우리는 자금에 대한 RIKEN 주셔서 감사합니다. P10 - Gal4은 알랭 빈센트 (Université 폴 Sabatier, 툴루즈, 프랑스)의 일종 선물했다.

1. 시약의 준비 시작하기 전에주의 사항 : 해부 및 immunohistochemical 얼룩은 자기 챔버에서 수행되고 유충은 특별히 모양의 곤충 핀을 사용하여 넘어뜨린 것입니다. 자기 챔버 건설, 이들 핀의 준비에 대한 자세한 지침은 관련 참고 문헌 14,15에서 찾을 수 있습니다. 간단히, 1x1cm의 사각형 구멍은 자성 시트 및 소형 챔버를 만들기 위해 시트의 뒤쪽에 부착된 coverslip으로 절단됩니다. 챔버의 측면은 에폭시 접착제로 봉인되며이 접착제는 챔버가 사용하기 전에 70 % 에탄올로 여러 번 씻어입니다 설정한 후. 해부의 곤충 핀은 필요한 모양 벤딩하여 준비하고 금속 탭을 14,15에 붙어 있습니다. 또는 금속 탭을 우리는 컷오프 노란색 팁 만든 핸들 거꾸로 플랫 헤드 스틸 그리기 핀을 사용합니다. 이 자기 ​​챔버 배열의 사용이 가까이 제어 ove 수 있습니다R 핀 위치와 조직 해부 동안 스트레칭. DA 뉴런의 조사관의 다른 하위 집합에 리포터 유전자 발현을 유발하기 위해서는 몇 가지 Gal4 행 (시모노 및 동료 16 요약)를 사용할 수 있습니다. 이 라인의 대부분은 공공 주식 센터에서 사용할 수 있습니다. 이 대표 프로토콜에서는, 우리는 DA 신경 세포의 대조되는 두 클래스가 공동으로 표시되는 라인의 immunostaining 수행 : 가장 간단한 클래스 I 및 가장 복잡한 수준의 IV (P10 - Gal4 17,18, UAS - mCD8 : : Kusabira - 오렌지 (KO)). <ol><li> 칼슘 + + 무료 HL3.1 염분 준비 <ol><li> MM 년 : 70 NaCl, 5 KCl, 20 MgCl 2, 10 NaHCO 3, 5 HEPES, 115 자당, 5 trehalose, 산도 7.2 19. 필터 소독 및 저장 4 ° C. 참고 : 칼슘 + + 무료 솔루션 해부하는 동안 근육 수축을 방지합니다. </li></ol></li><li> 2X PHEM 버퍼를 준비 <ol><li> MM 년 : 130 파이프, 60 HEPES, 20 EGTA, 4 MgSO4; 산도 7.0. 필터 소독 및 저장 4 ° C. 참고 : 산도가 7.0 접근 때까지이 자료는 용해되지 않습니다. </li></ol></li><li> 고정 바로 앞에 갓 정착액을 준비합니다. <ol><li> 50ml 팔콘 튜브에 25ml 솔루션을 먼저 믹스 2g의 paraformaldehyde, 100μl 1M NaOH와 10ml의 물을 준비하기 위해서. </li><li> 솔루션 분명히 때까지 흔드는와 55 ° C의 물을 욕조에 정착액 흔들어. (총 부피는 11.5에 대한 ML됩니다.) </li><li> 얼음 쿨 정착액. </li><li> 12.5ml 2X PHEM 버퍼를 추가합니다. </li><li> 1M HCL과 7.0 산도를 조정합니다. </li><li> 볼륨이 25ml가 될 때까지 물로 솔루션을 채우십시오. </li><li> 워트 먼지 종이를 사용하여 솔루션을 필터링합니다. </li></ol></li></ol> 2. 애벌레 해부 시작하기 전에 참고 : 감각 dendrites 이들 특히 미세 소관 네트워크를, 그리고 신경 쇠약 증세는 절개의 개시 이후 급속하게됩니다. 교류즉각적인 고정 다음 채 5 분 빠른 절개를 hieving하면이 프로토콜의 성공의 핵심 요인입니다. <ol><li> 물에 유충을 씻고 빠르게 머리카락 루프를 사용하여이 드롭으로 그들을 이동합니다. </li><li> 유리에 동양의 유충 등의 측면까지와 복부 쪽. 참고 :이 방향은 복부 클러스터 신경을 검사하는 것입니다. 지느러미 클러스터 신경을 검사하려면 방향을 반전. </li><li> 입 후크 근처 앞쪽에 끝을 핀에 가운데 곤충 핀을 사용합니다. 