우리는 S. Taneja - Bageshwar, K. Beifuss, S. Kedroske 및 HC 감사하고 싶습니다. 도움이 토론 샤오. 이 작품은 분자 및 세포 의학 TAMHSC학과에서 자금 업을 시작하여 기금을했다. 화합물 범위 및 회전 디스크는 부서와 학장의 TAMHSC 사무소에서 제공하는 자금을 구입했다. 일부 변종은 연구 자원을위한 국립 센터의 후원됩니다 Caenorhabditis 유전학 센터에 의해 제공되었다. pRF4 (rol - 6 (su1006)은) A. 화재의 선물했다.

1. DiI의 준비 얼룩 <ol><li> DMF 20 밀리그램 / ML DiI (Biotium, 주식 회사, 헤이워드, CA)의 주식 솔루션을 준비합니다. DiI가 민감한 빛이기 때문에 호일에 포장하여 빛으로부터 DiI를 보호합니다. </li><li> 각 인구 399.4 μL M9 0.6 μL DiI 주식을 추가하여 DiI의 작업 희석를 만듭니다. 이것은 30 μg / M9에 ML DiI의 최종 작업 희석를 제공해야합니다. 이것은 동시에 여러 인구를 얼룩에 확장할 수 있습니다. 호일에 튜브 (들)을 포장하여 빛으로부터 쉴드 DiI. </li></ol> 2. nematodes의 준비 <ol><li> 오염 nematodes의 인구를 포함하는 60mm 플레이트를 사용하십시오. 부드럽게 모든 애벌레와 성인 동물을 풀어야 접시의 표면에 걸쳐 원형 운동에 액체를 소용돌이로 M9 버퍼에서 0.5 % 트라 이튼의 솔루션 X - 100을 사용하는 접시에서 동물을 씻으십시오. 멸균 1.5 ML 튜브로 세척을 전송합니다. </li><li> 즉시 30 초 2000 RPM에서 동물을 돌린다. 많이 먹어 볼래요 제거하고 폐기튜브의 하단에있는 동물의 질량을 방해하지 않고 가능한 ernatant. </li><li> 잔여 트리톤 X - 100을 줄이기 위해, M9 버퍼, 스핀을 이용하여 동물 린스, 그리고 뜨는 제거합니다. 일단이 단계를 반복합니다. </li><li> 솔루션에서 동물 resuspend 수있는 튜브 및 와류 간략하게하기 위해 M9에서 일하는 DiI 솔루션 400 μL를 추가합니다. </li><li> 20 튜브를 흔들어 ° C 수평 가벼운 보호된 환경에서 350 rpm으로 3 시간 동안. 원하는 경우, 동물 얼룩 16 시간까지 incubated 수 있습니다. </li><li> 언바운드 색소의 양을 줄이기 위해 20 초 2000 RPM에서 동물을 돌린다. 제거하고 동물의 질량을 방해하지 않고 가능한 한 많은 뜨는으로 폐기하십시오. </li><li> 400 μL M9 버퍼와 OP50 E.와 씨앗 NGM 한천 플레이트의 박테리아가없는 부분에 액체를 부어 Resuspend 동물 대장균. 적어도 30 분 복구하는 동물을 허용합니다. 복구 기간 동안 동물 DiI의 얼룩 액체로부터 식품에 떨어져 크롤 링해야합니다. 이 단계는무료 DiI에서 배경 형광을 줄일 수 있습니다. </li></ol> 3. 표본을 장착하고 관찰 <ol><li> 압력솥이나 전자 레인지를 사용하여 물에 4 % 한천을 용융. </li><li> 유리 슬라이드에 실험실 테이프 겹겹이 두 가지로, 한천 패드의 균일한 두께를 보장하는 데 사용할 수있는 재사용 가능한 스페이서를 만듭니다. 