IM은 마리 퀴리 다시 통합 교부금에 의해 지원됩니다. 이 작품은 이스라엘 과학 기금 부여 번호에 의해 지원되었다. 1146년부터 1108년까지, 헬렌과 줄기 세포 과학의 웨이즈만 연구소에서 연구 및 레어 자선 재단 마틴 킴멀 연구소.

1. 부설 에그 <ol><li> 해부 - 5 일 : 성관계 여성은 효모와 보충 신선한 음식 2~4시간에 대한 알을 낳을 수 있습니다. 에 동기와 잘 개발 애벌레를, 그것이 중요하다 콩나물도하지 양식 (약 30 알 / 25mm 유리병). 일반적으로 7-16 여자들은 누워 얼마나 잘 따라 유리병마다 사용됩니다. </li></ol> 2. 애벌레 선택 <ol><li> 링어의 중간 (128mM NaCl, 2mM Kcl, 1.8mM CaCl 2, MgCl 4mm짜리 2, 35.5mM 자당, 5mM Hepes의 산도 6.9)로 가득한 9 잘 유리 해부 접시를 준비합니다. </li><li> 링어의 중간 얼음에 장소를 포함하는 여섯 잘 플레이트의 세포 strainers를 준비합니다. 또한, 우리는 나일론 메쉬가 장착되어 특별히 만든 금형을 사용합니다. </li><li> 고급 생물 학적 핀셋 (포셉)를 사용하여 유리 벽면에서 시간이 초과되었습니다 애벌레를 선택하고 링거스 포함 해부 접시에 놓으십시오. </li><li> 깨끗한 잘하는 여성 유충을 전송합니다. 우리가 그들의 생식선으로 남성에서 여성을 구분합니다. 남성 testes 쉽게 지방 신체의 뒷부분 세번째에 포함된 빅 클리어 타원으로 식별됩니다. 지방 신체의 같은 부분에 위치한 여성 난소는, 훨씬 작고, 분명히 둥근 분야로 확인할 수 있습니다. </li></ol> 3. 유충의 해부 <ol><li> 포셉과 두뇌로 단지 사후 유충 잡고 포셉의 두 번째 쌍의과 머리를 제거합니다. </li><li> 기관이 아래로 향하게하여, 등의 측면에 나머지 후부 부분을 넣으십시오. </li><li> 포셉 한 쌍의와 사후 spiracles하여 유충을 잡고 절반에 해당하는 애벌레 지방 기관이 나온다 때까지 다른 한쌍은 뒤로 표피를 압박할 수있는 동안 안으로 천천히 밀어. </li><li> 단단히 뒷부분 끝을 잡고 느슨하게 표피를 잡아 포셉의 다른 쌍을 사용합니다. 부드럽게 천천히 후부 끝부분을 끌어낼의 격차를 통해 표피와 연결된 소장 슬라이드 있도록. 이 과정의 끝에, 지방 시체가 완전히 소장 및 표피에서 분리해야합니다. 지방 신체의 앞쪽에 부분에서 대장을 분리합니다. 얼룩 쉽게 장착을 만들려면, 그것은 지방이 몸 그대로 유지됩니다 것이 중요합니다. </li><li> 선호 잘 포함 해부 애벌레에서 링어의 중간과 함께 철저하게 파스투레 피펫 젖었어. 이 외투 피펫 도움과를 지키는에서 지방 시체를 방지할 수 있습니다. 셀 스트레이너에 얼음 차가운 링어의 중간에 지방 시체를 전송하는 파스투레 피펫을 사용합니다. </li></ol> 4. 고정 및 스테 이닝 모든 단계 4에서 처음으로 항체와 부화를 제외하고, 상온에서 수행됩니다 ° C. <ol><li> 링어의 중간에 5 %의 포름 알데히드에 지방 시체를 품어. 부드러운 교반과 함께 20 분. </li><li> 1% PBT (1 % 트리톤 PBS에서 X - 100) 5 분 씻으십시오. 10 분이 단계 45 분 세 번째를 반복합니다. 1퍼센트 트리톤 X - 100은 애벌레 난소를 구멍과 항체가 그것을 뚫을 수 있도록해야합니다. </li><li> 부드러운 교반 1 시간 동안 0.3 % PBTB (0.3 % 트리톤 X - 100과 PBS에 1 % BSA)로 차단 </li><li> 4 박 이상 0.3 % PBTB에 희석 원하는 1 일 항체 ° 부드러운 동요와 C와 부화. 