최상의 결과를 얻으려면 끝에 가까운 핀을 삽입합니다. </li><li> 해부 실에서 HL3.1 식염수 한 방울을 넣으십시오. </li><li> microscissors와 유충의 가장 뒷부분 팁을 잘라. 참고 :이 단계는 microscissors (2.7 단계)에 대한 액세스를 허용합니다 유충의 사후 끝에 구멍을 엽니다. </li><li> 포셉 지금 유충의 사후 끝에 구멍 밖으로 삐져는 창자의 영역과 함께 잡아. 부드럽게 전체 창자를 꺼내. </li><li> 을 장소앞쪽에 향해 배면의 중간선에 따라 조리개와 컷을 통해 microscissors 중 하나 블레이드의 팁. </li><li> 코너 핀 사용하여 다음 앞쪽에 첫번째 사후 지금 무료로 모서리를, 핀. 동시에 부드럽게 애벌레 등심을 열고 스트레칭. </li></ol> 3. 고정, 차단 얼룩, 그리고 애벌레 필레의 장착 시작하기 전에주의 사항 : 모든 고정 및 얼룩 단계는 절개 챔버에서 수행됩니다. 이 과정에서,이 조직의 손상을 초래할 수 있으므로 유충을 잡고 곤충 핀을 노크하지 않도록주의합니다. 밖으로 건조에서 실험을 방지하기 위해 moistened 조직으로 둘러싸인 작은 파티 간다 구 컨테이너의 모든 얼룩 단계를 않습니다. <ol><li> 노란 팁을 사용하여 칼슘 + + 무료 HL3.1 버퍼를 기음. 즉시 다른 pipetteman을 사용하여 정착액을 추가합니다. </li><li> 부드럽게 칼슘 + + 무료 HL3.1 버퍼의 남은 흔적과 정착액를 섞어 아래 피펫과챔버 인치 즉시 대기음 후 챔버에 신선한 정착액을 추가합니다. </li><li> 20 분 상온에서 부화. </li><li> PBST에서 배의 상업 중심지 (0.1 %를 튼과 PBS에서 X - 100) 씻으십시오. </li><li> RT에서 20 분 대한 PBST로 5 % 염소 혈청과 차단. </li><li> PBST로 5 % 염소 혈청으로 희석 주 항체와 차단 솔루션을 교체합니다. 사용하는 기본 항체는 마우스 방지 α - Tubulin (DM1A)와 쥐 안티 CD8 (5H10) 모두 희석 1천분의 1입니다. 참고 : 조사는 (토론 참조) 마우스 방지 Futsch (22C10) 일부 상황에서는 20,21 희석 1,000분의 1과 마우스 방지 α - Tubulin (DM1A)를 대체하실 수 있습니다. </li><li> 4 (적어도 16h)을 밤새 품어 ° C. </li><li> PBST에서 배의 상업 중심지 씻으십시오. </li><li> PBST로 5 % 염소 혈청의 희석 보조 항체 솔루션을 추가합니다. 사용되는 이차 항체는 알렉사 형석 488 염소 안티 - 마우스 IgG와 Cy3 당나귀 안티 쥐 IgG 수 있습니다. 형광단을 방지하기 위해 적용 샘플을 유지22 사진 표백. </li><li> 두 시간 동안 RT에서 부화하거나, 또는 야간 4 ° C 상온에서 한 시간 따라갔다. </li><li> PBST에서 배의 상업 중심지 씻으십시오. </li><li> 슬라이드 아래 표피 사이드에있는 애벌레 필릿를 놓고, 80 % 글리세롤에 마운트하고, &#39;빠른&#39;마운트를 위해 매니큐어로 coverslip의 측면을 봉쇄. 참고 : 조직 개선 삭제하고 영구적으로 안정적인 예제, DPX에 마운트 이전 23 설명된대로. </li></ol> 4. 대표 결과 : 형광 염색법은 공촛점 현미경으로 조사되었다. 그림에서는 dendrite 아버 이내에 1-2, 다른 지점 다른 cytoskeletal 조직을했습니다. 그림 1은 1 일 instar 애벌레 단계에서 클래스 IV DA 신경 세포의 아버의 영역을 보여줍니다. KO는 및 보조 안티 CD8 항체 및 형광 (Cy3)을 사용하여 검색 : 전체 아버은 mCD8로 표시되어있다. Tubulin은 안티 α - Tubulin 항체 및 형석을 사용하여 감지escent 보조 (알렉사 형석 488). 주요 지점은 Tubulin 양성 반응이 일부 얇은 사이드 가지 Tubulin - 아무것도 없습니다. 영화 1 클래스 I DA 뉴런과 비슷한 얼룩을 통해 시리얼 섹션의 집합입니다. 