이 스페이서 슬라이드 총하십시오. </li><li> 이 스페이서 슬라이드 사이에 깨끗한 유리 슬라이드를 마련. 깨끗한 유리 슬라이드의 중심에 용융 4 % 한천의 피펫 약 150 μL (4 방울). 신속 한천 패드를 양식에 추가 슬라이드를 사용하여 용융 한천을 커버. 조심스럽게 장착 슬라이드의 상단을 중심으로 패드를 유지, 커버 슬라이드를 제거합니다. </li><li> 피펫 선충류 마취 (100 μm의 - 1 ㎜의 levamisole, 예를 들어) 5 μL에 대한 패드에. </li><li> 마운트 8-12 마취의 동물과 현미경 coverslip로 커버. </li><li> 적어도 40x objecti 장착되어 화합물이나 공촛점 현미경을 사용하여 동물을 관찰<html> 적 및 TRITC / DSRed (또는 다른 호환) 필터. DiI의 형광 여기 최대 549 NM이며 방사 최대 행 염료 (Biotium, 주식 회사, 헤이워드, CA)에 565 nm의 수 있습니다. </li></ol> 4. 대표 결과 야생 유형과 돌연변 C.의 DiI 얼룩은 표피 elegans. cuticular 표면 annuli 어떤 단계에서 원주 고랑로 구분하여 포함하고, 세로 능선이 alae했다. 각 발달 단계는 뚜렷한 작곡 2 cuticular 구조를하고 있습니다. alae와 annuli 모두 산등성이 또는 고랑이 찬란 애벌레와 성인 단계에 걸쳐, 표면 구성에 따라, 얼룩 및 최대이 방법을 사용하여 복구 후 하루에 보이는 남아 있습니다. 배경 형광 speckles 때로는 (그림 2 층 G) 관찰 아니지만 정기적으로 (그림 1, 그림 2A - E, H, I). 아르 모든 이미지는 언급 스피닝 디스크 공촛점거나, widefield (WF) 복합 현미경과 함께 찍은 사진. 엔트 &quot;&gt; <img alt="그림 1" src="/files/ftp_upload/3362/3362fig1.jpg" /> 그림 1. DiI는 얼룩 표피하고 환경 노출 뉴런. L2 단계 동물. 630x 확대. 모자이크 이미지 부품은 iVision - 맥 소프트웨어 (BioVision 기술, Exton, PA)를 사용하여 점령했다. 이미지는 어도비 포토샵 CS3 (어도비 시스템즈, 산호세, CA)를 사용하여 합류했다. 스케일 바 = 10 μm의. DiI도 찬란 얼룩 amphid하고 머리와 꼬리에 선충 감각 뉴런 각각 (화살표가 일부 표시). <img alt="그림 2" src="/files/ftp_upload/3362/3362fig2.jpg" /> 그림 2. DiI 찬란 얼룩 C. 이후 배아 발달의 모든 단계에서 elegans 표피. 630x 확대. 스케일 바 = 10 μm의. A) L1에서 야생 타입 동물의 얼룩, B) L2, C) dauer, D) L3, E) L4 및 F) 성인 단계. alae과 환상 산등성이가 찬란 (A, C) L1과 dauer 동물에 얼룩이 있습니다. L2 동물의 DiI는 얼룩 환상 산등성이를(B). 환상은 L3 및 L4 동물 (D, E)에 얼룩 고랑. alae의 고랑과 annuli는 성인 동물 (FH)에 얼룩이 있습니다. Alae는 동물의 길이 (화살표) 16를 실행하는 두 다섯, 또는 세 산등성이 (각각 L1, dauer, 또는 성인 동물에)의 구성됩니다. Annuli는 동물 주위 원주 산등성이 (화살촉, F와 J)를 만들 수 있습니다. 