이 단계는 일반적으로 롤러에 0.2 ML 튜브에서 수행됩니다. </li><li> 셀 strainers 다시 지방 시신을 전송합니다. </li><li> 부드러운 교반과 함께 0.3 % PBTB 3 회 30 분 각각을 씻으십시오. </li><li> 부드러운 교반 1 시간 동안 5% 정상 당나귀 혈청 보충 0.3 % PBTB로 차단합니다. </li><li> 적절한 보조 항체 부드러운 교반 2 시간 동안 차단 솔루션 (0.3 % PBT에서 5% 정상 당나귀 혈청과 보충 0.3 % PBTB)에 (제조 업체의 사양에 따라) 희석과 부화. 항체가 형광 경우이 시점 이후부터 어둠 속에 품어. 이 단계는 일반적으로 롤러에 0.2 ML 튜브에서 수행됩니다. </li><li> 삼십분 각 부드러운 동요와 0.3 % PBT와 3 번 씻으십시오. </li></ol> 5. 설치 <ol><li> 깨끗한 eppendorf 튜브에 지방 시체를 전송합니다. 매우 신중하게 떨어져 모든 액체를 제거하고 즉시 Vectashield 장착 미디어 커버. 난소가 지방 신체에서 분리되고있는 동안은 강화하지 않기 때문에 우리는 정기적으로 vectashield를 사용합니다. 최대 일주일까지 샘플 4 ° C에서 장착 매체에 저장할 수 있습니다. </li><li> 피펫 팁의 끝을 잘라 조심스럽게 현미경 슬라이드에 장착 미디어의 최소한의 볼륨 (30mm 커버 - 미끄럼 30 μl에 대한)와 지방 몸을 전송로를 사용합니다. </li><li> 지방 시체를 밖으로 확산 0.1 mm 직경의 핀을 잡고, 두 니켈 도금 핀 홀더를 사용하십시오. 난소는 지방 신체의 뒷부분 세번째에 위치하고 있습니다. 그들을 둘러싸고 지방 시체는 일반적으로 &#39;꽃&#39;과 같은 모양입니다. 이것은 난소의 위치를​​ 파악하는 데 도움을줍니다. 지방 신체의 나머지 부분에서 &#39;꽃&#39;을 밖으로 해부하다하고 후자를 폐기하십시오. </li><li> 지방 신체 surro을 제거주위 조심스럽게 두 개의 핀을 교차하여 생식선을 unding. 멀리 지방 바디 잔여물에서 슬라이드에 고립 생식선를 놓습니다. </li><li> 매니큐어와 표지 전표 날인과 함께 표지. </li><li> 공촛점 현미경을 사용하여 직접 시각화. 샘플은 최대 3 주 4 ° C에 저장할 수 있습니다. </li></ol> 6. 대표 결과 우리는 GSCs과 체세포의 틈새를 포함한 체세포의 난소 안에 여러 개의 세포 lineages의 설립에 따라 위의 프로토콜을 사용하고 있습니다. 이러한 목적을 위해 우리는 개발 생식선에있는 다른 세포 유형 간의 구분하는 구체적인 항체와 마커를 사용합니다. 여기 항체의 다른 조합을 물들일이 LL3 난소의 예를 보여줍니다. 그림 1A 함께 틈새 시장의 체세포를 형성 터미널 필라멘트 및 캡 세포 (녹색)를 강조 표시합니다. 그림 1B는 직접 세균 세포 (파란색)에 연락 혼합된 세포 (ICS, 마젠타), 보여줍니다. <img src="/files/ftp_upload/2537/2537fig1.jpg" alt="그림 1" /> 그림 1. LL3 난소. (A) 단클론 항체 1B1 (마젠타)는 체세포와 얼룩 fusome을 PGCs 이내 세포내 organelle을 (화살표) 설명합니다. 향상제 트랩 HH - lacZ (β - galactosidase 방지, 녹색)이 터미널 필라멘트에 표시됩니다. 필라멘트 체세포 캡 세포 (화살촉)의 기지에서 볼 수 있습니다. (B) 1B1 항체 (녹색)은 난소의 모든 체세포를 설명합니다. 안티 바사 (파란색)은 모든 PGCs를 표시합니다. PGCs 직접 혼합된 세포 (ICS, 안티 교통 체증, 마젠타)에게 문의하십시오. 바 (A와 B에 대한) 20 μm의 수 있습니다.

