우리는 플라이 주식을 제공하기위한 블루밍턴 초파리 증권 센터 감사합니다. 우리는 또한 항체를 제공하기 위해 발달 연구 하이 브리 도마 은행과 박사 린 쿨리 (예일 대학) 감사합니다. 특별한 감사는 원고에 그녀의 도움을 AhHyun 유 간다. 이 작품은 자연 과학 및 캐나다의 공학 연구 협의회 (크리거), 윌리엄과 에이다 이사벨 스틸 기금 (크리거) 및 건강 연구의 캐나다 연구소 (강화)에서 보조금에 의해 지원되었다.

1. 몸 벽 준비 . 참고 : 이전에 브렌트 등 10 또는 라마 찬드 및 Budnik (11, 12)에 설명 된대로 (몸 벽 근육에 분포 NMJs의 연구를위한) 셋째 령 애벌레 몸 벽의 준비하지만 일부 수정을 수행했다. <ol><li> 해부 <ol><li> 표준 절차 (13)를 사용하여 5~6일 25 ° C에서 플라이 주식과 십자가를 올립니다. </li><li> 미세 집게를 사용하여 유리 병 또는 병에서 세번째 령 유충 크롤링을 선택합니다. </li><li> 음식 입자를 제거하기 위해 인산 버퍼 식염수 (PBS)를 포함하는 작은 페트리 접시에 유충을 씻으십시오. </li><li> 실 가드 디스크에 하나의 애벌레를 놓고 얼음처럼 차가운 PBS 몇 방울에 몰입. 얼음처럼 차가운 PBS는 조작하기 쉽게 만드는 유충 기절 도움이됩니다 사용. 해부 전반에 걸쳐 준비가 항상 건조되는 것을 방지하기 위해 PBS에 빠져들되어 있는지 확인합니다. </li><li> 라의 위치를그 등쪽 두 기관 책자는 해부 현미경 (그림 2A)에서 볼 수 있습니다 너무 위로 향하게 RVA. 집게를 사용하면, minutien 핀을 파악 spiracles (그림 2B) 근처의 후방 끝 부분에 유충을 고정합니다. 다른 핀으로, 입 후크 근처 앞쪽 끝 부분에있는 표피 관통. 부드럽게 한 다음, 길이 유충을 스트레칭 (그림 2B)를 아래로 핀. </li><li> 미세 절제 가위를 사용하여 작은 구멍을 만드 핀 근처 후단을 꼬집어. 절개는 표피를 통과 할만큼 피상적해야한다. </li><li> 두 기관 책자 (그림 2C) 사이의 등쪽 정중선의 전체 길이를 따라 절단, 절개로 가위의 아래쪽 블레이드의 팁을 배치. 복부 몸 벽 근육 손상을 방지하기 위해 절단시 약간 위쪽으로 가위 블레이드를 가리 킵니다. </li><li> 약간 포스트 앞쪽에 작은 수평 절개를합니다eriorly - 위치 핀 (그림 2C). 다른 유사한 절개 약간 전방-배치 핀 (그림 2C) 후부합니다. 절개는 표피를 통과해야한다. 참고 : 유사합니다 결합 단계 1.1.7과 1.1.8에서 세 절개 &quot; <img fo:content-width="0.09in" src="/files/ftp_upload/52139/52139_letter_I.jpg" width="15px" /> 완료 할 때 &quot;, 즉, 애벌레 몸의 등 쪽의 왼손 및 오른손 플랩이 생성되어야한다. </li><li> 조심스럽게 집게와 내부 장기를 청소. PBS의 몇 가지 강력한 방울을 추가하면, 애벌레 몸에서 장기를 대체하여 쉽게 제거 할 수있게 도와 줄 것입니다. 그것은 몸 벽 근육에 손상을 원인이됩니다 애벌레 몸을 파고하지 마십시오. </li><li> 열린 애벌레 몸을 펼쳐라 아래 모서리 (그림 2D)를 핀. 피닝시 균일 텐션 사각형을 형성하도록 수평 및 수직 체벽 스트레칭 (SE전자 그림 모양에 대한 2G), 돌보는이 과정에서 몸 벽 근육이 찢어지지. </li><li> 남아있는 내부 장기 (그림 2E)를 제거 완료. </li></ol></li><li> 고정 및 투과성으로 <ol><li> 보우 인의 솔루션의 몇 방울에 고정 된 본체 벽을 담근다. 얼음에 15 분 동안 품어. 대안으로, 대안 고착성 등 4 % 파라 포름 알데히드 (PFA)를 사용한다; 30 분 동안 품어. </li><li> 트리톤과 인산 버퍼 식염수 (PBT)로 3 회 씻어. </li><li> 미세 핀셋으로 조심스럽게 핀을 제거하고 실리콘 화 0.65 ml의 마이크로 원심 분리 튜브에 그 모서리를 본체의 벽을 전송. </li><li> PLA를 위해 준비 될 때까지 4 ° C에서 PBT의 몸 벽을 저장합니다. 최적의 결과를 얻으려면 하루 해부의 두 내 몸 벽을 면역 염색 시작합니다. 참고 : 시약에 저장하고 신체의 모든 벽은 분석 기간 동안 동등하게 취급되도록, 다른 유전자형은 하나에 배치 할 수 있습니다튜브. 유전형 (예는도 2F 참조) 다르게 몸체 벽의 모서리를 절단함으로써 구별 될 수있다. </li></ol></li></ol> 2. 