이 작품은 RHF에 마가렛 M. 알켁 재단에 의해 지원되었다.

1 부 : 배아를 고정 <ol><li> 얼음 추위 depc - PBS로 해부 접시를 채우십시오. 얼음 계속. 포셉로 껍질을 감청하여 계란을 열고 조개 조각을 제거합니다. </li><li> 포셉과 알부민 두꺼운 제거합니다. 그 배가 위쪽으로 얼굴을 너무 거친 포셉과 난황을 기울이기. </li><li> 가위를 사용하여 배아 주위에 난황의 사각형 잘라. </li><li> 숟가락 사용, 얼음 추위 depc - PBS에 노른자와 장소에서 배아를 제거합니다. </li><li> 현미경, 세포막과 노른자를 제거하고 고정 요리로 전송할 수 있습니다. </li><li> 아래로 핀 depc - PBS를 제거합니다. depc - PBS에서 4% PFA로 교체하고 4 O / N를 수정 ° C. </li><li> 정착액 제거합니다. 얼음 추위 depc - PBS로 바꿉니다. 뭉툭한 끝 microcapillary 바늘이나 microdissection 나이프를 이용하여 프로브의 트래핑을 방지하기 위해, 신경계와 심장 충치 구멍. </li><li> 0.1 % 트윈 - 20 (PBT)를 2 배 10 MN depc - PBS 씻으십시오. 25 %, 50 %, PBT의 75 % 메탄올의 각 10 MN을 탈수. 100 % 메탄올 두 번 배아를 탈수. 메탄올에서 -20 ° C에 보관하십시오. </li></ol> 2 부 : Prehybridization <ol><li> 75 % 50 % PBT의 25 % 메탄올의 각 배아 10 MN을 Rehydrate. PBT에서 배 10 MN 씻으십시오. </li><li> 한편, 얼음 차가운 정착액 (PBT의 4 % paraformaldehyde)을 준비합니다. proteinase K 용액 (; [? 5g / ML] 마지막 PBT 3 ML / 유리병)와 PBT를 교체하십시오. 전체 유리병이 부드럽게 병을 회전하여 proteinase K 용액에 노출 있는지 확인합니다, 노출 시간에 대한 문제 해결을 참조하십시오. </li><li> RNase 무료 파스퇴르 피펫 제거 proteinase K를 사용하고 정착액 (2 ML)을 추가합니다. 즉시 정확히 20 MN을 위해 얼음에 놓으십시오. </li><li> 모든 RT의 세척은 nutator에서 수행됩니다. 2X PBT 10 미네소타, 그리고 50 %의 하이브리드화 버퍼를 포함하는 PBT에서 1X 10mn 씻으십시오. </li><li> 하이브 리다이 제이션 버퍼 1X 씻으십시오. 65 품어 ° 4-5 시간 2 ML의 하이브리드화 버퍼에 C. </li></ol> 파트 3 : 탐사선과 잡종 배아 <ol><li> 파고 및 FITC 결합 프로브 합성은 부록에 설명되어 있습니다. 강한 신호를 줄 것으로 예상 프로브, FITC 함유 세포핵과 synhesized되어야하며 약한 프로브 발굴 함유 세포핵과 함께 합성한다. </li><li> 2 ML의 약병을 사용하여 하이브 리다이 제이션 버퍼 Prewarm 프로브 (500 NG / ML 각). 즉시 프로브 솔루션 하이브 리다이 제이션 버퍼를 교체하십시오. 배아는 건조시키지 마십시오. 65 16 시간을위한 품어 ° C. </li><li> 하이브리드화 버퍼, 2 세척, 30 MN 65 ° C.와 각각의 프로브를 교체 65 (1 / 1 V / V /) 하이브리드화 버퍼 / MABT 15 MN 와시 ° C. </li></ol> 4 부 : 발굴 항체 보육 및 색상 반응 <ol><li> MABT에서 2x20 MN 씻으십시오. 2퍼센트 BBR / MABT 1 시간 씻으십시오. 2퍼센트에 5 시간을 씻어 BBR/20 % 히스 / MABT (버퍼를 차단). 차단 버퍼 희석 1:2000 파다 항체에 품어, 4 O / N ° nutator에 C. </li><li> MABT에서 배 10 미네소타 각각하고, 배 1 시간 MABT 각 씻으십시오. </li><li> NTMT에서 배 20 MN 씻으십시오. 10 ML의 NTMT에 200ul의 NBT / BCIP 기판을 추가합니다. 즉시 병을에서 NTMT를 제거하고 1-2 ML NBT / BCIP 버퍼로 바꿉니다. 어둠 속에 넣습니다. 20 MN 후 색상 반응을 모니터링, 20 MN의 간격으로 그 이후. </li><li> 색상 반응 (블루 나타납니다)에 따라, 10 MN을 위해 PBS에 세척 PBS로 가득 고정 접시에 아래 핀, 및 PBS의 4% PFA와 솔루션을 바꿉니다. 4 O / N ° C를 수정 </li><li> 새로운 섬광 유리병에 전송. 2x10 MN (1 % 트윈 - 20와 함께 PBS) PBST로 씻으십시오. MABT 2X 10 MN에 씻으십시오. </li><li> 63 ° C.에 MABT 30 MN의 부화 </li></ol> 제 5 부 : FITC 항체 보육 및 색상 반응 <ol><li> MABT에서 배 10 미네소타, 2 %에 1 시간을 씻어 BBR / MABT, 차단 버퍼에 5 시간 </li><li> 알칼리성 인산 가수 분해 효소 - 결합 백신 FITC - 항체 (1:500) 4 O / N ° C.