우리는이 프로토콜을 개발 Dörthe Jülich, 제니퍼 라운드와 앤드류 마라의 기여를 인정하고 싶습니다. NICHD, 미국의 암 사회와 천의 월에서 제공하는 연구 지원합니다.

1. 정치 <ol><li> 4 박 배아 ° C PBS에 4 % paraformaldehyde (PFA)와. 문제를 해결 </li><li> 삭제 수정하고 실온 (RT)에서 2 X PBS 5 분 각각 씻으십시오. </li><li> 시계 제조인의 집게를 사용하여 유리 우울증 플레이트의 PBS에서 수동으로 dechorionate 배아. 그들은 폴리 프로필렌의 pipettes에 충실 수 있으므로 dechorionation 후, 배아는 불을 광택 유리 파스퇴르 피펫을 사용하여 전송됩니다. </li><li> 오분 각 25 %의 일련의 50 %와 PBS의 75 % 메탄올을 통해 배아를 전송합니다. </li><li> 100 % 메탄올로 액체를 교체, 새로운 메탄올로 교체 후 5 분 부화합니다. </li><li> 1 시간 최소 -20 ° C에서 장소 배아. (우리는 일반적으로 현장 하이브 리다이 제이션의 표준은 년 이상 보관 배아에서 작동합니다. 하룻밤 배아를 품어,하지만 우리는 현장 하이브리드화에 형광의 높은 해상도가 장기간 보관하여 분실 여부를 검사하지 않았습니다.) </li><li> 75 % 오분 각각 50 %, RT에서 PBST에서 25 %의 메탄올을위한 배아를 씻으십시오. RT에서 오분 PBST에서 각각에 대해 두 번 씻으십시오. </li><li> RT에서 PBS에 4퍼센트 PFA에 20 분 다시 수정. </li><li> RT에서 오분 PBST에서 각각에 대해 두 번 씻으십시오. </li></ol> PFA에 관한 참고 : 우리는 (시약의 표 참조) -20 º C에서 aliquots에 4% PFA를 저장합니다. 초기 고정을 위해, 우리는 이전에 해동 적이있다는 PFA를 사용합니다. 이후 fixations 들어, 우리는 종종 이전 해동했다는 PFA를 사용합니다. 초기 고정이 절차를 성공적으로 얼룩을 위해 중요한 것 같습니다. 2. PROTEINASE 및 POSTFIXATION <ol><li> 배아를 permeabilize하는 3~12분에 대한 RT에서 proteinase K (PBST에 5μg/ml)와 다이제스트. (부화 시간은 젊은 단계 더 구분으로 태아의 나이에 따라 달라집니다. 또한 효소의 배치에 따라 다릅니다.) somitogenesis 스테이지 배아의, 우리가 일반적으로 3~4분에 대한 permeabilize. 이 부화하는 동안, 우리는 측면에 microcentrifuge 관하다. </li><li> PBST에서 잠시 씻어 ​​PBST로 5 분 한 번 씻는다. </li><li> RT에서 PBS에 4퍼센트 PFA에 20 분 다시 수정. </li><li> RT에서 PBST로 5 분 각각 두 번 씻으십시오. </li></ol> 3. PREHYBRIDIZATION <ol><li> 65 º C에서 5 분 동안 태아를 품어 HYB -. </li><li> HYB에 최소한 1 시간 동안 65 º C에서 Prehybridize +. </li></ol> 4. 이종 교잡 <ol><li> 제외한 모든 preHYB 50 μl를 제거하지만, 완전히 빠져들 배아를 보관해야합니다. </li><li> 부드럽게 튜브 flicking하여 배아와 혼합 각 riboprobe 1-2 μl (digoxygenin 및 플루오레신 - 레이블 riboprobes)를 추가합니다. 프로브의 양은 일반적으로 20μl 프로브 합성 반응에서 1 2μl입니다. </li><li> 플루오레신가 빛을 민감한 것을 감안할 때, 튜브는 알루미늄 호일에 싸서 또는 기타이 시점에서 최소한의 빛에 노출되어야합니다. </li><li> 65 º C.에서 하룻밤 배아를 품어 </li></ol> 5. 프로브 제거 참고이 시점 전달 부족 세제의 솔루션을 제공합니다. 세제의 제거는 얼룩 반응을 도와 나타나지만 배아는 오히려 끈적이 될 원인 않습니다. <ol><li> riboprobe를 제거합니다. </li><li> 50 % formamide/2xSSC 65 º C에서 2 X 30 분 씻으십시오. </li><li> 2 X SSC 65 º C에서 15 분 동안 씻으십시오. </li><li> 0.2 X SSC 65 º C 30 분 씻으십시오. </li></ol> 6. 안티 플루오레신 항체 보육 <ol><li> 1X maleic 산성 버퍼 플러스 2% 차단 시약 (시약의 표 참조)의 솔루션을 500μl의 RT 적어도 1 시간 차단합니다. </li><li> 차단 솔루션의 1:500 희석에로 로체가 제공하는 안티 - 플루오레신 - POD 항체를, 추가합니다. </li><li> 4 º C.에 밤새 품어 이 부화하는 동안, 우리는 측면에 microcentrifuge 관하다. </li><li> 1X maleic 산성 버퍼 4 X 20 분 씻으십시오. 오분 PBS에서 각각에 대해 두 번 씻으십시오. </li></ol> 7. 플루오레신 - LABELLED 프로브 탐지 <ol><li> TSA 플러스 플루오레신 솔루션 30-60분을 품어. (반응하십시오. 얼룩 솔루션을하기 전에 TSA 기판 스핀 다운 tyramide 시약에게 퍼어킨 엘머 증폭 희석제 버퍼에 1시 50분을 희석.)이 부화하는 동안, 우리는 측면에 microcentrifuge 관하다. 반응 시간은 각 프로브에 대해 경험적으로 결정되어야합니다. 불행히도, 얼룩 반응은 기판이 형광 그대로 시각적으로 모니터링할 수 없으며, 하나는 얼룩 반응에 걸쳐 유비 쿼터스 녹색 형광을 볼 수 있습니다. </li><li> 30 %, 50 %, 75 % 및 PBS의 100 % 메탄올 10 분만에 각 씻으십시오. </li><li> 첫 번째 퍼옥시데이즈를 inactivate 30 분 메탄올에 1% H 2의 해결책 0 2에서 품어. </li><li> 75 % 50 % PBS에 30 % 메탄올 10 분만에 각 씻으십시오. 