 통해 다시 한번 섹션을 탈수하고, 그들이 공기 건조 수 있습니다. </li><li> 하이브 리다이 제이션 솔루션의 적절한 볼륨을 준비하고 솔루션 (프로브 농도 : 각 프로브 1 NG / μl)를 모두 riboprobes를 추가합니다. 하이브 리다이 제이션 솔루션의 총 부피는 (섹션당 하이브 리다이 제이션 용액 16 μl) hybridized 수 섹션의 개수에 따라 결정됩니다. </li><li> 그 공기 방울 오버레이에게 조직을 보장하지 조직 및 coverslip 슬라이드에 하이브 리다이 제이션 솔루션의 적절한 볼륨을 적용합니다. </li><li> 이곳은 금속 슬라이드 홀더에 슬라이드. 미네랄 오일 욕조에 담가 소유자 ° C 야간 65으로 설정합니다. 그 coverslips (즉, 슬라이드 소유자의 측면은 미네랄 오일 컨테이너의 하단에 접촉해야합니다) 위쪽으로 직면하고 있는지 확인합니다. </li></ol> 5. 포스트 하이브 리다이 제이션 씻는다 <ol><li> 다음날, 조심스럽게 미네랄 오일 목욕에서 슬라이드 홀더를 제거하고 간단히 슬라이드에서 여분의 기름을 제거하기 위해 클로로포름에 린스. </li><li> 플레이스 5-10 분 2X SSPE 솔루션 슬라이드. Coverslips은 용액 중에 슬라이드에서 분리해야합니다. </li><li> 새로운 2X SSPE 솔루션에 슬라이드를 전송하고, 실온에서 1 시간 그들을 유지. </li><li> 2X SSPE 플러스 50 %의 포름 아미드를 포함하는 솔루션으로 섹션을 전송합니다. 이 솔루션의 온도는 야간 하이브 리다이 제이션 절차에 사용되는 온도를 일치해야합니다. 1.5 시간이 용액에 슬라이드를 유지. </li><li> 0.1X SSPE 솔루션 하이브리드화 단계의 동일한 온도로 미리 예열 섹션을 전송합니다. 30 분이 솔루션 섹션을 품어. 추가 30 분에 대해이 단계를 반복합니다. </li></ol> 6. Riboprobes의 감지 및 시각화 <ol><li> 어느하기 위해 0.3 % 과산화수소가 10 분 동안 추가되었습니다 TNT 버퍼에 슬라이드를 전송합니다. </li><li> TNT 버퍼, 3 회 (각 10 분)에 섹션을 씻으십시오. </li><li> DAKO 펜 사용하여 두뇌 섹션을 포함하는 주변 잘 그려. 중요한주의는 해당 섹션 건조하지 않도록하기 위해 행사되어야합니다. </li><li> 각 슬라이드에 TNB 버퍼 150 μl를 적용하고, 습기 챔버에서 30 분이 솔루션 섹션을 품어. </li><li> 틸팅 슬라이드로 초과 TNB 솔루션을 제거합니다. </li><li> 2 시간의 습기가 실내에 퍼옥시데이즈 - 복합 백신 발굴 항체 및 저장 슬라이드가 포함된 TNB 솔루션 150 μl을 적용합니다. 항체 농도 전에 dFISH을 수행하기 위해 관심의 각 사본에 대해 개별적으로 결정되어야합니다. </li><li> TNT 버퍼 부분을 씻어, 3 회, 10 분 각각. </li><li> 알렉사 각 슬라이드 594 - 복합 tyramide 작업 솔루션을 150 μl를 적용하고, 1 시간에 습기 챔버에 저장합니다. 이 솔루션은 제조 업체의 지시에 따라 준비되어야합니다. </li><li> TNT 버퍼 부분을 씻어, 3 회, 10 분 각각. </li><li> 30 분 - TNT 버퍼에 품어 섹션 어느 위해 0.3 % 과산화수소 10에 추가되었습니다. </li><li> TNT 버퍼 부분을 씻어, 3 회, 10 분 각각. </li><li> 슬라이드 당 TNB 버퍼 150 μl를 적용하고, 30 분 습한 실내에 보관하십시오. </li><li> 틸팅 슬라이드로 초과 TNB 솔루션을 제거합니다. </li><li> 2 시간의 습기가 실내에 퍼옥시데이즈 - 복합 안티 - 비오틴 항체 및 저장 슬라이드가 포함된 TNB 솔루션 150 μl를 추가합니다. </li><li> TNT 버퍼 부분을 씻어, 3 회, 10 분 각각. </li><li> 알렉사 150 μl 488 - 복합 슬라이드 1 시간을 위해 품어 섹션 tyramide 작업 솔루션을 적용합니다. 이 솔루션은 제조 업체의 권고에 따라 준비되어야합니다. </li><li> TNT 버퍼 부분을 씻어, 3 회, 10 분 각각. </li><li> 섹션 Hoechst 솔루션 (TNT 버퍼에 1:1000) 150 μl를 추가 2 분 습한 챔버에 보관하십시오. </li><li> TNT 버퍼에 섹션을 씻어, 3 회, 5 분마다. </li><li> 형광 호환 설치 매체 (예 : Vectashield 또는 antifade을 연장)와 Coverslip 섹션. </li></ol> 7. 대표 결과 <img src="/files/ftp_upload/2102/2102fig1.jpg" alt="그림 1" /> 그림 1. 우리는 여기 얼룩말 핀치 두뇌에서 얻은 대표 dFISH 결과를 보여줍니다. caudomedial nidopallium (NCM), 포유류의 청각 피질의 송버드 아날로그에서 얻은 photomicrographs 있습니다 표시됩니다. 뇌 섹션은 parvalbumin (A), 억제 신경의 하위 인구 마커에 대한 감독 biotinylated riboprobe와 hybridized 및 ribo 파고 - 라벨이 붙지만탐사선이 활동에 의존 유전자 zenk (B), 노래 위주의 뉴런에 안정적인 마커에 대한 감독. (A)와 (B) 청각 경험에 의해 활성화됩니다 억제 뉴런의 인구를 보여줍니다의 C) 중첩. 화살표와 화살촉이 두 riboprobes의 각 독점적으로 표시된 셀을 표시하고, 별표 관심의 두 사본을 공동 표현 대표 뉴런을 보여줍니다. 스케일 바 = 25 μm의.