에 품어 </li><li> MABT에서 배 10 미네소타 각각하고, 배 1 시간 MABT 각 씻으십시오. </li><li> 0.1 % 트윈 - 20 (TBST)으로 TBS 산도 8.45에서 배 20 MN에 씻으십시오. </li><li> TBST이 키트에서 제공하는 시약 1,2와 3를 사용하여 벡터 레드 작업 솔루션 (5 ML)를 준비합니다. 즉시 병을에서 TBST를 제거하고 벡터 레드 버퍼로 바꿉니다. 어둠 속에 넣습니다. 40 MN 후 색 반응, 그리고 이후 30 MN의 간격을 모니터링합니다. </li><li> 다음 색 반응 (적색 나타납니다), PBS로 전송 배아. </li></ol> 6 부 : 고정 및 처리 <ol><li> PBS를 포함하는 고정 접시에 전송하고, 4 RT 또는 O / N 20 MN에 대한 4퍼센트 PFA의 해결 ° C. </li><li> PBS, 2X 10 MN에 씻으십시오. 사진에 대한 처리하기 위해 PBS에 20 % 글리세롤 (이것은 태아가 반투명하게됩니다)을 전송할 수 있습니다. 챔버를 만들려면 슬라이드에 그들을 입력해, PVC 테이프 2 스트립 잘라. 블레이드를 사용하여 중앙에 사각형을 잘라 버릴거야. 광장 사용 포셉를 제거합니다. </li><li> 왁스 sectioning에 대한 처리하기 위해 메탄올의 등급 시리즈 (25 %, 50 %, 75 % 및 PBS 100 %, 10 MN 각)에서 탈수, PBS, 씻지 2X 10 MN로 다시 전송할 수 있습니다. </li><li> 프로파놀, 3mn로 바꿉니다. 1,2,3,4 - tetrahydronaphthalene로 교체, 20 MN은 Histoclear (2X 1 시간)으로 따라갔다. 1 시간에 대한 histoclear / 왁스 1분의 1 (V / V) 60 ° C로 바꿉니다. 왁스 처리로 교체조직학하십시오. </li></ol> 7 부 : 솔루션 -20에서 하이브 리다이 제이션 버퍼 저장 ° 팔콘에서 C, 50 ML : 25 ML의 포름 아미드, 3.25 ML (20xSSC), 0.5 ML (0.5M EDTA (에틸렌 다이아 민 테트라 초산)), 1 ML (10 % 트윈 - 20), 2.5 ML (1 % SDS) ; 125 UL (20mg/ml tRNA). 100ul (50 MG / ML 헤파린) MABT는 100mM maleic 산성을 준비, 산도 7.5 NaOH 알약과 함께. 150mM NaCl 및 0.1 % 트윈 - 20을 추가합니다. NTMT (사용 직전 만든), 50 ML : 1ml (5M NaCl), 2.5 ML (2M 트리스 - HCL pH9.5), 1.25 ML (2M MgCl 2), 5 ML (10 % 트윈 - 20). TBST : 0.1M 트리스 - HCL, 0.15M NaCl, 산도 8.45, 0.1 % 트윈 - 20. 8 부 : 문제 해결 <table border="1"><tbody><tr><th> 문제 </th><th> 원인 </th><th> 치료제 </th></tr><tr><td valign="top"> 태아가 웅크리고있다 </td><td valign="top"> 부족 탈수 / rehydration </td><td valign="top"> 연속 탈수 / rehydration 단계 동안 10 MN 간격을 유지 </td></tr><tr><td valign="top"> 프로브 신호가 없습니다 </td><td valign="top"> 프로브는 병에 강등 RNase 오염입니다 </td><td valign="top"> 전체 유리병이 2 단계에서 Proteinase K 용액에 노출되었는지 확인합니다 1퍼센트 아가로 오스 겔에 프로브를 실행 </td></tr><tr><td valign="top"> 프로브 마음 레이블 충치 신경 튜브입니다 </td><td valign="top"> 프로브가 갇혀있어 </td><td valign="top"> 2 단계에서는 모든 충치가 뇌를 사용 microdissection 나이프 있는지 확인하거나 microcapillary </td></tr><tr><td valign="top"> 배아는 다음과 하이브리드화 (3 단계)를 분해 </td><td valign="top"> Proteinase K 단계가 너무 깁니다 하이브 리다이 제이션 온도가 너무 높습니다 왼쪽 </td><td valign="top"> Proteinase K은 2 MN (단계 5-7), 3 MN (80-10 단계), 4 MN가 (11-13 단계), (단계 3 + 4) 이상 1mn보다 왼쪽해서는 안됩니다 높은 T O에 잡종을하지 마십시오 65 O C 이상 </td></tr></tbody></table> 파트 9 : 대표 결과 대표 배아은 아래와 같습니다 : (A) 엠브료 (무대 HH7)이 파다 - 삭스 프로브 (파란색)와 FITC - 델타 - 1 프로브 (적색)으로 표시됩니다. (B) 엠브료 (무대 HH8)이 파다 - Pax6 프로브 (파란색)와 FITC - 쉬 프로브 (적색)으로 표시됩니다. NF, 신경 폴드, ANP, 앞쪽에 신경 판, PSM, presomitic mesoderm, N, notochord, NT, 신경 튜브). DRS의 실험실에서 프로브 선물. C. Tabin, M. 굴딩스, D. 헨리크, 그리고 J. 브리 스코. <img src="/files/ftp_upload/old/904_2008_10_20_1/904_Figure-01.png" alt="" width="303" height="300" />