<ol>
	<li>Fuller, M. T., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+and+female+Drosophila+germline+stem+cells:+two+versions+of+immortality.">Male and female Drosophila germline stem cells: two versions of immortality.</a> Science. 316, 402-404 (2007).</li><li>Li, L., Xie, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+niche:+structure+and+function.">Stem cell niche: structure and function.</a> Annual review of cell and developmental biology. 21, 605-631 (2005).</li><li>Chen, D., McKearin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+circuitry+controlling+a+stem+cell+niche.">Gene circuitry controlling a stem cell niche.</a> Curr Biol. 15, 179-184 (2005).</li><li>Kai, T., Spradling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+empty+Drosophila+stem+cell+niche+reactivates+the+proliferation+of+ectopic+cells.">An empty Drosophila stem cell niche reactivates the proliferation of ectopic cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 4633-4638 (2003).</li><li>Kai, T., Spradling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiating+germ+cells+can+revert+into+functional+stem+cells+in+Drosophila+melanogaster+ovaries.">Differentiating germ cells can revert into functional stem cells in Drosophila melanogaster ovaries.</a> Nature. 428, 564-569 (2004).</li><li>Lopez-Onieva, L., Fernandez-Minan, A., Gonzalez-Reyes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jak/Stat+signalling+in+niche+support+cells+regulates+dpp+transcription+to+control+germline+stem+cell+maintenance+in+the+Drosophila+ovary.">Jak/Stat signalling in niche support cells regulates dpp transcription to control germline stem cell maintenance in the Drosophila ovary.</a> Development. 135, 533-540 (2008).</li><li>Song, X., Zhu, C. H., Doan, C., Xie, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+stem+cells+anchored+by+adherens+junctions+in+the+Drosophila+ovary+niches.">Germline stem cells anchored by adherens junctions in the Drosophila ovary niches.</a> Science. 296, 1855-1857 (2002).</li><li>Tazuke, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+germline-specific+gap+junction+protein+required+for+survival+of+differentiating+early+germ+cells.">A germline-specific gap junction protein required for survival of differentiating early germ cells.</a> Development. 129, 2529-2539 (2002).</li><li>Xie, T., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=decapentaplegic+is+essential+for+the+maintenance+and+division+of+germline+stem+cells+in+the+Drosophila+ovary.">decapentaplegic is essential for the maintenance and division of germline stem cells in the Drosophila ovary.</a> Cell. 94, 251-260 (1998).</li><li>Xie, T., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+niche+maintaining+germ+line+stem+cells+in+the+Drosophila+ovary.">A niche maintaining germ line stem cells in the Drosophila ovary.</a> Science. , 290-328 (2000).</li><li>Guo, Z., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glypican+Dally+is+required+in+the+niche+for+the+maintenance+of+germline+stem+cells+and+short-range+BMP+signaling+in+the+Drosophila+ovary.">The glypican Dally is required in the niche for the maintenance of germline stem cells and short-range BMP signaling in the Drosophila ovary.</a> Development. 136, 3627-3635 (2009).</li><li>Wang, X., Harris, R. E., Bayston, L. J., Ashe, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+IV+collagens+regulate+BMP+signalling+in+Drosophila.">Type IV collagens regulate BMP signalling in Drosophila.</a> Nature. 455, 72-77 (2008).</li><li>Godt, D., Laski, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cell+rearrangement+and+cell+recruitment+in+Drosophila+ovary+morphogenesis+and+the+requirement+of+bric+a+brac.">Mechanisms of cell rearrangement and cell recruitment in Drosophila ovary morphogenesis and the requirement of bric a brac.</a> Development. 121, 173-187 (1995).