다음 십분 PBS에서 각각에 대해 두 번 씻는다. 그것은 중요하다는 것을 충족의 모든hanol이 제거됩니다. </li></ol> TYRAMIDE 신호 증폭주의 사항 : 우리는 퍼어킨 엘머 TSA 키트를 사용하여왔다. 우리는 알렉사 - Tyramide 기판은 퍼어킨 엘머 증폭 희석제 버퍼를 사용하여 잘 얼룩 발견하지만, 우리는 Invitrogen / 분자 프로브 키트와 함께 제공되는 얼룩 버퍼를 사용하여 성공을 없었어요. 마지막으로, 우리는 플루오레신와 알렉사 - 647은 영향을받지있는 동안 Cy5 형광이 이후 메탄올 / H 2 O 2 치료에 의해 ​​제거 것으로 나타났습니다. Cy3 또한 부정적인 그것이 구조적으로 관련되어 Cy5로 메탄올 / H 2 O 2 치료에 의해 ​​영향을받을 수 있습니다. 이러한 이유로, Cy3과 Cy5 TSA 반응은 현장 프로토콜에서 두 형광의 두 번째 얼룩 반응을 위해 사용해야합니다. 8. 안티 Digoxygenin 항체 인큐베이션 <ol><li> 1X maleic 산성 버퍼 플러스 2% 차단 시약의 용액에 RT 적어도 1 시간 동안 다시 배아를 차단합니다. </li><li> 위의 솔루션을 차단에서 1:1000 희석에 로체 제공한으로 방지 발굴 POD 항체 추가 </li><li> 4 º C.에 밤새 품어 이 부화하는 동안, 우리는 측면에 microcentrifuge 관하다. </li><li> 1X maleic 산성 버퍼 4 X 20 분 씻으십시오. 오분 PBS에서 각각에 대해 두 번 씻으십시오. </li></ol> 9. Digoxygenin - LABELLED 프로브 탐지 <ol><li> (얼룩 솔루션을하기 전에 TSA 기판 스핀 다운. 반응의 경우, 증폭 희석제 버퍼에 tyramide 시약 1시 50분을 희석) TSA에 30~60분 플러스 Cy5 솔루션을 품어이 부화하는 동안, 우리는 측면에 microcentrifuge 관하다. 반응 시간은 각 프로브에 대해 경험적으로 결정되어야합니다. </li><li> 십분 PBST 각 세 번 씻으십시오. </li></ol> 10. PROPIDIUM의 요오드화물 얼룩 <ol><li> 오분 2 X SSC에 각각에 대해 두 번 씻으십시오. </li><li> 37 30 분 배아를 품어 ° 10 μl RNAse (100μg/ml의 최종 농도에 대한)과 함께 50 μl 2 X SSCT에서 C. </li><li> 삼분 RT에서 2 X SSC에 각 6 번 씻으십시오. </li><li> 2 X SSC에 330μg/ml propidium 요오드화물의의 솔루션에 8 분 동안 배아를 얼룩. </li><li> 삼분 RT에서 2 X SSC에 각 6 번 씻으십시오. </li><li> RT에서 PBS에 4퍼센트 PFA에 20 분 수정. </li><li> RT에서 PBST로 5 분 각각 두 번 씻으십시오. </li></ol> 11. 설치 <ol><li> 십분 25% 각과 PBST에 50 % 글리세롤을위한 배아를 품어. 4 º C.에 75 % 글리세롤에서 하룻밤 지우기 </li><li> 우리는 해부하다와 deyolk 배아과 평면 현미경 슬라이드에 그들을 탑재합니다. 난황은 상당한 배경 형광을 생성할 수 있습니다. 절개는 배아가 gylcerol에 ovenight 해제 후 훨씬 쉽습니다. </li></ol> 솔루션 : <table border="1"><tbody><tr><td> PBST </td><td> PBS 플러스 0.1 % 트윈 </td><td></td></tr><tr><td> SSCT </td><td> SSC 플러스 0.1 % 트윈 </td><td></td></tr><tr><td> HYB - </td><td> 50 % 포름 아미드 5xSSC 0.1 % 트윈 - 20 </td><td></td></tr><tr><td> HYB + </td><td> HYB - 5mg/ml torula (효모) RNA 50μg/ml 헤파린 </td><td style="width: 300px;"> torula RNA는 RNA로의 proteinase K의 소화에 의해 준비가되어 이후 페놀 -, 페놀 - 클로로포름 - 및 클로로포름 - extraction.The RNA가 시켰던 및 DEPC - 처리된 물속에 녹아있다. </td></tr><tr><td> 1 X maleic 산성 버퍼 </td><td> 150mM maleic 산, 100mM NaCl (산도 7.5) </td><td></td></tr></tbody></table><img src="/files/ftp_upload/1229/Brend_Holley_Fig1.png" alt="그림 1" width="600" height="688" /> 그림 1. 원위치 하이브리드화의 전체 마운트 대표 결과가 이중 형광. (AC). 이미지는 10 - 체절 단계에서 zebrafish 배아의 후부 지역을 통해 하나의 공촛점 섹션을 표시합니다. 보기 맨 앞쪽에와 지느러미입니다. propidium 요오드화물의와 핵의 얼룩진 색 파란색입니다. (A) deltaC의 mRNA는 digoxygenin - 표시 riboprobe 및 TSA - Cy5 시약을 사용하여 검색했습니다. (B) her1 유전자의 mRNA 베꼈는데 것은 플루오레신 - 표시 riboprobe 및 TSA - 플루오레신 시약과 함께 발견되었다. (C) 녹색과 적색 채널을 병합하면 unambiguously 두 유전자의 서로 다른 또는 중복 유전자의 표현의 영역을 식별합니다. (D, E) her1 표현의 상세는이 절차의 높은 해상도를 보여주는. mRNA의 Subcellular 지방화 명확하게 분별 수 있습니다. 화살촉은 핵에 얼룩의 점에 의해 밝혀 적극적인 전송을 전시 세포를 나타냅니다. 화살표는 mRNA의 세포질 지방화를 보여주는 세포를 나타냅니다. her1 및 deltaC에 대한 (F, G) 더블 얼룩이 두 유전자 (흰색 화살촉) 또는 별도로 유전자 (빨간색과 녹색 화살촉) 중 하나를 해독 아르 세포를 보여줍니다.