성인 돌연변이 동물의 표피는 적당한 cuticular 조직 결함 (GH)를 표시합니다. G) alae의 산등성이가 (WF) 불연속 있습니다. H) 정원 외의 alae의 산등성이는 융합과 분기 또는 (WF) bifurcated 있습니다. 콜라겐 유전자 돌연변이 alae와 환상 조직 결함 (IJ)를 나타냅니다. I) 유전자 변형 동물에 overexpressing pRF4 (rol - 6 (su1006)), 동물의 길이 각도로 alae의 거짓말의 산등성이. 유전자 변형 동물 overexpressing pRF4 (rol - 6 (su1006))에 J) Annuli이 불규칙 패턴을 표시합니다. <img alt="그림 3" src="/files/ftp_upload/3362/3362fig3.jpg" /> 그림 3. 통근자알 형태학의 구조 DiI의 얼룩에 의해 불이됩니다. 630x 배율은. 스케일 바 = 10 μm의. DiI 또한 A) 성인 남녀 추니 외음부, B) 성인 남성 꼬리 광선 및 팬 등 다른 외부 기능을 강조하고, C) 남녀 추니 꼬리 스파이크 (이 돌연변이 배경에 갈래). <img alt="그림 4" src="/files/ftp_upload/3362/3362fig4.jpg" /> 그림 4. 표피는 환경 노출된 뉴런보다 DiI와 얼룩이 오래 걸립니다. 630x 확대. 스케일 바 = 10 μm의. 얼룩 두 시간 후, amphid (표시되지 않음) 및 선충 감각 뉴런은 (화살표) 충분히 스테인드 수 있습니다. 대조적으로, 젊은 동물의 표피는 일부 패치 (화살촉)에 얼룩 있습니다. <table border="1"><tbody><tr><td> 빨래 </td><td> 얼룩 솔루션 (+ DiI) </td><td> 배양 시간 </td><td> 표피 자국 </td></tr><tr><td> M9 + 0.5 % TRiton X - 100 </td><td> M9 + 0.5 % 트리톤 X - 100 </td><td> 2 시간 </td><td> 아니 </td></tr><tr><td> M9 + 0.5 % 트리톤 X - 100 </td><td> M9 + 0.5 % 트리톤 X - 100 </td><td> 3 시간 </td><td> 아니 </td></tr><tr><td> M9 + 0.5 % 트리톤 X - 100 </td><td> M9 </td><td> 2 시간 </td><td> 일부의 </td></tr><tr><td> M9 + 0.5 % 트리톤 X - 100 </td><td> M9 </td><td> 3 시간 </td><td> 예 </td></tr><tr><td> M9 + 0.5 % 트리톤 X - 100 </td><td> H 2 0 </td><td> 2 시간 </td><td> 일부의 </td></tr><tr><td> M9 + 0.5 % 트리톤 X - 100 </td><td> H 2 0 </td><td> 3 시간 </td><td> 예 </td></tr></tbody></table> 표 1. 다른 조건 하에서 Cuticular 얼룩. 다양한 인큐베이션 솔루션 및 시간은 동물 cuticular 얼룩을 최적화하기 위해 테스트되었습니다. H 2 0, 멸균 증류수. 부분 얼룩하지만 성인 표피의 얼룩 C, 애벌레의 표피의 곳곳에 얼룩 (그림 4)을 나타냅니다onsistently.

