저자는 지적 지원, 물자 지원, 종목 아키라 치바을 인정하고 싶습니다. 덧글에 대한 줄리아 달만에 감사합니다.

1. 일 플라이 <ol><li> 실내 온도 10에서 표준 옥수수 가루 한천-당밀 효모 매체에 파리를 유지한다. </li><li> 십자가를 들어, 우화의 6 시간 내 처녀를 분리. 원하는 유전자형의 남성을 횡단 한 후, 변경 사항은 모든 3-4일 새로운 유리 병에 파리. </li></ol> 주 : 이들 실험의 경우, 인 Gal4 라인 A9는 날개에서 도입 유전자의 발현을 구동하는 데 사용 하였다. 플라이 주식 블루밍턴 주식 센터에서 얻을 수 있습니다. 이 실험에 사용 된 주식은 A9-인 Gal4 (BL 번호 8761), His2Av-GFP (BL 번호 5941), SCO는 / CYO HsCre (BL 번호 1092), UAS - ChRFP - 욕조 (BL 번호 25773), 11 lollibow 있습니다. 2. 선택 및 번데기의 장착 <ol><li> 무대 번데기 하나 흰색 prepupae (WPP)로 수집하고 적절한 연령에 도달 할 때까지 25 ° C에서 그들을 유지하거나 형태 학적 기준 (12)을 사용하여. </li><li> 세포 분열, 최근 머리 외전을받은 선택 번데기를 관찰하십시오.이 시점에서 때까지 그냥 우화 (에서&gt; 96 시간) 전에, 번데기는 움직이지 장기간 경과 관찰을 허용하고 있습니다. </li><li> 먼저 물을 붓에 그들을 괴롭히는 부드럽게 한 후 접착제를 느슨하게 할 수 있도록 잠시 대기, 물에 적신 붓으로 터치하여 유리 병에서 번데기를 제거합니다. </li><li> 부드럽게 침샘 접착제와 음식 입자를 제거하기 위해 물에 붓으로 그들을 괴롭히는 선택한 번데기를 씻어. </li><li> 커버 슬립 바닥 (번호 1 ½ 커버 슬립)와 25mm 페트리 접시에 깨끗한 번데기를 전송합니다. </li><li> 그 조직이 coverslip에 가장 가까운 수 있도록 지원 등의 점토 또는 치과 왁스 하나의 얇은 스트립을 사용하여, 번데기를 탑재합니다. 참고 : 여기에 설명 된 연구에서, 번데기 날개, 다리, 복부 histoblasts 또는 지느러미 notum이 관찰되었다. 실제로, 번데기 케이스의 표면의 20 μM 내의 조직 관찰 가능하다. </li><li> 동양 번데기주의 깊게 있도록 Tissu은또한 E는 유리 커버 슬립의 표면에 평행하다. </li><li> 번데기가 장착되면, 번데기 및 커버 슬립 사이의 공간에 티 오디 글리콜 (TDG)의 얇은 층을 전송하기 위해 붓을 사용합니다. </li></ol> 참고 : TDG는, 표면 산란을 감소 오일의 굴절률과 일치하고, 산소가 조직 (13)을 투과 할 수있다. 그들은 세포 행동의 빠른 중단의 원인이 산소의 조직을 박탈하는 경향 오일의 사용은 권장되지 않습니다. 3. 영상 참고 : 영상의 경우, 공 초점 현미경 confocality가 번데기 케이스의 강렬한 조명에 의한 어둡게 배경의 대부분을 제거 필수 될 가능성이 높습니다. <ol><li> 번데기 개발과 생존에 대한 조명의 영향을 최소화하기 위해 공 초점에 대한 설정을 조정합니다. 그렇게하려면, 여진 강도 및 컬렉션의 감도 간의 균형. 영상 위스콘신의 일반적인 구성라이카 SP5는 공진 스캐너 (8,000 Hz에서), 2 %로 설정 레이저 파워 (20 % 전력의 10 % 투과율), 120 μm의 작은 구멍 세트, 라인이 8로 설정 평균을 사용하는 일을. 설정 실험 조건에 따라 달라질 수 있습니다. </li></ol> 참고 : 미세 규모가 초점 심도를 제한하지만 성공적으로,이 방법에 사용 된 40X 및 63X 기름 침지 렌즈 (0.1 mm 작동 거리와) 같은 다양한 해결하려면. 4. 분석 프레임을 분석하기 위해, Z-스택이나 영화보기, 측정, 및 프리젠 테이션 (14) 파일을 수정하기위한 효과적인 도구가 피지에 데이터 파일을 가져옵니다. 예를 들어 유사 분열을 완료하는 시간은 telophase 할 의향로부터 셀을 보면서 단계별로 프레임 샘플링 간격 및 다차원 이미지 브라우저를 사용하여 측정 하였다. x-, Y-및 Z-치수 측정 도구를 사용하여 측정 할 수있다. 소프트웨어와 그 사용에 대한 자세한 지침은참조 : http://fiji.sc/Fiji을.