<ol>
	<li>Streit, A., Berliner, A. J., Papanayotou, C., Sirulnik, A., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+neural+induction+by+FGF+signaling+before+gastrulation.">Initiation of neural induction by FGF signaling before gastrulation.</a> Nature. 406, 74-78 (2000).</li><li>Rodr&#237;guez Esteban, C., Capdevila, J., Economides, A. N., Pascual, J., Ortiz, A., Izpis&#250;a Belmonte, J. 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+novel+Cer-like+protein+Caronte+mediates+the+establishment+of+embryonic+left-right+asymmetry.">The novel Cer-like protein Caronte mediates the establishment of embryonic left-right asymmetry.</a> Nature. 401, 243-251 (1999).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+the+organizer+after+radical+ablation+of+Hensen's+node+and+the+anterior+primitive+streak+in+the+chick+embryo.">Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo.</a> Development. 122, 3263-3273 (1996).</li><li>Kawakami, M., Nakanishi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+an+endogenous+PKA+inhibitor,+PKIalpha,+in+organizing+left-right+axis+formation.">The role of an endogenous PKA inhibitor, PKIalpha, in organizing left-right axis formation.</a> Development. 128, 2509-2515 (2001).</li><li>Basch, M. L., Bronner-Fraser, M., Garcia-Castro, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+the+neural+crest+occurs+during+gastrulation+and+requires+Pax7.">Specification of the neural crest occurs during gastrulation and requires Pax7.</a> Nature. 441, 218-222 (2006).</li></ol>

The authors have nothing to disclose.