<ol>
	<li>Cox, G. N., Kusch, M., Edgar, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cuticle+of+Caenorhabditis+elegans:+its+isolation+and+partial+characterization.">Cuticle of Caenorhabditis elegans: its isolation and partial characterization.</a> The Journal of Cell Biology. 90, 7-17 (1981).</li><li>Cox, G. N., Staprans, S., Edgar, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cuticle+of+Caenorhabditis+elegans.+II.+Stage-specific+changes+in+ultrastructure+and+protein+composition+during+postembryonic+development.">The cuticle of Caenorhabditis elegans. II. Stage-specific changes in ultrastructure and protein composition during postembryonic development.</a> Dev. Biol. 86, 456-470 (1981).</li><li>Hall, D., Altun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans Atlas. , (2008).</li><li>Page, A. P., Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cuticle.">The cuticle.</a> The C. elegans Research Community. , (2007).</li><li>Kramer, J. M., Johnson, J. J., Edgar, R. S., Basch, C., Roberts, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sqt-1+gene+of+C.+elegans+encodes+a+collagen+critical+for+organismal+morphogenesis.">The sqt-1 gene of C. elegans encodes a collagen critical for organismal morphogenesis.</a> Cell. 55, 555-565 (1988).</li><li>Mende, N. v. o. n., Bird, D. M., Albert, P. S., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dpy-13:+a+nematode+collagen+gene+that+affects+body+shape.">dpy-13: a nematode collagen gene that affects body shape.</a> Cell. 55, 567-576 (1988).</li><li>Blaxter, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cuticle+surface+proteins+of+wild+type+and+mutant+Caenorhabditis+elegans.">Cuticle surface proteins of wild type and mutant Caenorhabditis elegans.</a> The Journal of Biological Chemistry. 268, 6600-6609 (1993).</li><li>Costa, M., Draper, B. W., Priess, J. R. <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+actin+filaments+in+patterning+the+Caenorhabditis+elegans+cuticle.">The role of actin filaments in patterning the Caenorhabditis elegans cuticle.</a> Dev. Biol. 184, 373-384 (1997).</li><li>Sapio, M. R., Hilliard, M. A., Cermola, M., Favre, R., Bazzicalupo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zona+Pellucida+domain+containing+proteins,+CUT-1,+CUT-3+and+CUT-5,+play+essential+roles+in+the+development+of+the+larval+alae+in+Caenorhabditis+elegans.">The Zona Pellucida domain containing proteins, CUT-1, CUT-3 and CUT-5, play essential roles in the development of the larval alae in Caenorhabditis elegans.</a> Dev. Biol.. 282, 231-245 (2005).</li><li>Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cuticle+collagen+genes.+Expression+in+Caenorhabditis+elegans.">Cuticle collagen genes. Expression in Caenorhabditis elegans.</a> Trends Genet. 16, 21-27 (2000).</li><li>Kramer, J. M., French, R. P., Park, E. C., Johnson, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+rol-6+gene,+which+interacts+with+the+sqt-1+collagen+gene+to+determine+organismal+morphology,+encodes+a+collagen.">The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen.</a> Mol. Cell Biol. 10, 2081-2089 (1990).</li><li>Thein, 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=Caenorhabditis+elegans+exoskeleton+collagen+COL-19:+an+adult-specific+marker+for+collagen+modification+and+assembly,+and+the+analysis+of+organismal+morphology.">Caenorhabditis elegans exoskeleton collagen COL-19: an adult-specific marker for collagen modification and assembly, and the analysis of organismal morphology.</a> Dev. Dyn. 226, 523-539 (2003).</li><li>McMahon, L., Muriel, J. M., Roberts, B., Quinn, M., Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+sets+of+interacting+collagens+form+functionally+distinct+substructures+within+a+Caenorhabditis+elegans+extracellular+matrix.">Two sets of interacting collagens form functionally distinct substructures within a Caenorhabditis elegans extracellular matrix.</a> Molecular Biology of the Cell. 14, 1366-1378 (2003).</li><li>Link, C. D., Ehrenfels, C. W., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+expression+of+male+copulatory+bursa+surface+markers+in+Caenorhabditis+elegans.">Mutant expression of male copulatory bursa surface markers in Caenorhabditis elegans.</a> Development. 103, 485-495 (1988).</li><li>Tong, Y. G., Burglin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditions+for+dye-filling+of+sensory+neurons+in+Caenorhabditis+elegans.">Conditions for dye-filling of sensory neurons in Caenorhabditis elegans.</a> J. Neurosci. Methods. 188, 58-61 (2010).</li><li>Singh, R. N., Sulston, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+observations+on+moulting+in+Caenorhabditis+elegans.">Some observations on moulting in Caenorhabditis elegans.</a> Nematologica. 24, 63-71 (1978).</li><li>Collet, J., Spike, C. A., Lundquist, E. A., Shaw, J. E., Herman, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+osm-6,+a+gene+that+affects+sensory+cilium+structure+and+sensory+neuron+function+in+Caenorhabditis+elegans.">Analysis of osm-6, a gene that affects sensory cilium structure and sensory neuron function in Caenorhabditis elegans.</a> Genetics. 148, 187-200 (1998).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li></ol>