<ol>
	<li>Raff, J. W., Jeffers, K., Huang, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+Fzy/Cdc20+and+Fzr/Cdh1+in+regulating+the+destruction+of+cyclin+B+in+space+and+time.">The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the destruction of cyclin B in space and time.</a> J. Cell Biol. 157, 1139-1149 (2002).</li><li>Stramer, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+wound+inflammation+in+Drosophila+embryos+reveals+key+roles+for+small+GTPases+during+in+vivo+cell+migration.">Live imaging of wound inflammation in Drosophila embryos reveals key roles for small GTPases during in vivo cell migration.</a> J. Cell Biol. 168, 567-573 (2005).</li><li>Clark, I. B., Jarman, A. P., Finnegan, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+Drosophila+gonad+formation+reveals+roles+for+Six4+in+regulating+germline+and+somatic+cell+migration.">Live imaging of Drosophila gonad formation reveals roles for Six4 in regulating germline and somatic cell migration.</a> BMC Dev. Biol. 7, 52 (2007).</li><li>Fuger, P., Behrends, L. B., Mertel, S., Sigrist, S. J., Rasse, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+synapse+development+and+measuring+protein+dynamics+using+two-color+fluorescence+recovery+after+photo-bleaching+at+Drosophila+synapses.">Live imaging of synapse development and measuring protein dynamics using two-color fluorescence recovery after photo-bleaching at Drosophila synapses.</a> Nat. Protoc. 2, 3285-3298 (2007).</li><li>Siller, K. H., Serr, M., Steward, R., Hays, T. S., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+Drosophila+brain+neuroblasts+reveals+a+role+for+Lis1/dynactin+in+spindle+assembly+and+mitotic+checkpoint+control.">Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/dynactin in spindle assembly and mitotic checkpoint control.</a> Mol. Biol. Cell. 16, 5127-5140 (2005).</li><li>Roy, S., Hsiung, F., Kornberg, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+Drosophila+cytonemes+for+distinct+signaling+pathways.">Specificity of Drosophila cytonemes for distinct signaling pathways.</a> Science. 332, 354-358 (2011).</li><li>Gho, M., Bellaiche, Y., Schweisguth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+the+Drosophila+microchaete+lineage:+a+novel+intrinsically+asymmetric+cell+division+generates+a+glial+cell.">Revisiting the Drosophila microchaete lineage: a novel intrinsically asymmetric cell division generates a glial cell.</a> Development. 126, 3573-3584 (1999).</li><li>Ninov, N., Martin-Blanco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+epidermal+morphogenesis+during+the+development+of+the+adult+abdominal+epidermis+of+Drosophila.">Live imaging of epidermal morphogenesis during the development of the adult abdominal epidermis of Drosophila.</a> Nat. Protoc. 2, 3074-3080 (2007).</li><li>Edgar, B. A., Lehner, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+control+of+cell+cycle+regulators:+a+fly's+perspective.">Developmental control of cell cycle regulators: a fly's perspective.</a> Science. 274, 1646-1652 (1996).</li><li>Wirtz, R. A., Semey, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+kitchen:+equipment,+media+preparation,+and+supplies.">The Drosophila kitchen: equipment, media preparation, and supplies.</a> Drosophila Information Service. 58, 176-180 (1982).</li><li>Boulina, M., Samarajeewa, H., Baker, J. D., Kim, M. D., Chiba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+multicolor-labeled+cells+in+Drosophila.">Live imaging of multicolor-labeled cells in Drosophila.</a> Development. 140, 1605-1613 (2013).</li><li>Bainbridge, S. P., Bownes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staging+the+metamorphosis+of+Drosophila+melanogaster.">Staging the metamorphosis of Drosophila melanogaster.</a> J. Embryol. Exp. Morphol. 66, 57-80 (1981).</li><li>Staudt, T., Lang, M. C., Medda, R., Engelhardt, J., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2,2'-thiodiethanol:+a+new+water+soluble+mounting+medium+for+high+resolution+optical+microscopy.">2,2'-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy.</a> Microsc. Res. Tech. 70, 1-9 (2007).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat. Methods. 9, 676-682 (2012).</li><li>Meyer, E. J., Ikmi, A., Gibson, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interkinetic+nuclear+migration+is+a+broadly+conserved+feature+of+cell+division+in+pseudostratified+epithelia.">Interkinetic nuclear migration is a broadly conserved feature of cell division in pseudostratified epithelia.</a> Curr. Biol. 21, 485-491 (2011).</li><li>Sauer, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis+in+the+neural+tube.">Mitosis in the neural tube.</a> J. Comp. Neurol. 62, 377-405 (1935).</li><li>Livet, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+strategies+for+combinatorial+expression+of+fluorescent+proteins+in+the+nervous+system.">Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.</a> Nature. 450, 56-62 (2007).</li></ol>