면역 조직 화학 참고 :. 셋째 령 애벌레 몸 벽의 면역 염색은 이전 브렌트 등 14 라마 찬드 및 Budnik (11)에 설명 된대로 수행하지만, 일부 수정 11,14와했다. 참고 : 달리 명시되지 않는 실내 온도에서 교반과 함께 모든 단계를 수행합니다. <ol><li> 블로킹 <ol><li> 10 분마다 PBT로 3 회 보디 벽을 씻는다. </li><li> 1 시간 동안 1 % 소 혈청 알부민 (BSA)로 차단합니다. </li></ol></li><li> 면역 염색 <ol><li> 관심 두 단백질에 대해 마우스 및 토끼 일차 항체와 본체의 벽을 인큐베이션을 실온에서 2 시간 또는 밤새 39 ° C (1 %의 BSA에 희석). 250 토끼이 경우 1:10 마우스 항 굴림 및 1을 사용NTI-HTSM (15, 16). 하지 마우스 나 토끼에서 만들어진 마커에 대한 항체도 포함 할 수있다 - 예를 들어, 1을 사용 : 200 염소 항 - HRP는 신경 세포막을 윤곽을 그리다. </li><li> 10 분마다 PBT로 3 회 세척 할 것. </li><li> 실온에서 2 시간 또는 밤새 39 ° C (1 %의 BSA에 희석) 마커를 검출하기 위해 형광 접합 된 이차 항체와 함께 배양한다. HRP 염소 항 - 항체를 검출하기 위해 200-FITC 접합 항 - 염소 : 예 1을 사용 - PLA 신호 이후 적색 형광 가시화 된 바와 같이, 형광 물질은 다른 마커를 검출하기 위해 사용되어야한다. 때문에 빛에 민감한 시약의 사용, 이후이 시점에서 어둠 속에서 튜브를 유지. 참고 : PLA는 공 초점 현미경 아래에서 빨강 채널에서 시각 신호와, 마우스 및 토끼에서 제기 된 주요 항체 사이에 수행 할 수에 사용되는 키트는 할 수 있습니다. 원하는 경우, 다른 키트는 분석이 주역으로 수행 할 수 있도록 그 사용할 수 있습니다RY 항체는 다른 종에서 발생하고, 신호는 다른 채널에 가시화. </li></ol></li></ol> 3. 근접 내고 분석 참고 : 달리 명시되지 않는 실내 온도에서 교반과 함께 모든 단계를 수행합니다. <ol><li> PLA 프로브 <ol><li> 10 분마다 PBT로 3 회 보디 벽을 씻는다. </li><li> 37 ° C에서 2 시간 동안 (5 희석 1 % BSA 각 1) PLA의 프로브를 품어. 이 경우, 5-10 몸체 벽의 적당한 침지 및 혼합을 보장하기 PLA 프로브 항 - 마우스 마이너스 40 μL, PLA 프로브 항 - 토끼 PLUS 40 μL와 1 % BSA 120 μl를 사용한다. PLA 프로브 1시 25분 희석율 여전히 (이 실험 예) 적절한 신호 대 잡음 비율을 초래할 수있다. </li></ol></li><li> 결찰 <ol><li> 5 분 각각에 대해 두 번 세척 버퍼와 신체 벽을 씻으십시오. </li><li> 1 시간에 대한 연결 용액 (결찰 완충액 리가 아제 1시 40분 희석)과 인큐베이션t 37 ° C의. 이 경우, 본체 5-10 벽의 적절한 혼합 및 침지를 위해 리가 아제 5 μL, 5 결찰 완충액 40 μL 및 고순도의 물 155 μl를 사용한다. </li></ol></li><li> 확대 <ol><li> 2 분 각각에 대해 두 번 세척 버퍼와 신체 벽을 씻으십시오. </li><li> 37 ° C에서 2 시간 동안 증폭 용액 (증폭 완충액 폴리머 라제 1:80 희석)으로 배양한다. 이 경우, 본체 5-10 벽의 적절한 혼합 및 침지를 위해 폴리머 라제 2.5 μL, 5 증폭 완충액 40 μL 및 고순도 157.5 μL의 물을 사용한다. </li></ol></li><li> 이미징을위한 준비 <ol><li> 10 분 각각에 대해 두 번 세척 버퍼 B와 몸 벽을 씻으십시오. </li><li> 1 분에 대해 한 번 0.01x 워시 버퍼 B 씻으시오. </li><li> 장착하기 전에 적어도 30 분 동안 장착 솔루션의 몇 방울의 평형, 또는 4 ℃에서 하룻밤 저장합니다. </li><li> 미세 집게를 사용하여 조심스럽게 플랫폼 슬라이드 w에 몸 벽을 전송i 번째 자신의 손톱이 아래를 향. 행과 mountant의 하락 또는 두 내에서 같은 방향으로 몸 벽을 배치합니다. 다음 투명 매니큐어와 슬라이드를 밀봉, 공기 방울을 발생하지 않도록 준비를 돌보는 동안 22mm X 40mm coverslip에 배치합니다. </li><li> 공 초점 이미징을위한 준비가 될 때까지 -20 ° C에서 어둠 속에서 슬라이드를 저장합니다. </li></ol></li></ol>