그림 2는 1 일 instar 애벌레 단계에서 Futsch와 CD8에 대한 항체 물들일 클래스 IV DA 신경 세포의 아버의 영역을 보여줍니다. 주요 지점 Futsch - 긍정, 일부 얇은 사이드 가지 Futsch - 아무것도 없습니다. 그림 3. 애벌레 바디 벽 Tracheae은 복잡한 미세 소관 조직을 보여줍니다. 영화 2, 3 바디 벽의 근육과 기관을 통해 얼룩의 시리얼 섹션을 표시합니다. <img alt="그림 1" src="/files/ftp_upload/3662/3662fig1.jpg" /> 그림 1. 그림. 1 1 일 instar 애벌레 단계에서 클래스 IV DA 신경 세포의 아버의 영역을 보여줍니다. : 전체 아버은 mCD8로 표시되어있다 : KO 및 안티 - CD8 항체 및 형광 보조 (Cy3)을 사용하여 발견했습니다. Tubulin은 안티 - &amp; - Tubuli를 사용하여 감지N 항체 및 형광 보조 (알렉사 488). 기본에 패널 AC 순차적 공촛점 Z - 섹션 (0.5μm), C&#39; - C &quot;와 (과 노란색 화살촉) 또는 microtubules가 강조 표시됩니다 (보라색 화살촉)가없는 지점의 패널 C. 예제에서 단일 항체 얼룩. 레드 화살촉 하이라이트 microtubules 상피 세포. <img alt="그림 2" src="/files/ftp_upload/3662/3662fig2.jpg" /> 그림 2. 그림. 2 1 일 instar 애벌레 단계에서 Futsch와 CD8에 대한 항체 물들일 클래스 IV 다 신경 세포의 아버과 비슷한 영역을 보여줍니다. : 전체 아버은 mCD8로 표시되어있다 : KO 및 안티 - CD8 항체 및 형광 보조 (Cy3)을 사용하여 발견했습니다. Futsch는 안티 Futsch 항체 및 형광 보조 (알렉사 488)를 사용하여 감지됩니다. 주요 지점 Futsch - 긍정, 일부 얇은 사이드 가지 Futsch - 아무것도 없습니다. 와 (과 노란색 화살촉) 또는 (보라색 화살촉) Futsch하지 않고 가지의 예제가 강조 표시됩니다. 그림 3. 기관은 위에서 설명한 안티 Tubulin 프로토콜을 사용하여 얼룩. 이 유충은 셋째 instar하고 이전에 23,24을 설명한대로 해부 했어요. 영화 3 사각형으로 표시된 필드에서 확대 시리얼 섹션을 보여줍니다. 영화 1. 직렬 부분은 내가 신경 클래스의 돌기 아버 걸쳐 Tubulin의 얼룩을 추적. KO (마젠타) 및 보조 안티 CD8 항체 및 형광 (Cy3)을 사용하여 검색 : 전체 아버은 mCD8로 표시되어있다. Tubulin은 (녹색) 반 α - Tubulin 항체 및 형광 보조 (알렉사 형석 488)를 사용하여 감지됩니다. 규모 : 비디오 이미지의 한쪽이 섹션에서 46.88μm에 해당합니다. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie1.avi">동영상을 보려면 여기를 클릭하십시오.</a> 몸매에 Tubulin의 얼룩을 추적 영화 2. 직렬 섹션삼분의 일 instar의 유충의 Y 벽의 근육. Tubulin은 반 α - Tubulin 항체 및 형광 보조 (알렉사 형석 488)를 사용하여 감지됩니다. 규모 : 비디오 이미지의 한쪽이 섹션에서 46.88μm에 해당합니다. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie2.avi">동영상을 보려면 여기를 클릭하십시오.</a> 동영상 3. Figure.3에 사각형으로 표시된 삼분의 일 instar의 유충의 시체가 벽에 기관에서 Tubulin의 얼룩을 추적 시리얼 섹션. Tubulin은 반 α - Tubulin 항체 및 형광 보조 (알렉사 형석 488)를 사용하여 감지됩니다. 규모 : 비디오 이미지의 한쪽이 섹션에서 46.88μm에 해당합니다. <a href="http://www.jove.com/files/ftp_upload/3662/YalginMooreMovie3.avi">동영상을 보려면 여기를 클릭하십시오.</a>