<ol>
	<li>Schulte-Merker, S., Ho, R. K., Herrmann, B. G., N&#252;sslein-Volhard, 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+protein+product+of+the+zebrafish+homologue+of+the+mouse+T-gene+is+expressed+in+nuclie+of+the+germ+ring+and+the+notochord+of+the+early+embryo.">The protein product of the zebrafish homologue of the mouse T-gene is expressed in nuclie of the germ ring and the notochord of the early embryo.</a> Development. 116, 1021-1027 (1992).</li><li>Jowett, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+in+situ+hybridization+techniques+in+zebrafish.">Double in situ hybridization techniques in zebrafish.</a> Methods. 23, 345-358 (2001).</li><li>J&#252;lich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beamter/deltaC+and+the+role+of+Notch+ligands+in+the+zebrafish+somite+segmentation,+hindbrain+neurogenesis+and+hypochord+differentiation.">beamter/deltaC and the role of Notch ligands in the zebrafish somite segmentation, hindbrain neurogenesis and hypochord differentiation.</a> Dev Biol. 286, 391-404 (2005).</li><li>Mara, A., Schroeder, J., Chalouni, C., Holley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priming,+Initiation+and+Synchronization+of+the+Segmentation+Clock+by+deltaD+and+deltaC.">Priming, Initiation and Synchronization of the Segmentation Clock by deltaD and deltaC.</a> Nat Cell Biol. 9, 523-530 (2007).</li><li>Lecuyer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+mRNA+localization+reveals+a+prominent+role+in+organizing+cellular+architecture+and+function.">Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.</a> Cell. 131, 174-187 (2007).</li></ol>