</li><li>Sahut-Barnola, I., Godt, D., Laski, F. A., Couderc, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+ovary+morphogenesis:+analysis+of+terminal+filament+formation+and+identification+of+a+gene+required+for+this+process.">Drosophila ovary morphogenesis: analysis of terminal filament formation and identification of a gene required for this process.</a> Developmental biology. 170, 127-135 (1995).</li><li>Song, X., Call, G. B., Kirilly, D., Xie, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+controls+germline+stem+cell+niche+formation+in+the+Drosophila+ovary.">Notch signaling controls germline stem cell niche formation in the Drosophila ovary.</a> 134, 1071-1080 (2007).</li><li>Zhu, C. H., Xie, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+expansion+of+ovarian+germline+stem+cells+during+niche+formation+in+Drosophila.">Clonal expansion of ovarian germline stem cells during niche formation in Drosophila.</a> Development. 130, 2579-2588 (2003).</li><li>Ward, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+signal+to+the+niche+through+the+Notch+pathway+in+the+Drosophila+ovary.">Stem cells signal to the niche through the Notch pathway in the Drosophila ovary.</a> Curr Biol. 16, 2352-2358 (2006).</li><li>Gilboa, L., Lehmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repression+of+primordial+germ+cell+differentiation+parallels+germ+line+stem+cell+maintenance.">Repression of primordial germ cell differentiation parallels germ line stem cell maintenance.</a> Curr Biol. 14, 981-986 (2004).</li><li>Gilboa, L., Lehmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soma-germline+interactions+coordinate+homeostasis+and+growth+in+the+Drosophila+gonad.">Soma-germline interactions coordinate homeostasis and growth in the Drosophila gonad.</a> Nature. 443, 97-100 (2006).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 비디오는 후반 세번째 instar 애벌레의 난소의 격리 및 얼룩 프로토콜을 보여줍니다. 일상적으로 안정이 프로토콜을 수행하려면, 관심은 다음 사항에 지불되어야합니다 <ol><li> 동기화 및 잘 발달 애벌레의 경우, 이상의 크롤 링은 피해야합니다. </li><li> 작은 반투명 난소의 손실을 피하기 위해 지방 몸이 그대로 해부하다에 있는지 확인하십시오. 이것은 (두 번째 혹은 세 instar의 시작) 이하 난?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>NaCl</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Kcl</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hepes</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Albumin Bovine Fraction V</td>
<td>&nbsp;</td>
<td>MP Biomedicals</td>
<td>160069</td>
<td>&nbsp;</td>
<tr>
<td>Dumont biology tweezers 5 dumstar polished</td>
<td>&nbsp;</td>
<td>FST Fine Science Tools</td>
<td>11295-10</td>
<td>&nbsp;</td>
<tr>
<td>Nickel plated pin holder</td>
<td>&nbsp;</td>
<td>FST Fine Science Tools</td>
<td>26018-17</td>
<td>&nbsp;</td>
<tr>
<td>s.s minutien pins 0.1mm diam, 10mm long</td>
<td>&nbsp;</td>
<td>FST Fine Science Tools</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>9 well plates 85X100 mm, 22mm o.d.x7mm deep</td>
<td>&nbsp;</td>
<td>CORNING</td>
<td>7220-85</td>
<td>&nbsp;</td>
<tr>
<td>Stereo Microscope MZ 16.5 with a standard transmitted light base TL ST</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 well plates</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>3516</td>
<td>&nbsp;</td>
<tr>
<td>Slides</td>
<td>&nbsp;</td>
<td>Menzel Glaser GmbH</td>
<td>798</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>2940-223</td>
<td>&nbsp;</td>
<tr>
<td>Mounting media</td>
<td>&nbsp;</td>
<td>Vectashield</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>FAL352350</td>
<td>&nbsp;</td>
<tr>
<td>1B1 antibody</td>
<td>&nbsp;</td>
<td>Developmental Studies Hybridoma Bank</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Traffic Jam</td>
<td>&nbsp;</td>
<td>Laboratory of Dr. Dorothea Godt</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Vasa</td>
<td>&nbsp;</td>
<td>Laboratory of Dr. Ruth Lehmann</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anti &beta;-Galactosidase</td>
<td>&nbsp;</td>
<td>Cappel</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Secondary Antibodies</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