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Neurosci. 23, 6171-6175 (2003).</li><li>Ye, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+dendritic+and+axonal+development+by+the+novel+Kruppel-like+factor+Dar1.">Differential regulation of dendritic and axonal development by the novel Kruppel-like factor Dar1.</a> J. Neurosci. 31, 3309-3319 (2011).</li><li>Parrish, J. Z., Xu, P., Kim, C. C., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+bantam+functions+in+epithelial+cells+to+regulate+scaling+growth+of+dendrite+arbors+in+drosophila+sensory+neurons.">The microRNA bantam functions in epithelial cells to regulate scaling growth of dendrite arbors in drosophila sensory neurons.</a> Neuron. 63, 788-802 (2009).</li><li>Sugimura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+developmental+modes+and+lesion-induced+reactions+of+dendrites+of+two+classes+of+Drosophila+sensory+neurons.">Distinct developmental modes and lesion-induced reactions of dendrites of two classes of Drosophila sensory neurons.</a> J. Neurosci. 23, 3752-3760 (2003).</li><li>Budnik, V., Gorczyca, M., Prokop, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selected+methods+for+the+anatomical+study+of+Drosophila+embryonic+and+larval+neuromuscular+junctions.">Selected methods for the anatomical study of Drosophila embryonic and larval neuromuscular junctions.</a> Int. Rev. Neurobiol. 75, 323-365 (2006).</li><li>Sullivan, W., Ashburner, M., Hawley, 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=.">.</a> Drosophila Protocols. , (2000).</li><li>Shimono, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidendritic+sensory+neurons+in+the+adult+Drosophila+abdomen:+origins,+dendritic+morphology,+and+segment-+and+age-dependent+programmed+cell+death.">Multidendritic sensory neurons in the adult Drosophila abdomen: origins, dendritic morphology, and segment- and age-dependent programmed cell death.</a> Neural. Dev. 4, 37-37 (2009).</li><li>Colomb, S., Joly, W., Bonneaud, N., Maschat, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+concerted+action+of+Engrailed+and+Gooseberry-Neuro+in+neuroblast+6-4+is+triggering+the+formation+of+embryonic+posterior+commissure+bundles.">A concerted action of Engrailed and Gooseberry-Neuro in neuroblast 6-4 is triggering the formation of embryonic posterior commissure bundles.</a> PLoS One. 3, 2197-2197 (2008).</li><li>Dubois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collier+transcription+in+a+single+Drosophila+muscle+lineage:+the+combinatorial+control+of+muscle+identity.">Collier transcription in a single Drosophila muscle lineage: the combinatorial control of muscle identity.</a> Development. 134, 4347-4355 (2007).</li><li>Feng, Y., Ueda, A., Wu, 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=A+modified+minimal+hemolymph-like+solution,+HL3.1,+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae.">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> J Neurogenet. 18, 377-402 (2004).</li><li>Hummel, T., Krukkert, K., Roos, J., Davis, G., Klambt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Futsch/22C10+is+a+MAP1B-like+protein+required+for+dendritic+and+axonal+development.">Drosophila Futsch/22C10 is a MAP1B-like protein required for dendritic and axonal development.</a> Neuron. 26, 357-370 (2000).</li><li>Zipursky, S. L., Venkatesh, T. R., Teplow, D. B., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+development+in+the+Drosophila+retina:+monoclonal+antibodies+as+molecular+probes.">Neuronal development in the Drosophila retina: monoclonal antibodies as molecular probes.</a> Cell. 36, 15-26 (1984).</li><li>Brent, J., Werner, K., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Larval+NMJ+Immunohistochemistry.">Drosophila Larval NMJ Immunohistochemistry.</a> J. Vis. Exp. (25), e1108-e1108 (2009).</li><li>Karim, M. R., Moore, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+Analysis+of+Drosophila+Larval+Peripheral+Sensory+Neuron+Dendrites+and+Axons+Using+Genetic+Mosaics.">Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics.</a> J. Vis. Exp. (57), e3111-e3111 (2011).</li><li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Larval+NMJ+Dissection.">Drosophila Larval NMJ Dissection.</a> J. Vis. Exp. (24), e1107-e1107 (2009).</li><li>Tao, J., Rolls, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrites+have+a+rapid+program+of+injury-induced+degeneration+that+is+molecularly+distinct+from+developmental+pruning.">Dendrites have a rapid program of injury-induced degeneration that is molecularly distinct from developmental pruning.</a> J. Neurosci. 31, 5398-5405 (2011).</li><li>Yamamoto, M., Ueda, R., Takahashi, K., Saigo, K., Uemura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+axonal+sprouting+and+dendrite+branching+by+the+Nrg-Ank+complex+at+the+neuron-glia+interface.">Control of axonal sprouting and dendrite branching by the Nrg-Ank complex at the neuron-glia interface.</a> Curr. Biol. 16, 1678-1683 (2006).</li><li>Mattie, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+microtubule+growth,++TIPs,+and+kinesin-2+are+required+for+uniform+microtubule+polarity+in+dendrites.">Directed microtubule growth, +TIPs, and kinesin-2 are required for uniform microtubule polarity in dendrites.</a> Curr. Biol. 20, 2169-2177 (2010).</li><li>Pawson, C., Eaton, B. A., Davis, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formin-dependent+synaptic+growth:+evidence+that+Dlar+signals+via+Diaphanous+to+modulate+synaptic+actin+and+dynamic+pioneer+microtubules.">Formin-dependent synaptic growth: evidence that Dlar signals via Diaphanous to modulate synaptic actin and dynamic pioneer microtubules.</a> J. Neurosci. 28, 11111-11123 (2008).</li><li>Williams, D. W., Tyrer, M., Shepherd, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+and+tau+reporters+disrupt+central+projections+of+sensory+neurons+in+Drosophila.">Tau and tau reporters disrupt central projections of sensory neurons in Drosophila.</a> J. Comp. Neurol. 428, 630-640 (2000).</li></ol>