The protocol presented here works well with probes that give a clean strong signal after staining for 30-45 minutes in a typical alkaline phosphatase-mediated reaction. Prior to performing the fluorescent in situ hybridization protocol, we always test our probes using the standard non-fluorescent protocol (provided in supplemental material along with the probe synthesis protocol). We have had less success with the fluorescent in situ hybridization protocol when using weaker probes, but it may be possible in some cases....

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Paraformaldehyde 16% solution, EM Grade</td>
<td>&nbsp;</td>
<td>Electron Microscopy Services</td>
<td>15710</td>
<td>Dilute to 4% in 1.33x PBS (final 1x). Store in 2ml aliquots at &#x2013;20&deg;C</td>
</tr>
<tr>
<td>Proteinase K, recombinant PCR grade</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>03115879001</td>
<td>Make a 20mg/ml stock solution in water. Store in 1ml aliquots at &#x2013;20&deg;C</td>
</tr>
<tr>
<td>Blocking Reagent</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11096176001</td>
<td>Make a 10% stock solution in 1x maleic acid buffer. Store in 50ml aliquots at &#x2013;20&deg;C. Stable over multiple freeze/thaws.</td>
</tr>
<tr>
<td>Anti-Fluorescein-POD, Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11426346910</td>
<td>Aliquot and store at -20&deg;C. Keep one working aliquot at 4&deg;C.</td>
</tr>
<tr>
<td>Anti-Digoxigenin-POD, Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11207739910</td>
<td>As above.</td>
</tr>
<tr>
<td>TSA Plus Fluorescein system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL741001KT</td>
<td>Prepare each vial of TSA reagent only when needed. Dissolve in 60&mu;l DMSO. Store at 4&deg;C.</td>
</tr>
<tr>
<td>TSA Plus Cyanine5 system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL745001KT</td>
<td>As above</td>
</tr>
<tr>
<td>TSA Plus Cyanine3 system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL744001KT</td>
<td>As above</td>
</tr>
<tr>
<td>TSA Kit #16 AlexaFluor647 tyramide (plus HRP-goat-anti-rabbit IgG)</td>
<td>&nbsp;</td>
<td>Molecular Probes /Invitrogen</td>
<td>T20926</td>
<td>Dissolve tyramide reagent in 150&mu;l DMSO, aliquot and store at &#x2013;20&deg;C.</td>
</tr>
<tr>
<td>RNase, DNase free</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11119915001</td>
<td>Supplied as 500&mu;g/ml solution</td>
</tr>
<tr>
<td>Propidium Iodide</td>
<td>&nbsp;</td>
<td>Molecular Probes /Invitrogen</td>
<td>P-3566</td>
<td>Supplied as 1mg/ml solution in water.</td>
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		</div>