dissection and staining of drosophila larval ovaries

많은 기관 embryogenesis 동안에 성인 생활 동안 유지 보수 또는 수리를 위해 자신의 개발을위한 줄기 세포에 따라 달라집니다. 그들이 환경과 상호 작용하는 방법에 줄기 세포 형태, 방법을 이해하는 것은 개발, 항상성 및 질병을 이해 그러므로 매우 중요합니다. 과일 파리 Drosophila melanogaster의 난소는 체세포 지원 전지 (틈새) 1, 2와 세균 라인 줄기 세포 (GSCs)의 상호 작용에 대한 영향력 모델로 재직하고 있습니다. 유전자를 조작하는 능력을 결합 틈새와 GSCs의 알려진 위치, 줄기 세포와 그 틈새 3-12 사이의 상호 작용의 다양한 명료하게하다하는 연구자를 허용하고 있습니다. GSC 유지 및 분화를 조절하는 메커니즘에 대한 정보의 풍부한에도 불구하고, 상대적으로 조금은 GSCs 및 체세포의 틈새 시장 개발 중에 양식 방법에 대한 알려져있다. 그의 세포 컴포넌트 단자 필라멘트와 캡 세포 (그림 1), 세 번째 애벌레 instar 13-17 동안 양식을 포함 약 18 체세포의 틈새. GSCs는 원시 배아 세포 (PGCs)에서 발생한. PGCs 일찍 애벌레 단계에서 세포 분열 따위에 의해 번식하지만 틈새의 형성 다음 PGCs의 하급 집단은 GSCs 7, 16, 18, ​​19이됩니다. 함께, 체세포의 틈새 세포와 GSCs은 유기체의 일생 동안 계란을 생산하는 기능 단위를합니다. GSC 단위의 형성에 관한 많은 질문도 답이 남아 있습니다. 이러한 전구체 틈새와 줄기 세포 엽 성의 전구 물질에 대한 세포, 또는 그들이 GSCs가로의가 PGCs 내에서 비​​대칭 세대 간의 조정하는 프로세스는 최고의 유충에서 공부하실 수 있습니다. 그러나 애벌레 난소 개발 방식 연구 물리적으로 도전합니다. 첫째, 애벌레 난소 작은 수 있습니다. 비록 늦은 애벌레 단계에서 그들은 전반에만 100μm 수 있습니다. 또한, 난소 투명하고 하얀 지방이 신체에 박혀있다. 여기 형광 항체와 얼룩 다음 늦은 세번째 instar (LL3) Drosophila의 유충에서 난소를 분리를위한 단계별 절차를 설명합니다. 우리는, 난소의 위치를​​ 침몰하지 티슈 얼룩 및 세척하고, 항체가 조직에 침투되었는지 확인하는 등의 문제 몇 가지 기술적인 솔루션을 제공합니다. 이 프로토콜은 이전의 애벌레 단계와뿐만 아니라 애벌레 testes에 적용할 수 있습니다.

Watch this Scientific Journal Video about Dissection and Staining of Drosophila Larval Ovaries at JoVE.com

Dissection and Staining of Drosophila Larval Ovaries

틈새와 줄기 세포를 개발하는 동안 양식 얼마나 실용적인 의미가있는 중요한 질문이다. 에<em&gt; Drosophila</em&gt; 난소 배아 라인 줄기 세포 및 체세포의 틈새는 애벌레 개발하는 동안 형식입니다. 이 동영상은 늦은 세번째 instar (LL3)에서 여성 생식선을, 해부하다 얼룩 및 마운트하는 방법을 보여줍니다<em&gt; Drosophila</em&gt; 애벌레.

해부 그리고 스테 이닝 Drosophila 애벌레 난소

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

dissection-and-staining-of-drosophila-larval-ovaries

Research

JoVE Journal

Biology

Dissection and Staining of Drosophila Larval Ovaries

26.0K 조회수.  Weizmann Institute of Science. 틈새와 줄기 세포를 개발하는 동안 양식 얼마나 실용적인 의미가있는 중요한 질문이다. 에 Drosophila 난소 배아 라인 줄기 세포 및 체세포의 틈새는 애벌레 개발하는 동안 형식입니다. 이 동영상은 늦은 세번째 instar (LL3)에서 여성 생식선을, 해부하다 얼룩 및 마운트하는 방법을 보여줍니다 Drosophila 애벌레.

비디오: Dissection and Staining of Drosophila Larval Ovaries

Dissection of Drosophila Ovaries

Drosophila 난소의 해부

연구

생물학

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Drosophila Melanogaster에서 애벌레 CNS의 해부

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

Drosophila 애벌레 NMJ의 해부

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

Drosophila 애벌레 NMJ Immunohistochemistry

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

라이브 해부 Drosophila 태아 : 항체 스테 이닝에 의해 돌연변이 컬렉션을 검사 늘씬한 방법

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

애벌레의 심장 해부, 소년과 성인 Zebrafish, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

늦게 단계 배아와 애벌레의 초음파 - 촉진 면역 형광 염색<em&gt; 초파리</em&gt; 조직<em&gt; 제자리에서</em

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

해부하고, 상상 디스크의 면역 염색에서<em&gt; 초파리</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

해부와의 설치<em&gt; 초파리</em&gt; 번데기 아이 디스크

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

에서 애벌레 활동의 측정<em&gt; 초파리</em&gt; 활동 모니터

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...