<ol>
	<li>Woods, D. F., Bryant, P. 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+discs-large+tumor+suppressor+gene+of+Drosophila+encodes+a+guanylate+kinase+homolog+localized+at+septate+junctions.">The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions.</a> Cell. 66, 451-464 (1991).</li><li>Yamanaka, T., Ohno, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Lgl/Dlg/Scribble+in+the+regulation+of+epithelial+junction,+polarity+and+growth.">Role of Lgl/Dlg/Scribble in the regulation of epithelial junction, polarity and growth.</a> Front Biosci. 13, 6693-6707 (2008).</li><li>Humbert, P. O., 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=Control+of+tumourigenesis+by+the+Scribble/Dlg/Lgl+polarity+module.">Control of tumourigenesis by the Scribble/Dlg/Lgl polarity module.</a> Oncogene. 27, 6888-6907 (2008).</li><li>Lahey, T., Gorczyca, M., Jia, X. X., Budnik, V. <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+tumor+suppressor+gene+dlg+is+required+for+normal+synaptic+bouton+structure.">The Drosophila tumor suppressor gene dlg is required for normal synaptic bouton structure.</a> Neuron. 13, 823-835 (1994).</li><li>Ataman, B., Budnik, V., Thomas, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolding+proteins+at+the+Drosophila+neuromuscular+junction.">Scaffolding proteins at the Drosophila neuromuscular junction.</a> Int Rev Neurobiol. 75, 181-216 (2006).</li><li>Thomas, U., Kobler, O., Gundelfinger, E. D. <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+larval+neuromuscular+junction+as+a+model+for+scaffold+complexes+at+glutamatergic+synapses:+benefits+and+limitations.">The Drosophila larval neuromuscular junction as a model for scaffold complexes at glutamatergic synapses: benefits and limitations.</a> J Neurogenet. 24, 109-119 (2010).</li><li>Matsuoka, Y., Li, X., Bennett, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adducin:+structure,+function+and+regulation.">Adducin: structure, function and regulation.</a> Cell Mol Life Sci. 57, 884-895 (2000).</li><li>Wang, S., 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=Drosophila+adducin+regulates+Dlg+phosphorylation+and+targeting+of+Dlg+to+the+synapse+and+epithelial+membrane.">Drosophila adducin regulates Dlg phosphorylation and targeting of Dlg to the synapse and epithelial membrane.</a> Dev Biol. 357, 392-403 (2011).</li><li>Soderberg, O., 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=Characterizing+proteins+and+their+interactions+in+cells+and+tissues+using+the+in+situ+proximity+ligation+assay.">Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay.</a> Methods. 45, 227-232 (2008).</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), (2009).</li><li>Ramachandran, P., Budnik, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+staining+of+Drosophila+larval+body-wall+muscles.">Immunocytochemical staining of Drosophila larval body-wall muscles.</a> Cold Spring Harb Protoc. 2010, (2010).</li><li>Ramachandran, P., Budnik, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Drosophila+larval+body-wall+muscles.">Dissection of Drosophila larval body-wall muscles.</a> Cold Spring Harb Protoc. , (2010).</li><li>Ashburner, M. <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: A Laboratory Manual. , (1989).</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), (2009).</li><li>Parnas, D., Haghighi, A. P., Fetter, R. D., Kim, S. W., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+postsynaptic+structure+and+protein+localization+by+the+Rho-type+guanine+nucleotide+exchange+factor+dPix.">Regulation of postsynaptic structure and protein localization by the Rho-type guanine nucleotide exchange factor dPix.