zebrafish whole mount high-resolution double fluorescent in situ hybridization

전체 마운트<em&gt; 현장에서</em&gt; 하이브 리다이 제이션은 발달 생물학에서 가장 널리 사용되는 기법 중 하나입니다. 여기서 우리는 고해상도 이중 형광을 제시<em&gt; 현장에서</em단일 유전자의 정확한 표현 패턴을 분석하는 두 유전자의 표현 도메인의 중복을 결정&gt; 하이브리드화 프로토콜. 프로토콜은 표준의 수정된 버전입니다<em&gt; 현장에서</em알칼리성 인산 가수 분해 효소와 같은 NBT / BCIP 빠른 레드와 같은 기판을 사용하여&gt; 하이브 리다이 제이션<sup&gt; 1,2</sup&gt;. 이 프로토콜은 tyramide 신호 증폭 (TSA)과 함께 표준 digoxygenin 및 플루오레신 라벨 프로브를 활용<sup&gt; 3</sup&gt;. 상용 TSA 키트는 녹색에서 멀리 빨간색 형광 방출로 유연하게 실험적인 디자인이 같은 propidium 요오드화물 또는 단백질에 대한 형광 immunohistochemistry 등 다양한 핵 얼룩,와 함께 사용할 수 있습니다. TSA 빠르게 covalently 표시 안티 센스 riboprobe의 인근에 moieties, 일반적으로 티로신 잔류물에 바인딩 반응 형광 기판을 생산하고 있습니다. 그 결과 얼룩 패턴은 mRNA의 subcellular 지방화에 고해상도 아르는 공촛점 현미경을 스캐닝 레이저를 사용하여 관찰할 수<sup&gt; 3,4</sup&gt;. 하나는 염색체 loci에서 초기 기록을 관찰 핵 및 세포질 얼룩을 구분하여 mRNA의 대뇌 피질의 현지화 등 다른 패턴을 시각화 수 있습니다. 의 연구<em&gt; Drosophila</em&gt; mRNAs의 약 70 %가 자주 인코딩 단백질의 기능과 상관 관계 subcellular 지방화의 특정 패턴을 전시 것을 나타냅니다<sup&gt; 5</sup&gt;. 3D 공촛점 데이터 세트의 컴퓨터 지원 재건과 결합하면, 우리의 프로토콜은 전체 척추의 배아에서 하위 셀룰러 해상도 mRNA 분포의 상세한 분석을 수 있습니다.

Watch this Scientific Journal Video about Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization at JoVE.com

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

현장 하이브리드화의 전체 마운트 발달 생물학에서 가장 널리 사용되는 기법 중 하나입니다. 여기, 우리는 단일 유전자의 정확한 표현 패턴을 분석하는 두 유전자의 표현 도메인의 중복을 결정하는 현장 하이브리드화 프로토콜을 두 번 형광 높은 해상도를 제시한다. 우리는 조직 조직을 강조하기 위해 propidium 요오드화물 핵 카운터 얼룩이 포함됩니다.

Zebrafish 전체 마운트 고해상도 더블 형광등 현장에서 하이브 리다이 제이션

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

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JoVE Journal

Biology

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

23.1K 조회수.  Yale University. 현장 하이브리드화의 전체 마운트 발달 생물학에서 가장 널리 사용되는 기법 중 하나입니다. 여기, 우리는 단일 유전자의 정확한 표현 패턴을 분석하는 두 유전자의 표현 도메인의 중복을 결정하는 현장 하이브리드화 프로토콜을 두 번 형광 높은 해상도를 제시한다. 우리는 조직 조직을 강조하기 위해 propidium 요오드화물 핵 카운터 얼룩이 포함됩니다.

비디오: Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

조기 칙 태아의 현장 하이브리드화에서 더블 전체 산

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

연구

생물학

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

칙의 전체 마운트 항체 스테 이닝을위한 방법

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

전체 마운트를 사용하여 현장에서</em분자 생물학과 Organismal를 링크> 하이브 리다이 제이션

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

전체 산<em&gt; 현장에서</emE11.5 마우스 태아에 E8.5의&gt; 하이브리드

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

RNA 형광하여 단일 세포 유전자 전사의 분석<em&gt; 현장에서</em&gt; 하이브리드 (물고기)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

형광등과 결합 염색체 복제 타이밍<em&gt; 현장에서</em&gt; 하이브리드

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

전체 마운트 RNA 형광<em&gt; 현장에서</em의&gt; 하이브리드<em&gt; Drosophila</em&gt; 태아

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em&gt; 현장에서</em전체 마운트 태아 및 세포 조직학에서&gt; 하이브리드는 해양 Elasmobranchs을위한 적응

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

높은 해상도 전체 마운트<em&gt; 제자리에서</em유전자 발현 및 기능을 연구하는 제브라 피쉬 배아에서&gt; 하이브리드

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...