저자가 공개하는 게 없다.

분할 세포의 기능, His2AvGFP 발현 세포의 공 촛점 분석을 통해 초파리의 살아있는 번데기 날개의 유사 분열을 관찰하기위한 간단한 준비에 필요한 개발을 시각화, 측정, 정량화한다. 이 방법은 번데기 날개의 세포주기가 유사 분열을 입력 상피 세포의 꼭대기의 표면에 그 핵의 움직임에 pseudostratified 상피 세포의주기를 셀에 강한 유사성을 맺는 것을 문서화하는 데 사용되었다. telophase에 따라...

<html> 식초 파리, 초파리 melanogaster의는. 초파리 연구를 표현, 최저 돌연변이 등의 유전자 조작의 세련된 형태를 허용 유전자 실험의 풍부한 역사를 가지고 생물학의 여러 측면을 연구하기위한 기초가 튼튼한 모델입니다. 형광 단백질 라벨의 도래와 함께,이 레퍼토리는 살아있는 동물의 세포와 단백질의 연구를 포함하도록 확장했다. 플라이 배아는 생체 내 1-3 깊은, 고해상도 영상을 허용 작은 광학적으로 알 수있는 바와 같이 이러한 연구를위한 훌륭한 시스템이다. 플라이 개발의 다른 단계 진통제 4, 해부 및 단기 문화 5,6, 또는 영상 7,8의 표피에있는 윈도우의 생성을 필요로하는, 덜 다루기 쉬운적일 수있다. 이러한 조작은 보통 장기 동물 발달을 손상 또는 짧은 기간에 촬상을 제한 방식으로 동물에 영향을 미친다. 대부분의 배아 줄기 세포주기가 절단되기 때문에 세포주기 규제 유사 유전자의 새로운 변이를 연구하는 엔트 &quot;&gt;, 그것은. 세포주기의 타이밍과 충실도를 연구하는 적절한 준비를 찾을 수 필수적이었다 (SM 또는 S-G2-M) 및 연구에서 돌연변이는 이후 단계까지, 그것은 번데기 조직에서 세포주기를 관찰하는 것이 중요하다고 결함을 표시하지 않습니다. 번데기에있는 상피 세포가 전형적인 G1-S-G2-M 세포주기와이 단계의 번데기가이 근육의 움직임 9 할 수 없다. 조작에 대한 초기 시작 지점 번데기 경우의 명백한 불투명도에도 불구하고 Histone2AV-GFP를 표현 그대로 전체 번데기를. 포함이 그대로 준비가 생체 내 이미징에서 장기적으로 우수한 것으로 판명.이 기술은 간단합니다 학부 연구원이 정기적으로 초파리에서 세포 및 발달 생물학의 측면을 연구하는 데 사용할 아직 해상도가 마이크로 미터 스케일 기능의 차별을 할 수 있도록 충분히 괜찮 충분히. 이 방법에서는, 시간, 분, 또는 초 이상의 이벤트의 관측은 단순히 시계열 파라미터를 조정함으로써 가능하다. 파란색, 녹색, 노란색, 오렌지색과 적색 형광 단백질, 또는 이들의 조합을 사용하여 비디오,되었습니다. 중요한 경우, 치료조차 장기간 이미징 동물의 개발이나 생존에 영향이 없으며 레이저의 강도를 최소화하도록 수행된다.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fly stuff fly pad</td>
			<td style="width:71px;">Genesee scientific</td>
			<td style="width:119px;">59-114</td>
			<td style="width:167px;">for fly anesthetization</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CO2 gas</td>
			<td style="width:71px;">Airgas south</td>
			<td style="width:119px;">CD50</td>
			<td>For fly anesthetization</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Regulator</td>
			<td style="width:71px;">Airgas south</td>
			<td style="width:119px;"> </td>
			<td>CO2 regulator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fly vials</td>
			<td style="width:71px;">Genesee scientific</td>
			<td>32-113RLBF</td>
			<td>Fly culture</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Drosophila lines:A9-Gal4 (Bl#8761), His2Av-GFP (Bl#5941), Sco/CyO HsCre (Bl#1092), UAS-ChRFP-Tub (Bl#25773)</td>