우리는 공개 아무것도 없어.

그것을 달성하는 방법 복잡한 세포 모양 이해하기 위해서는 정확하게 분석 미세 소관 기관 수 있도록 중요합니다. 여기 돌기 arborization의 신경 감각 dendrites의 분석 미세 소관 기관에 대한 강력한 immunohistological 라벨링 방법을 설명합니다. 얼룩 감각 신경뿐만 아니라,이 방법은 기관, 근육 및 다른 신체 벽면 조직의 강력한 immunohistological 얼룩을 구현합니다. 우리는 DA의 뉴런의 ...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 수 </th><th> 댓글 (선택 사항) </th></tr><tr><td> 집게 </td><td> 뒤몽 </td><td> 11251-20 </td><td></td></tr><tr><td> Microscissors </td><td> FST </td><td> 15000-08 </td><td></td></tr><tr><td> 마우스 방지 α - tubulin (복제 : DM1A) </td><td> 시그마 </td><td> T9026 </td><td> 희석 1,000분의 1 </td></tr><tr><td> 마우스 방지 Futsch (복제 : 22C10) 표면에 뜨는 </td><td> 발달 연구 하이 브리 도마 은행 </td><td> 22C10 </td><td> 희석 1,000분의 1 </td></tr><tr><td> 쥐 방지 CD8 (복제 : 5H10) </td><td> Caltag </td><td> MCD0800 </td><td> 희석 1,000분의 1 </td></tr><tr><td> 알렉사 형석 488 안티 - 마우스 IgG </td><td> Invitrogen </td><td> A - 11001 </td><td> 희석 5백분의 1 </td></tr><tr><td> Cy3 안티 쥐 IgG </td><td> 잭슨 Immunoresearch </td><td> 712-166-150 </td><td> 희석 200분의 1 </td></tr></tbody></table>

immunohistological labeling of microtubules in sensory neuron dendrites, tracheae, and muscles in the drosophila larva body wall