현장 하이브리드화 방식의 2 색 전체 마운트는 관심 중 하나 또는 두 개의 유전자를 사용하여 유전자 발현의 공간 및 시간적 패턴 모두를 결정하는 데 사용됩니다. 응용 프로그램은 새로운 유전자의 유전자 발현 패턴의 평가 (예 : 1,2,뿐만 아니라 유전자 발현 다음과 모욕의 변화 (배아 조작 3, 구슬 4 및 RNA 또는 DNA의 electroporation 5 구성)을 포함합니다.

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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>Eggs</td>
<td>Animal</td>
<td>Charles River </td>
<td>Premium Fertile</td>
<td>&nbsp;</td>
<tr>
<td>Stereomicroscope</td>
<td>Microscope</td>
<td>Leica Microsystems</td>
<td>MZ9.5</td>
<td>&nbsp;</td>
<tr>
<td>Marsh Automatic Incubator</td>
<td>Tool</td>
<td>Lyon</td>
<td>RX</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization Incubator</td>
<td>Tool</td>
<td>Hybaid</td>
<td>Micro-4</td>
<td>&nbsp;</td>
<tr>
<td>Adams Nutator orbital mixer</td>
<td>Tool</td>
<td>Fisher</td>
<td>14-062</td>
<td>&nbsp;</td>
<tr>
<td>Curved Forceps (1)</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>72991-4C</td>
<td>&nbsp;</td>
<tr>
<td>Fine scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14161-10</td>
<td>&nbsp;</td>
<tr>
<td>Spoon</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>10370-18</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Capillary Pipette</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>70950-12</td>
<td>&nbsp;</td>
<tr>
<td>Microcapillary tube</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1049-1PAK</td>
<td>Pull using vertical micropipette puller; blunt end with fine forceps</td>
</tr>
<tr>
<td>Microdissecting knife</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10056-12</td>
<td>Use to puncture cavities prior to in situ hybridization</td>
</tr>
<tr>
<td>Minuten pins 0.2mm diam</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-20</td>
<td>Keep pins together using a magnetic stirrer;</td>
</tr>
<tr>
<td>Sylgard 184 Silicon Elastomer Curing Agent and Base</td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>0001986475</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>Diethylpyrocarbonate (depc)</td>
<td>Reagent</td>
<td>Acros Organics</td>
<td>10025025</td>
<td>Add 1ml depc to 1l PBS; shake; autoclave</td>
</tr>
<tr>
<td>16% PFA</td>
<td>Reagent</td>
<td>Electron Microscopy Sciences</td>
<td>15710</td>
<td>&nbsp;</td>
<tr>
<td>Tween-20</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P1379-100ML</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P2308-5MG</td>
<td>Stock at 10 mg/ml in depc-H2O in 10ul aliquots. Store at -20C.</td>
</tr>
<tr>
<td>Formamide, deionized</td>
<td>Reagent</td>
<td>Ambion</td>
<td>9342</td>
<td>&nbsp;</td>
<tr>
<td>Yeast tRNA-25 mg</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15401-011</td>
<td>Stock at 20 mg/ml in depc-H2O in 125ul aliquots. Store at -20C</td>
</tr>
<tr>
<td>Heparin Sodium salt</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H4784-250MG</td>
<td>Stock at 50 mg/ml in depc-H2O in 100ul aliquots. Store at -20C.</td>
</tr>
<tr>
<td>SSC x20 pH5.5</td>
<td>Reagent</td>
<td>Fisher</td>
<td>PR-V4261</td>
<td>&nbsp;</td>
<tr>
<td>EDTA (0.5M)</td>
<td>PR-V4231</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>SDS</td>
<td>Reagent</td>
<td>Fisher</td>
<td>BP166-100</td>
<td>&nbsp;</td>
<tr>
<td>Maleic acid</td>
<td>Reagent</td>
<td>Fluka</td>
<td>63189</td>
<td>&nbsp;</td>
<tr>
<td>Boehringer Blocking Reagent</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11096176001</td>
<td>Use at 2% in MABT. To make, heat with MABT for 20 mn at 60C. Can make a stock and store in 1-5 ml aliquots at 20C.</td>
</tr>
<tr>
<td>Heat inactivated sheep serum</td>
<td>Reagent</td>
<td>Jackson Immuno</td>
<td>013-000-121</td>
<td>Dissolve in 10 ml H2O; heat 550C, 30 mn. Aliquot at 1ml, -20C.</td>
</tr>
<tr>
<td>Anti-Digoxigenin-AP, Fab fragments</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11093274910</td>
<td>&nbsp;</td>
<tr>
<td>NBT/BCIP stock solution</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11681451001</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Fluorescein-AP, Fab fragments</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11426338910</td>
<td>&nbsp;</td>
<tr>
<td>2M Tris</td>
<td>BP1759-500</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vector Red Alkaline Phosphatase Substrate Kit</td>
<td>Reagent</td>
<td>Vector Laboratories</td>
<td>SK-5100</td>
<td>&nbsp;</td>
<tr>
<td>1,2,3,4-tetrahydronaphthalene</td>
<td>Reagent</td>
<td>Sigma</td>
<td>429325-100ML</td>
<td>Under hood</td>
</tr>		
</tbody>
</table>
</div>