</a> Neuron. 32, 415-424 (2001).</li><li>Petrella, L. N., Smith-Leiker, T., Cooley, 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+Ovhts+polyprotein+is+cleaved+to+produce+fusome+and+ring+canal+proteins+required+for+Drosophila+oogenesis.">The Ovhts polyprotein is cleaved to produce fusome and ring canal proteins required for Drosophila oogenesis.</a> Development. 134, 703-712 (2007).</li><li>Pielage, J., Bulat, V., Zuchero, J. B., Fetter, R. D., 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=Hts/Adducin+controls+synaptic+elaboration+and+elimination.">Hts/Adducin controls synaptic elaboration and elimination.</a> Neuron. 69, 1114-1131 (2011).</li><li>Spradling, A. C., 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=The+Berkeley+Drosophila+Genome+Project+gene+disruption+project:+Single+P-element+insertions+mutating+25%+of+vital+Drosophila+genes.">The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes.</a> Genetics. 153, 135-177 (1999).</li><li>Bokoch, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+the+p21-Activated+Kinases.">Biology of the p21-Activated Kinases.</a> Annu Rev Biochem. 72, 743-781 (2003).</li><li>Bahri, S., 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=The+leading+edge+during+dorsal+closure+as+a+model+for+epithelial+plasticity:+Pak+is+required+for+recruitment+of+the+Scribble+complex+and+septate+junction+formation.">The leading edge during dorsal closure as a model for epithelial plasticity: Pak is required for recruitment of the Scribble complex and septate junction formation.</a> Development. 137, 2023-2032 (2010).</li><li>Albin, S. D., 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=Coordinating+structural+and+functional+synapse+development:+postsynaptic+p21-activated+kinase+independently+specifies+glutamate+receptor+abundance+and+postsynaptic+morphology.">Coordinating structural and functional synapse development: postsynaptic p21-activated kinase independently specifies glutamate receptor abundance and postsynaptic morphology.</a> J Neurosci. 24, 6871-6879 (2004).</li><li>Koles, K., Budnik, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+in+neuromuscular+junction+development.">Wnt signaling in neuromuscular junction development.</a> Cold Spring Harb Perspect Biol. 4, (2012).</li><li>Packard, M., 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=The+Drosophila+Wnt,+wingless,+provides+an+essential+signal+for+pre-+and+postsynaptic+differentiation.">The Drosophila Wnt, wingless, provides an essential signal for pre- and postsynaptic differentiation.</a> Cell. 111, 319-330 (2002).</li><li>Weibrecht, I., 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=Proximity+ligation+assays:+a+recent+addition+to+the+proteomics+toolbox.">Proximity ligation assays: a recent addition to the proteomics toolbox.</a> Expert Rev Proteomics. 7, 401-409 (2010).</li><li>Thymiakou, E., Episkopou, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+signaling+effector-complexes+downstream+of+bmp4+using+PLA,+a+proximity+ligation+assay.">Detection of signaling effector-complexes downstream of bmp4 using PLA, a proximity ligation assay.</a> J Vis Exp. (49), (2011).</li><li>Maglione, M., Sigrist, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+forest+tree+by+tree:+super-resolution+light+microscopy+meets+the+neurosciences.">Seeing the forest tree by tree: super-resolution light microscopy meets the neurosciences.</a> Nat Neurosci. 16, 790-797 (2013).</li><li>Wang, S., 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=.">.</a> Biol. Open. 3 (12), 1196-1206 (2014).</li></ol>