복잡한 세포 형태의 차이가 달성되는 방법을 이해하기 위하여, 정확하게 미세 소관 조직을 수행하는 것이 중요합니다. Drosophila 애벌레 바디 벽이 세포와 조직 morphogenesis을 연구하는 모델 몇 가지 세포 유형이 포함되어 있습니다. 예를 들어 tracheae는 튜브 morphogenesis 1, Drosophila의 유충의 감각 뉴런은 일반의 해설과 모수 석의 차별화 2-5와 퇴보 6 신경 세포 수준의 특정 메커니즘에 대한 기본 시스템이되고있는 돌기 arborization (DA)를 검사하는 데 사용됩니다 . dendrite 가지의 모양은 신경 세포 클래스 사이에 크게 다릅니다, 심지어는 하나의 신경 세포 7,8의 다른 지점 사이에 있습니다. DA의 뉴런의 유전자 연구는 차동 cytoskeletal 조직이 돌기 지점 모양 4,9-11의 형태학의 차이를 기초 수있는 것이 좋습니다. 우리에게 강력한 면역 라벨 방법을 제공DA 감각 신경 dendrite 아버의 생체내의 미세 소관 기관 (그림 1, 2, 영화 1) ssay. 이 프로토콜은 먼저 instar의 유충의 해부와 immunostaining, 활성 감각 신경 세포의 dendrite의 파생물 및 분기 조직 12,13을 발생하는 단계를 보여줍니다. 감각 뉴런을 얼룩뿐만 아니라,이 방법은 강력한 근육의 미세 소관 조직의 라벨 (영화 2, 3),기도 (그림 3, 영화 3), 기타 신체 벽의 조직을 실현하고 있습니다. 그런 메커니즘을 조사하면 신체 벽에 현장에서 미세 소관 조직을 분석하고자 조사에 대한 가치가 컨트롤 조직 및 세포 모양.

Watch this Scientific Journal Video about Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall at JoVE.com

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

등의 연결을 dendrites 같은 복잡한 세포 모양은, 개발하는 동안 달성하는 방법 이해하기 위하여, 정확하게 분석 미세 소관 기관 수 있도록 중요합니다. 여기 돌기 arborization의 신경 감각 dendrites의 미세 소관 조직, 기관, 근육을 검토하는 강력한 immunohistological 라벨링 방법을 설명 및 기타<em&gt; Drosophila</em&gt; 유충 바디 벽 조직.

Immunohistological 감각 신경 세포의 Dendrites에 Microtubules의 라벨링, Tracheae, 그리고 근육에 Drosophila 유충 바디 벽

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

immunohistological-labeling-microtubules-sensory-neuron-dendrites

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JoVE Journal

Neuroscience

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

12.3K 조회수.  RIKEN Brain Science Institute. 등의 연결을 dendrites 같은 복잡한 세포 모양은, 개발하는 동안 달성하는 방법 이해하기 위하여, 정확하게 분석 미세 소관 기관 수 있도록 중요합니다. 여기 돌기 arborization의 신경 감각 dendrites의 미세 소관 조직, 기관, 근육을 검토하는 강력한 immunohistological 라벨링 방법을 설명 및 기타 Drosophila 유충 바디 벽 조직.

비디오: Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

손상의 감각 오르간 전구체 세포 라이브 셀 이미징 Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

연구

신경과학

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

의 형태학의 분석 Drosophila</em유전 모자이크 사용하기> 애벌레 주변 감각 신경 세포의 Dendrites 및 Axons

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

의 Proprioceptors의 시각화<em&gt; Drosophila</em&gt; 애벌레와 Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

역행 형광 라벨이 확인 된 신경 세포의 표적 세포 외 단일 단위 기록을 허용<em&gt; 생체 내</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

의 중추 신경계에 단일 세포의 라벨<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

에있는 후각 수용체 뉴런의 전체 마운트 Immunolabeling<em&gt; 초파리</em&gt; 안테나

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

의 컴퓨터 추적하여있는 분석 후각 행동<em&gt; 초파리</em&gt; 네 사분면의 후각에

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

간단한 한 단계 성인의 전체 마운트 준비에 대한 해부 프로토콜<em&gt; 초파리</em&gt; 두뇌

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

천연 폴리페놀 커큐민을 사용하여 뇌 조직에서 아밀로이드 플라크의 라벨링 및 이미징

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

뇌 슬라이스와 기본 세포 배양에서 피라미드 뉴런의 탄도 라벨링

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...