<ol>
	<li>Jin, L., Lloyd, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization:+methods+and+applications.">In situ hybridization: methods and applications.</a> J Clin Lab Anal. 11, 2-9 (1997).</li><li>Stoler, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization.">In situ hybridization.</a> Clin Lab Med. 10, 215-236 (1990).</li><li>Komminoth, P., Werner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+and+signal+amplification:+approaches+to+increase+the+sensitivity+of+in+situ+hybridization.">Target and signal amplification: approaches to increase the sensitivity of in situ hybridization.</a> Histochem Cell Biol. 108, 325-3233 (1997).</li><li>Kessler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+digoxigenin:anti-digoxigenin+(DIG)+technology--a+survey+on+the+concept+and+realization+of+a+novel+bioanalytical+indicator+system.">The digoxigenin:anti-digoxigenin (DIG) technology--a survey on the concept and realization of a novel bioanalytical indicator system.</a> Mol Cell Probes. 5, 161-205 (1991).</li><li>Panoskaltsis-Mortari, A., Bucy, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+with+digoxigenin-labeled+RNA+probes:+facts+and+artifacts.">In situ hybridization with digoxigenin-labeled RNA probes: facts and artifacts.</a> Biotechniques. 18, 300-307 (1995).</li><li>Qian, X., Lloyd, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+signal+amplification+methods+for+in+situ+hybridization.">Recent developments in signal amplification methods for in situ hybridization.</a> Diagn Mol Pathol. 12, 1-13 (2003).</li><li>Mello, C. V., Jarvis, E. D., Denisenko, N., Rivas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+song-regulated+genes+in+the+brain+of+songbirds.">Isolation of song-regulated genes in the brain of songbirds.</a> Methods Mol Biol. 85, 205-217 (1997).</li><li>Pinaud, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+neurons+participate+in+the+brain's+response+to+birdsong+auditory+stimulation.">GABAergic neurons participate in the brain's response to birdsong auditory stimulation.</a> Eur J Neurosci. 20, 1318-1330 (2004).</li><li>Velho, T. A., Pinaud, R., Rodrigues, P. V., Mello, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-induction+of+activity-dependent+genes+in+songbirds.">Co-induction of activity-dependent genes in songbirds.</a> Eur J Neurosci. 22, 1667-1678 (2005).</li><li>Tremere, L. A., Jeong, J. K., Pinaud, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estradiol+shapes+auditory+processing+in+the+adult+brain+by+regulating+inhibitory+transmission+and+plasticity-associated+gene+expression.">Estradiol shapes auditory processing in the adult brain by regulating inhibitory transmission and plasticity-associated gene expression.</a> J Neurosci. 29, 5949-5963 (2009).</li><li>Pinaud, R., Mello, C. V., Velho, T. A., Wynne, R. D., Tremere, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+two+mRNA+species+at+single-cell+resolution+by+double-fluorescence+in+situ+hybridization.">Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization.</a> Nat Protoc. 3, 1370-1379 (2008).</li><li>Pinaud, R., Jeong, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplex+fluorescence+in+situ+hybridization+in+the+study+of+gene+co-regulation+in+the+vertebrate+brain.+Methods+Mol+Biol.">Duplex fluorescence in situ hybridization in the study of gene co-regulation in the vertebrate brain. Methods Mol Biol.</a> 611, 115-129 (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

우리는 척추 두뇌가 neurochemically 및 기능 구성 방법 연구, 어떻게 behaviorally - 관련 감각 자극에 미치는 영향 성인 뇌 8-10에서 뉴런의 게놈 기기 확인하기 위해이 프로토콜을 사용하고 있습니다. 우리는 성공적으로 마우스, 쥐 그리고 노래하는 새는에서 뇌 조직에서이 방법을 사용하지만,이 프로토콜은 척추 동물의 배열, 그리고 아마도이 아닌 신경 조직에서 얻은 뇌 부분에 쉽게 적용할 수?...