저자가 공개하는 게 없다.

이 보고서는 PLA가 초파리 애벌레 NMJ에 적용 할 수있는 방법을 보여줍니다. 분석은 본 NMJ에서 내인성 단백질 - 단백질 상호 작용을 검출하기위한 목적 유생 체벽 근육 제제에 대해 수행된다. 이 기술로, 굴림 및 HTS 서로 매우 밀접, 따라서 구체적으로 시냅스 영역 (27)에서, 복합체에 존재하는 것으로 도시된다. 이 결과의 지원으로, 이전의 연구는 다음과 같은 데이터와의 연관성의 증?...

큰 초파리 디스크 (굴림)은 세포막의 특정 사이트에서 큰 단백질 복합체의 조립을 조율 도움이 단백질 비계의 막 관련 구 아닐 키나제 가족의 보존 회원입니다. 종양 억제 단백질, 굴림은 상피 apicobasal 극성 1,2,3를 결정하는 중요한 역할로 원래 확인했다. 굴림 또한 유생 4시 글루타메이트 운동 신경의 신경 근육 접합 (NMJ)에서 중요한 발판 모듈 역할을한다. DLG는 애벌레 NMJ에서 다양한 역할을 수행하고, 그 pleiotropism 여러 단백질 5,6와 연결하는 능력에 의존한다. 하나는 이러한 단백질은 후진타오 - 리 타이 샤오 (HTS), 주로 7 골격 액틴 스펙 트린을 조절하는 역할에 관해서 설명 된 포유류 adducins에 상동입니다. 이것은 이전과 굴림 HTS 시험관에 기초하여 서로 착체를 형성 할 수 있음을 보여왔다 </EM&gt; 전체 성인 플라이 해물 (8)을 포함하는 공동 면역 침전 실험. 이러한 결과의 단점 중 하나는, 그러나, 그들이 어디에이 복잡한 형태를 나타내지 않는다는 것이다. 면역 조직 화학 염색의 사용으로, 굴림과 HTS의 분포는 애벌레 NMJs의 시냅스 막에 겹쳐 관찰되지만,이 지역 8 복잡한에? 최근에 보여 여기에서 더 자세히 설명, 근접 내고 분석 (PLA)는 애벌레 NMJ 27 구체적으로 굴림과 HTS 사이의 현장 연관을 찾기 위해 사용됩니다. PLA는 주로 9 동일계 단백질 - 단백질 상호 작용을 검출 할 수있는 세포 및 조직 배양에 사용되는 비교적 새로운 기술이다. 이 분석에서, 관심의 단백질에 대해 두 일차 항체 종 특이 이차 항체의 쌍의 검출 올리고 뉴클레오타이드에 접합되는 프로브를 PLA, 명명 (도 1A, B). 두 단백질의 경우S는 첨부 PLA 프로브 사이의 거리가 두 개의 추가 커넥터 올리고 뉴클레오티드 (도 1C)의 하이브리드 화를 통해 가교 될 수있다 (수십 나노 미터 내의 IE) 서로 근접하게된다. 이 형태에서, 올리고 뉴클레오티드 커넥터의 자유 단부가 서로 접촉 할 정도로 가까운 및 닫힌 원형 DNA 분자가 라이 게이션 동일계에서 (도 1D)에 형성 될 수있다. 