			<td style="width:71px;">Bloomington Stock center</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass bottom dishes #1 1/2</td>
			<td style="width:71px;">WillCo Wells BV</td>
			<td style="width:119px;"> </td>
			<td>For microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thiodiethylene Glycol</td>
			<td style="width:71px;">Fluka</td>
			<td align="right" style="width:119px;">88559</td>
			<td>mountant</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Modeling clay</td>
			<td style="width:71px;">art supply store</td>
			<td style="width:119px;"> </td>
			<td>Support to position pupae against</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Paintbrushes</td>
			<td style="width:71px;">art supply store</td>
			<td style="width:119px;"> </td>
			<td>To manipulate flies</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fine Forceps, Inox #5</td>
			<td style="width:71px;">Fine science tools</td>
			<td style="width:119px;">11252-20</td>
			<td>Dumont #5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">computer</td>
			<td style="width:71px;">any</td>
			<td style="width:119px;"> </td>
			<td>8 Gb RAM for image/movie analysis</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">Fiji software</td>
			<td style="width:71px;"> </td>
			<td style="width:119px;">Free ware http://fiji.sc/Fiji</td>
			<td>Image analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Confocal microscope</td>
			<td style="width:71px;"> </td>
			<td style="width:119px;"> </td>
			<td>Any fast scanning confocal should be sufficient</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">20x dry, and 40x or 63x oil immersion lenses</td>
			<td style="width:71px;">any</td>
			<td style="width:119px;"> </td>
			<td>For imaging tissue, cellular and subcellular features</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immersion oil (non fluorescent)</td>
			<td style="width:71px;"> </td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo microscope</td>
			<td style="width:71px;">any</td>
			<td style="width:119px;"> </td>
			<td>For fly manipulation</td>
		</tr>
	</tbody>
</table>