솔루션 <ol><li> 칼럼 세척 버퍼 : 50 ML DEPC - 처리된 물 10 MM 트리스 - HCL, 0.15 M NaCl, 0.05 MM EDTA (에틸렌 다이아 민 테트라 초산), 50 μg / μl tRNA, 0.1 % SDS. </li><li> 열 차단 버퍼 : 50 ML DEPC - 처리된 물 10 MM 트리스 - HCL, 50 MM NaCl, 0.1 MM EDTA (에틸렌 다이아 민 테트라 초산). </li><li> TNT 버퍼 : 60 ML 1 M 트리스 - HCL, 5 M NaCl 18 ML과 트리톤의 1.8 ML DEPC - 처리된 물 600 ML에서 X - 100. </li><li> TNB 버퍼 : 100 MM 트리스 - HCL, 8.3 μg / μl BSA, 0.15 M NaCl 3 % 트리톤 DEPC - 처리된 물에 X - 100. </li><li> TE 버퍼 : 10 MM 트리스 - HCL, pH를 7.5 플러스 1 ㎜ EDTA (에틸렌 다이아 민 테트라 초산), 산도 7.5. </li><li> 아세틸화 솔루션 : triethanolamine 플러스 DEPC - 처리된 물 200 ML에 아세트산 무수물 0.5 ML의 2.7 ML. </li><li> 하이브 리다이 제이션 솔루션 : 50 % 포름 아미드, 2X SSPE, 2 μg / μl tRNA, 1 μg / μl BSA 및 1 μg / DEPC - 처리된 물에 μl 폴리. 이 섹션마다 16리터이 필요합니다. </li></ol>

double fluorescence in situ hybridization in fresh brain sections

여기서 우리는 두 형광의 수정된 버전을 설명<em&gt; 현장에서</em&gt; 하이브 리다이 제이션 (dFISH) 메소드 신선한 냉동 뇌 부분에 대한 관심의 두 mRNAs 검출에 최적화된. 우리 그룹은 성공적으로 유전자 공동 규제를 공부하기 위해이 방법을 사용하고 있습니다. 더 구체적으로, 우리는 단세포 해상도의 해부 조직 neurochemical 특성, 및 중앙 감각 회로의 감각적 경험의 영향을 탐험이 dFISH 방법을 사용했습니다. 이 프로토콜은 쥐, 쥐 및 노래하는 새는에서 뇌 조직에서 검증된되었지만 쉽게 다른 척추 종 적용할뿐만 아니라 이외의 신경 조직의 배열로 될 예정입니다. 이 영화에서 우리는이 절차의 주요 단계에 대한 자세한 데모를 제공합니다.

Watch this Scientific Journal Video about Double Fluorescence in situ Hybridization in Fresh Brain Sections at JoVE.com

Double Fluorescence in situ Hybridization in Fresh Brain Sections

이 프로토콜은 아닌 방사성을 포함<em&gt; 원위치</em척추 두뇌의 얇은 부분에서, 단일 셀 해상도로 두 사본 종의 동시 확인 가능&gt; 하이브 리다이 제이션 절차.

더블 형광 현장에서</em신선한 두뇌 섹션> 하이브 리다이 제이션

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

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Neuroscience

Double Fluorescence in situ Hybridization in Fresh Brain Sections

16.0K 조회수.  University of Rochester. 이 프로토콜은 아닌 방사성을 포함 원위치 하이브 리다이 제이션 절차.

비디오: Double Fluorescence in situ Hybridization in Fresh Brain Sections

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

Lipophilic 염료를 결합, 현장에서 하이브리드화, Immunohistochemistry, 그리고 조직학

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

연구

신경과학

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

작은 동물의 만성 신경 레코딩을위한 초경량 동력 Microdrive의 설계 및 조립

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

이후 Wholemount을위한 완벽한 척수 손상 및 뇌 해부 프로토콜<em&gt; 제자리에서</em&gt; 애벌레 바다 장어의 하이브리드

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

고해상도를 사용하여 뇌 섹션에서 Axonally 지역화 된 mRNA를 검출<em&gt; 현장에서</em&gt; 하이브리드

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

빠른<em&gt; 현장에서</em세로토닌 증후군 쥐의 파라 포름 알데히드 - 접두사 뇌에 올리고 뉴클레오티드 프로브를 사용하여&gt; 하이브리드

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

더블 형광 결합<em&gt; 현장에서</em&gt; 마우스 뇌 섹션에서 세 가지 유전자의 발현의 검출을위한 Immunolabelling와 하이브리드

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

RNA에 의한 Locust Antenna 내의 악취 수용체 유전자의 국산화<em&gt; 현장에서</em&gt; 하이브리드 화

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

신선한 인간의 뇌 조직에서 뇌 모세 혈관의 절연

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Section(신선 냉동 또는 고정 마우스 뇌 절편에서 Multiplex Fluorescence In Situ Hybridization과 형광 면역조직화학의 결합)

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.

더블 형광 현장에서 하이브 리다이 제이션