원형 DNA 분자는 PLA 프로브 (그림 1E)에 결합 올리고 뉴클레오티드 중 하나에 의해 준비가 끝났다 현장에서 롤링 원 증폭을위한 템플릿 역할을합니다. 그 결과 증폭, concatemeric DNA 제품 내에서 시퀀스는 다음 형광 표지, 보완 올리고 뉴클레오티드 프로브 (그림 1 층)로 시각화 할 수 있습니다. 증폭 된 DNA는 PLA 프로브 중 하나, 단백질 - 단백질 상호 작용 withi의 세포 내 위치 파악에 부착되는 방식 때문에NA 조직을 용이하게 확인 될 수있다. 여러 가지 방법이 일반적으로, 이러한 공동 면역 침전 등의 체외 기술을 포함하여 단백질 - 단백질 상호 작용을 검출 (-풀다운 분석 및 효모 두 하이브리드 심사, 및 포스터 공명 에너지 전달 (FRET) 및 생체 분자 형광 보완 등의 생체 기술에 사용됩니다 BiFC). 시험 관내 기법 함정 상호 내생 발생되는 경우에, 상기 생체 기술 내인성 대응의 기본 동작을 반영하지 않을 융합 단백질의 인공 표현을 수반하면서 이들이 식별하지 않는다는 것이다. PLA의 하나의 주요 장점은 FR에 비교되는 신호를 생성하는 데 필요한의 근사 정도, 조직 내에서 서로 가까운 위치에 있고 가능성 착체를 형성 내인성 단백질 인터랙의 세포 내 위치 파악을 결정하는 능력이ET와 BiFC. PLA 인해 항체 인식 및 DNA 증폭의 결합에 높은 특이성과 민감도와의 상호 작용을 검출 할 수있다. 따라서, 분석은 상호 작용의 정확한 위치를 드러내 puncta의 형태의 이산, 밝은 신호를 생성 할 수있다. 또한, 부족하게 보이는 항원을 검출 할 수있다. 마지막으로, PLA는 수행 할 수있는 비교적 간단한 기술 없으며,이를 완료하는 데 표준 면역 절차보다 더 오래 걸립니다. 따라서, PLA는 종종 긴 준비 시간과 광범위한 문제 해결 퍼진 다른 단백질 - 단백질 상호 작용 분석을 통해 기술적 이점을 제공한다. 이 프로토콜은 PLA가 반응계 내에서 내인성 단백질 - 단백질 상호 작용을 검출하기위한 목적 초파리 애벌레 NMJ 어떻게 적용될 수 있는지를 보여준다. 여기에, PLA는 굴림과 HTS가 실제로 NMJs의 시냅스 부위에 복잡한에 존재하는 것으로 표시됩니다 애벌레 몸 벽 근육 준비에 수행. PLA는 이전에 애벌레 NMJ을 연구하는 데 사용되지 않은, 초파리의 조직이 분석을 사용한 출판 논문의 단지 소수는 현재이 있습니다. 그것은 초파리 사회에 PLA의 추가 노출이 다른, 더 일반적으로 사용되는 단백질 - 단백질 상호 작용 분석을 보완하기 위해 추가 도구로 그 사용이 증가 될 것으로 기대된다.