우리는 공개 아무것도 없어.

여기에 제시 DiI의 얼룩 방법은 C. elegans의 표피를 시각화하기 위해 상대적으로 신속하고 편리하게 얻을 수 있습니다. 일반적으로 이미지의 환경 노출 감각 뉴런 15,17하는 데 사용하는 방법을 repurposing 및 최적화함으로써, DiI는 찬란 alae와 환상 구조 (그림 1 및 2)뿐만 아니라 외음부, 남성 꼬리, 그리고 남녀 추니 꼬리 스파이크를 모두 얼룩하는 데 사용할 수 있습니다 (그림 3). 우리는 ...

<table border="1"><tbody><tr><td> 시약 </td><td> 동의어 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> DiI </td><td> 1,1 &#39;- Dioctadecyl - 3, 3,3&#39;, 3&#39; - tetramethylindocarbocyanine 퍼클로레이트 </td><td> Biotium 주식 회사 </td><td> 60,010 </td><td> 주식 희석 : DMF 20 밀리그램 / ML 작업 희석 : 30 밀리그램 / ML </td></tr><tr><td> DMF </td><td> Dimethylformamide </td><td> 시그마 - 올드 리치 주식 회사 </td><td> D4551 </td><td></td></tr><tr><td> 소라 고둥 X - 100 </td><td> Octylphenoxypolyethoxyethanol </td><td> VWR 인터내셔널, LLC. </td><td> EM - 9410 </td><td></td></tr><tr><td> M9 </td><td> 22mM KH 2 PO 4 42mM 나 2 HPO 4, 86mM NaCl, 1mM MgSO 4 </td><td></td><td></td><td></td></tr><tr><td> NGM &lt;/ TD&gt; <td> 선충류 성장 매체 </td><td> IPM 과학 주식 회사 </td><td> 11006-501 </td><td> NGM 한천 프로토콜 18 다음 준비할 수 </td></tr><tr><td> 한천 - 한천 </td><td></td><td> EMD 화학 주식 회사 </td><td> 1.01614 </td><td> 물에 4% </td></tr><tr><td> Levamisole </td><td> Levamisole 하이드로 클로라이드 </td><td> 시그마 - 올드 리치 주식 회사 </td><td> 31,742 </td><td> 100 μm의 - 필요에 따라 1 ㎜의 levamisole </td></tr><tr><td> 현미경 슬라이드 </td><td></td><td> VWR 인터내셔널, LLC. </td><td> 16005-106 </td><td></td></tr><tr><td> 현미경 커버 안경 </td><td></td><td> VWR 인터내셔널, LLC. </td><td> 16004-302 </td><td></td></tr><tr><td> 컴파 운드 범위 </td><td></td><td> 칼 자이스 혈구, 주식 회사 </td><td> A1m </td><td> 실험의 필요에 맞게 목표를 사용하여 </td></tr><tr><td> TRITC 또는 기타 호환 필터 </td><td> 및nbsp; </td><td> 채도 기술 주식 회사 </td><td> 49,005 </td><td> 동부 표준시 - DSRed (TRITC/Cy3)는 필터 세트를 sputtered </td></tr></tbody></table>

visualization of caenorhabditis elegans cuticular structures using the lipophilic vital dye dii

C.의 표피 elegans는 동물 1-4 외관을 둘러싸는 매우 강한 구조입니다. 표피는 환경에서 동물을 보호뿐만 아니라 몸 모양을 결정하고 운동성 4-6에서 역할을뿐만 아니라. 표피 세포에 의해 분비 여러 레이어 바깥쪽의 지질 층 7 포함하여 표피를 구성. 표피의 주변 산등성이는 annuli 패턴 동물의 길이라는 개발 8 모든 단계 동안 존재하고 있습니다. Alae는 L1, dauer, 그리고 성인 단계 2,9를 포함하여 발달의 특정 단계 동안 존재하는 세로 능선입니다. cuticular 콜라겐 조직에 영향을 유전자에 돌연변 cuticular 구조와 동물 신체 형태 5,6,10,11을 변경할 수 있습니다. DIC 광학과 복합 현미경을 사용하여 cuticular 이미지가 강조 가능한, 현재 방법이지만 cuticular 구조 fluores 포함센트 transgene 표현 12, 항체 염색법 13, 그리고 전자 현미경 1. 분류 밀 배아 agglutinin (WGA)도 cuticular glycoproteins을 시각화하는 데 사용되었지만 미세한 cuticular 구조 14 해결 제한됩니다. 형광 염료를 사용하여 표면의 얼룩 cuticular을 준수하지만, 세부 15 특징 적이되었습니다. 우리는 살아 C로 표피를 시각화하는 방법을 제시 일반적으로 C로 사용되는 적색 형광 염료 lipophilic DiI를 (1,1 &#39;- dioctadecyl - 3, 3,3&#39;, 퍼클로레이트을 3&#39;이 - tetramethylindocarbocyanine) 사용 elegans elegans은 환경 노출 뉴런을 시각화합니다. DiI의 얼룩에 대한이 최적화된 프로토콜은 고해상도 형광 annuli의 시각화, alae, 외음부, 남성 꼬리, 그리고 C로 남녀 추니 꼬리 증가를위한 간단하고 강력한 방법입니다 elegans.

Watch this Scientific Journal Video about Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI at JoVE.com

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

우리는 사는 표피를 시각화하는 방법을 제시<em&gt; C. elegans</em&gt; 일반적으로 사용되는 적색 형광 염료 lipophilic DiI를 (1,1 &#39;- dioctadecyl - 3, 3,3&#39;, 퍼클로레이트을 3&#39;이 - tetramethylindocarbocyanine) 사용<em&gt; C. elegans</em&gt; 환경 노출된 뉴런을 시각화합니다. 이 최적화된 프로토콜로, alae와 환상 cuticular 구조 DiI로 얼룩진 및 복합 현미경을 사용하여 관찰하고 있습니다.