double whole mount in situ hybridization of early chick embryos

여자의 난자는 초기 배아 발달의 연구에 귀중한 도구입니다. 투명성, 접근성 및 조작 용이성, 그건 뇌, 신경 튜브, 체절과 심장 primordia 형성에 유전자 발현을 연구를위한 이상적인 도구가합니다. 이 비디오는 현장 하이브리드화 2 색 전체 마운트에있는 다른 단계를 보여주는, 첫째, 배아는 난자에서 해부하고 paraformaldehyde에서 해결되었습니다. 둘째, 배아는 prehybridization에 대한 처리됩니다. 배아는 다음 두 가지 프로브, 팔 결합 하나와 FITC에 결합하여 하나 hybridized입니다. 하룻밤 하이브리드화 다음, 배 (胚)가 발굴 결합 항체와 incubated입니다. 발굴 기판에 대한 컬러 반응이 수행하고, 관심 지역은 파란색으로 나타납니다. 배아는 다음 FITC 항체와 결합 incubated입니다. 배아는 FITC와 색상 반응을 처리하고, 관심 지역은 빨간색으로 나타납니다. 마지막으로, 배아는 고정 및 처리 phtograph과 sectioning입니다. 문제 해결 가이드도 제공됩니다.

Watch this Scientific Journal Video about Double Whole Mount in situ Hybridization of Early Chick Embryos at JoVE.com

Double Whole Mount in situ Hybridization of Early Chick Embryos

이 비디오는 현장 하이브리드화, 2 개의 다른 유전자의 공간과 시간적 표현식 패턴이 젊은 여자는 배아에서 시각 될 수있는 방법 2 색 전체 마운트를 보여줍니다. 이 방법은 원래 데이비드 윌킨슨, 도밍고스 헨리크, 필 잉엄 데이비드 이쉬 - Horowicz에 의해 도입되었다.

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 ...

double-whole-mount-in-situ-hybridization-of-early-chick-embryos

Research

JoVE Journal

Biology

12.4K 조회수.  Institute of Biosciences and Technology - Texas A&M Health Science Center. 이 비디오는 현장 하이브리드화, 2 개의 다른 유전자의 공간과 시간적 표현식 패턴이 젊은 여자는 배아에서 시각 될 수있는 방법 2 색 전체 마운트를 보여줍니다. 이 방법은 원래 데이비드 윌킨슨, 도밍고스 헨리크, 필 잉엄 데이비드 이쉬 - Horowicz에 의해 도입되었다.

비디오: Double Whole Mount in situ Hybridization of Early Chick Embryos

Method for Culture of Early Chick Embryos ex vivo (New Culture)

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  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

조기 칙 태아 예 생체내 (새로운 문화)의 문화에 대한 방법

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

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 ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

칙 태아의 단일 세포 Transfection

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

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 ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

초기 여자는 배아에서 Morphogenetic 조직 Deformations 추적

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

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 ...