<table border="0" cellpadding="0" cellspacing="0" width="630">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Name of Material/Equipment</td>
			<td style="width:120px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:191px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Forceps (fine #5)</td>
			<td style="width:120px;">Almedic</td>
			<td>A10-704</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="133">
			<td height="133" style="height:133px;width:211px;">Sylgard Disc</td>
			<td style="width:120px;">World Precision Instruments</td>
			<td>SYLG184</td>
			<td style="width:191px;">Mix elastomer base and curing agent in a 10:1 ratio. Set for 30 min. Pour into a mold (e.g. use a 12-well cell culture plate). Let cure for at least 24 hr. Adhere to the lid of a 60x15mm petri dish lid when dissecting.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Minutien Pins (0.0125 mm tip diameter)</td>
			<td style="width:120px;">Fine Science Tools</td>
			<td>26002-10</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">Microdissection Scissors (ultra fine)</td>
			<td style="width:120px;">Fine Science Tools</td>
			<td style="width:111px;">15200-00</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:211px;">Platform Slides</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Glue two 22x22mm coverslips onto a microscope slide with clear nail polish, leaving a &#60;20mm gap in between for sample mounting.</td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:211px;">w1118</td>
			<td style="width:120px;">Bloomington Drosophila Stock Center</td>
			<td style="width:111px;">3605</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:211px;">hts01103</td>
			<td style="width:120px;">Bloomington Drosophila Stock Center</td>
			<td style="width:111px;">10989</td>
			<td style="width:191px;">Stock was re-balanced over a GFP balancer so that homozygous mutants can be selected based on the absence of GFP signal.</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">1x PBS (Phosphate Buffered Saline): 3mM NaH2PO4, 7mM Na2HPO4, 130mM NaCl, pH 7.0</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">NaH2PO4 (Caledon Laboratories - 8180-1), Na2HPO4 (Caledon - 8120-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Bouin&#39;s Solution</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">HT10132</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="114">
			<td height="114" style="height:114px;width:211px;">4% PFA (Paraformaldehyde): 4% PFA in 1x PBS</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">PFA (Anachemia Science - 66194-300). See doi:10.1101/pdb.rec9959 Cold Spring Harb Protoc 2006 for instructions on how to make the solution.&#160;</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">1x PBT (Phosphate Buffered Saline with Triton): 1x PBS with 0.01% Triton</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Triton X-100 (Sigma-Aldrich - T8787)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">1% BSA (Bovine Serum Albumin): 1% BSA in 1x PBT</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">BSA (Bioshop Canada - ALB001). Store at 4 &#176;C.</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">mouse anti-Dlg (Discs large)</td>
			<td style="width:120px;">Developmental Studies Hybridoma Bank</td>
			<td>4F3</td>
			<td style="width:191px;">Use at a 1:10 dilution in 1% BSA.</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">rabbit anti-HtsM (Hu-li tai shao)</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Provided by Dr. Lynn Cooley (Yale University). Use at a 1:250 dilution in 1% BSA.</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">rabbit anti-Pak (p21-activated kinase)</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Provided by Dr. Nicholas Harden (Simon Fraser University). Use at a 1:500 dilution in 1% BSA.</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">mouse anti-Wg (Wingless)</td>
			<td style="width:120px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:111px;">4D4</td>
			<td style="width:191px;">Use at a 1:5 dilution in 1% BSA.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">goat anti-Hrp (Horseradish peroxidase)</td>
			<td style="width:120px;">Jackson&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; ImmunoResearch</td>
			<td style="width:111px;">123-065-021</td>
			<td style="width:191px;">Use at a 1:200 dilution in 1% BSA.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">FITC-conjugated donkey anti-goat</td>
			<td style="width:120px;">Jackson&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; ImmunoResearch</td>
			<td style="width:111px;">705-095-003</td>
			<td style="width:191px;">Use at a 1:200 dilution in 1% BSA.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ PLA Probe anti-mouse MINUS</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO92004</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ PLA Probe anti-rabbit PLUS</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO92002</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ Detection Reagents Red</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td>DUO92008</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">1x Wash Buffer A: 0.01M Tris, 0.15M NaCl, 0.05% Tween 20, pH 7.4</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82049</td>
			<td style="width:191px;">Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1), Tween 20 (Fisher Scientific - BP337)</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">1x Wash Buffer B: 0.2M Tris, 0.1M NaCl, pH 7.5</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82049</td>
			<td style="width:191px;">Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">0.01x Wash Buffer B: 2mM Tris, 1mM NaCl, pH 7.5</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82049</td>
			<td style="width:191px;">Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ Mounting Medium with DAPI</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82040</td>
			<td style="width:191px;"></td>
		</tr>
	</tbody>
</table>