의 시각화 Caenorhabditis Elegans</emLipophilic 바이탈 염료, DiI를 사용하기> 표피 구조

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

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Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

17.0K 조회수.  Texas A&M University System Health Science Center, College of Medicine. 우리는 사는 표피를 시각화하는 방법을 제시 C. elegans 일반적으로 사용되는 적색 형광 염료 lipophilic DiI를 (1,1 '- dioctadecyl - 3, 3,3', 퍼클로레이트을 3'이 - tetramethylindocarbocyanine) 사용 C. elegans 환경 노출된 뉴런을 시각화합니다. 이 최적화된 프로토콜로, alae와 환상 cuticular 구조 DiI로 얼룩진 및 복합 현미경을 사용하여 관찰하고 있습?...

비디오: Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

측정 Caenorhabditis 엘레간스</em96 웰 플레이트에 Microtiter> 수명

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

연구

생물학

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

기본<em&gt; Caenorhabditis elegans</em&gt; 방법 : 동기화 및 전망

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

사용<em&gt; 예쁜 꼬마 선충</em&gt; 다세포 생물의 단백질 항상성을 연구하는 모델 시스템으로

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

의 재배<em&gt; 예쁜 꼬마 선충</em&gt; 실험실에서 세 가지 차원에서

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

혈관 시각화를위한 DiI 관류<em&gt; 암 브리 스 토마 멕 시카 눔</em

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

예쁜꼬마선충의 세포구조와 세포세포형태를 연구하기 위한 정량적 접근법

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

조직별 샤페론 상호 작용을 위해 검사하기 위해 Caenorhabditis elegans를 사용

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Hand Dissection of Caenorhabditis elegans Intestines

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, aging, immunity, and environmental response. Critical interactions between the C. elegans host and its environment converge within the intestine, where gut microbiota concentrate. Therefore, the ability to isolate intestine tissue away from the rest of the worm is necessary to assess intestine-specific processes. This protocol describes a method for hand dissecting adult C. elegans intestines. The procedure can be performed in fluorescently labeled strains for ease or training purposes. Once the technique is perfected, intestines can be collected from unlabeled worms of any genotype. This microdissection approach allows for the simultaneous capture of host intestinal tissue and gut microbiota, a benefit to many microbiome studies. As such, downstream applications for the intestinal preparations generated by this protocol can include but are not limited to RNA isolation from intestinal cells and DNA isolation from captured microbiota. Overall, hand dissection of C. elegans intestines affords a simple and robust method to investigate critical aspects of intestine biology.

Hand Dissection of Caenorhabditis elegans Intestines

The present protocol describes a procedure for isolating intestines from adult Caenorhabditis elegans nematode worms by hand for input in genomics, proteomics, microbiome, or other assays.

Caenorhabditis elegans 내장의 손 해부

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, ...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

모델 선충 Caenorhabditis elegans의 자연 미생물 총의 분리 및 특성화

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures the interaction of homologous chromosomes is the assembly of the synaptonemal complex (SC) in meiotic prophase I. Even though there is little sequence homology between protein components within the SC among different species, the general structure of the SC has been highly conserved during evolution. In electron micrographs, the SC appears as a tripartite, ladder-like structure composed of lateral elements or axes, transverse filaments, and a central element. 
However, precisely identifying the localization of individual components within the complex by electron microscopy to determine the molecular structure of the SC remains challenging. By contrast, fluorescence microscopy allows for the identification of individual protein components within the complex. However, since the SC is only ~100 nm wide, its substructure cannot be resolved by diffraction-limited conventional fluorescence microscopy. Thus, determining the molecular architecture of the SC requires super-resolution light microscopy techniques such as structured illumination microscopy (SIM), stimulated-emission depletion (STED) microscopy, or single-molecule localization microscopy (SMLM). 
To maintain the structure and interactions of individual components within the SC, it is important to observe the complex in an environment that is close to its native environment in the germ cells. Therefore, we demonstrate an immunohistochemistry and imaging protocol that enables the study of the substructure of the SC in intact, extruded Caenorhabditis elegans germline tissue with SMLM and STED microscopy. Directly fixing the tissue to the coverslip reduces the movement of the samples during imaging and minimizes aberrations in the sample to achieve the high resolution necessary to visualize the substructure of the SC in its biological context.

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

Super-resolution microscopy can provide a detailed insight into the organization of components within the synaptonemal complex in meiosis. Here, we demonstrate a protocol to resolve individual proteins of the Caenorhabditis elegans synaptonemal complex.

Caenorhabditis elegans 생식계열 내 시냅톤 복합체의 초고해상도 현미경

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures ...

의 시각화 Caenorhabditis Elegans 표피 구조