imaging through the pupal case of drosophila melanogaster

세포 및 발달 생물학 모델로 초파리의 오랜 사용 도구의 배열을 굴복했다. 함께, 이러한 기술은 방법 론적 다양한 각도에서 세포 및 발달 생물학의 분석을 사용할 수있다. 라이브 영상은 세포 분열 또는 세포 운동성 동적 셀 프로세스를 관찰하기위한 새로운 방법이다. 그것은 실시간 이미징을 사용하여 반응계 내에서 유사 분열을 관찰하는 것이 필수적이되었다에 uncharacterized 추정 세포주기 단백질에 돌연변이를 갖는 격리. 초파리에있는 대부분의 라이브 영상 연구 때문에 그들의 작은 크기와 광학 선명도의 조작과 관찰에 액세스 할 수있는 배아 단계에 초점을 맞추고있다. 이 간극 단계 중 하나 또는 모두를 결여에 그러나, 이들 단계에서 세포주기가 드물다. 대조적으로, 초파리의 번데기 날개의 세포는 전형적인 세포주기를 가지고 번데기 전개 약 20 시간에 걸쳐 급격한 유사 분열주기를 겪는다. 내가 용이dentify 및 현장에서 유사 분열을 잡을 수있는 적절한 단계의 번데기를 분리. 설치 그대로 번데기 실험은 세포와 동물 생존에 미치는 영향을 최소화하면서 몇 시간 동안 실행할 수 있도록 이미징 동안 취급 용이성 및 내구성의 최상의 조합을 제공했다. 이 방법은 작은, 또는보다 작은 염색체를 비행 기능을 관찰 할 수 있습니다. 현미경 설정 및 설치의 세부 사항 조정, 준비의 확장을 막 인접 셀의 역학과 같은 튜 불린과 같은 형광 표지 단백질을 시각화 할 수있었습니다. 이 방법은 모든 시험 형광 단백질을 작동 시간 규모의 다양한 통해 서브 마이크론 규모의 기능을 캡처 할 수 있습니다. 기존의 공 초점 현미경으로 번데기의 외부 20 μm의 제한하지만, 생체 내에서 번데기 조직에서 단백질과 세포 역학을 관찰하는이 방법은 이러한 조직에서 세포 및 발달 생물학의 연구에서 일반적으로 유용 할 수 있습니다.

이러한 현상 초파리 눈 또는 현상 척추 중추 신경계의 심실 층으로서 pseudostratified 상피 세포는, 핵 운동을 겪는, 세포주기에 맞춰, interkinetic 핵 옮기는 칭했다. 핵은 기저 표면 또는 근처에와 핵이 혀끝의 표면 (15, 16)에 도달하면 세포가 유사 분열을 입력 할 때 DNA 복제가 발생합니다. 번데기 날개 세포는 머리 외전 후 첫 몇 시간 동안 빠른 속도로 나누어 단층 상피 세포를 형성한다....

Watch this Scientific Journal Video about Imaging Through the Pupal Case of Drosophila melanogaster at JoVE.com

Imaging Through the Pupal Case of Drosophila melanogaster

이 논문은 직접 번데기를 통해 이미지 세포 행동에 빠른 스캐닝 공 초점 현미경을 사용하는 방법을 보여줍니다. 그대로 번데기 경우를 떠남으로서,이 방법은 직접 공부하기 어려운 초파리 개발의 단계에서 관찰하고 역동적 인 세포 프로세스의 측정을 할 수 있습니다.

의 번데기 케이스를 통해서 영상<em&gt; 초파리</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

imaging-through-the-pupal-case-of-drosophila-melanogaster

Research

JoVE Journal

Biology

Imaging Through the Pupal Case of Drosophila melanogaster

13.2K 조회수.  University of Miami.  이 논문은 직접 번데기를 통해 이미지 세포 행동에 빠른 스캐닝 공 초점 현미경을 사용하는 방법을 보여줍니다. 그대로 번데기 경우를 떠남으로서,이 방법은 직접 공부하기 어려운 초파리 개발의 단계에서 관찰하고 역동적 인 세포 프로세스의 측정을 할 수 있습니다. 

비디오: Imaging Through the Pupal Case of Drosophila melanogaster

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 ...

연구

생물학

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

라이브 영상 Drosophila melanogaster 배아 Hemocyte 마이 그 레이션

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

의 입식 이미징 Drosophila 태아

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

성인에서 Oenocytes의 해부 Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

의 분리 Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

Drosophila Pupal의 복부 Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

의 수명 측정<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

해부, 이미징 및 약물 학적 치료 : 초파리의 번데기 고환 및 단일 남성 생식 줄 낭종의 생체 내 문화

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

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 ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

(A)의 유지 보수<em&gt; 노랑 초파리</em&gt; 인구 케이지

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...