detection of in situ protein-protein complexes at the drosophila larval neuromuscular junction using proximity ligation assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

야생 형의 제 3 령 애벌레 NMJs에서 굴림은 주로 유형의 종말보다 굴림 면역 수준이 형 IB의 종말에서 더 두드러 인 상태, I 형 글루타메이트 종말의 시냅스 막에서 발견된다 (그림 3A) 4. HTS는 근육에 걸쳐 존재하지만 HTS 면역 수준이 두 유형 I 종말 동일 표시와 시냅스 부위에 집중하고, 또한 (그림 3A &#39;) 8,17 시냅스 발견된다. 굴림과 HTS의 분포가 크게 시냅?...

Watch this Scientific Journal Video about Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay at JoVE.com

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

이 프로토콜은 근접 내고 분석은 초파리 애벌레 신경 근육 접합부에서 현장 단백질 - 단백질 상호 작용을 검출 할 수있는 방법을 보여줍니다. 이 기술, 디스크 크고 후진타오 - 리 타이 샤오는 시냅스 지역에서 복잡한, 이전에 공동 면역 통해 식별 관계를 형성하기 위해 표시됩니다.

검출<em&gt; 현장에서</em상기&gt; 단백질 - 단백질 복합체<em&gt; 초파리</em근접 내고 분석을 사용하기&gt; 애벌레 신경 근육 접합부

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

detection-situ-protein-protein-complexes-at-drosophila-larval

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JoVE Journal

Neuroscience

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

13.0K 조회수.  Simon Fraser University.  이 프로토콜은 근접 내고 분석은 초파리 애벌레 신경 근육 접합부에서 현장 단백질 - 단백질 상호 작용을 검출 할 수있는 방법을 보여줍니다. 이 기술, 디스크 크고 후진타오 - 리 타이 샤오는 시냅스 지역에서 복잡한, 이전에 공동 면역 통해 식별 관계를 형성하기 위해 표시됩니다. 

비디오: Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

3 애벌레 Segmental 신경의 시각화 차 Instar Drosophila 애벌레 준비

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

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신경과학

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Drosophila 애벌레 Segmental Axons 이내 시냅틱 Vesicles의 생체내 시각화에

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

의 활성 영역의 해부 및 이미징 Drosophila 신경근육학 융티온

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

연속 신경 근육 분기점에서 싱글 Schwann 세포를 윤곽을 그리다하는 사진 표백

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

상기 표현형의 특성을 : 정량이 중요한 이유<em&gt; 초파리</em&gt; 애벌레 신경 근육 학 분기점

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

높은 처리량 및 다중 파라 메트릭 정량화를위한 ​​두 알고리즘<em&gt; 초파리</em&gt; 신경 근육 접합 형태학

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

초파리 애벌레 신경 근육 학 교차로에서 시 냅 스 전류의 초점 Macropatch 녹음

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The well characterized larval neuromuscular junction is often used for such studies. However, rapid larval development followed by muscle histolysis and nervous system remodeling during metamorphosis makes this model problematic for the study of slow age-dependent degenerative changes like those occurring in amyotrophic lateral sclerosis. Alternatively, adult flies live for 90 days and the adult leg can be used to study motor neuron changes over the course of adult lifespan using in vivo fluorescent imaging through the cuticle. Here, we describe a leg dissection technique coupled with immunocytochemistry, which allows for the study of molecular changes at the neuromuscular junction of identified adult leg motor neurons. These techniques can be coupled with a myriad of antibodies labeling both pre- and post-synaptic structures. Together these procedures allow a more complete characterization of slow age-dependent changes in adult flies and can be applied across multiple motor neuron disease models.

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

We describe a dissection technique that preserves the architecture of the neuromuscular junction and enables a detailed immunocytochemical study of motor neurons in the adult Drosophila leg.

확인된 모터 뉴런을 위한 신경근육 정션에서 변경을 검출하기 위하여 Drosophila 성인 다리의 해부 및 면역 조직 화학

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The ...

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting the expression of G protein-coupled receptors (GPCRs). Formalin-fixed jugular-nodose ganglia were processed with the RNAscope technology to simultaneously detect the expression of two representative GPCRs (cholecystokinin and ghrelin receptors) in combination with one marker gene of either nodose (paired-like homeobox 2b, Phox2b) or jugular afferent neurons (PR domain zinc finger protein 12, Prdm12). Labeled ganglia were imaged using confocal microscopy to determine the distribution and expression patterns of the aforementioned transcripts. Briefly, Phox2b afferent neurons were found to abundantly express the cholecystokinin receptor (Cck1r) but not the ghrelin receptor (Ghsr). A small subset of Prdm12 afferent neurons was also found to express Ghsr and/or Cck1r. Potential technical caveats in the design, processing, and interpretation of multiplex ISH are discussed. The approach described in this article may help scientists in generating accurate maps of the transcriptional profiles of vagal afferent neurons.

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

Multiplex in situ hybridization (ISH) was employed to simultaneously visualize the transcripts for two G protein-coupled receptors and one transcription factor in the entire vagal ganglionic complex of the adult mouse. This protocol could be used to generate accurate maps of the transcriptional profiles of vagal afferent neurons.

시투 혼성화에서 멀티플렉스를 사용하여 마우스 Vagal afferent 뉴런에서 G 단백질 결합 수용체 발현의 검출

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting ...