Name: immunohistological labeling of microtubules in sensory neuron dendrites, tracheae, and muscles in the drosophila larva body wall
Uploaded: 2011-11-10T13:00:00
Description: 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

Vi tackar RIKEN för finansiering. P10-Gal4 var en sorts gåva av Alain Vincent (Université Paul Sabatier, Toulouse, Frankrike).

1. Beredning av reagens Anteckningar innan du börjar: Dissection och immunhistokemisk färgning utförs i en magnetisk kammare och larven är låst ner med speciellt formade insekter stift. Detaljerade anvisningar om byggandet av en magnetisk kammare, och beredning av dessa stift finns i referenser 14,15. I korthet är en 1x1cm fyrkantigt hål skärs i en magnetisk plåt och ett täckglas som fästs på baksidan av bladet för att göra en liten kammare. Sidorna av...

<ol>
	<li>Schottenfeld, J., Song, Y., Ghabrial, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tube+continued:+morphogenesis+of+the+Drosophila+tracheal+system.">Tube continued: morphogenesis of the Drosophila tracheal system.</a> Curr. Opin. Cell. Biol. 22, 633-639 (2010).</li><li>Gao, F. B., Brenman, J. E., 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=Genes+regulating+dendritic+outgrowth,+branching,+and+routing+in+Drosophila.">Genes regulating dendritic outgrowth, branching, and routing in Drosophila.</a> Genes Dev. 13, 2549-2561 (1999).</li><li>Corty, M. M., Matthews, B. J., Grueber, 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=Molecules+and+mechanisms+of+dendrite+development+in+Drosophila.">Molecules and mechanisms of dendrite development in Drosophila.</a> Development. 136, 1049-1061 (2009).</li><li>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=Intrinsic+mechanisms+to+define+neuron+class-specific+dendrite+arbor+morphology.">Intrinsic mechanisms to define neuron class-specific dendrite arbor morphology.</a> Cell. Adh. Migr. 2, 81-82 (2008).</li><li>Grueber, W. B., 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=Tiling+of+the+Drosophila+epidermis+by+multidendritic+sensory+neurons.">Tiling of the Drosophila epidermis by multidendritic sensory neurons.</a> Development. 129, 2867-2878 (2002).</li><li>Nishimura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+Behaviors+and+Segmental+Coordination+During+Larval+Locomotion+Is+Disrupted+by+Nuclear+Polyglutamine+Inclusions+in+a+New+Drosophila+Huntington's+Disease-Like+Model.">Selection of Behaviors and Segmental Coordination During Larval Locomotion Is Disrupted by Nuclear Polyglutamine Inclusions in a New Drosophila Huntington's Disease-Like Model.</a> J. Neurogenet. 24, 194-206 (2010).</li><li>Ramon y Cajal, 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> Histology of the nervous system of man and vertebrates, 1995 translation. , (1911).</li><li>London, M., Hausser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+computation.">Dendritic computation.</a> Annu. Rev. Neurosci. 28, 503-532 (2005).</li><li>Jinushi-Nakao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knot/Collier+and+cut+control+different+aspects+of+dendrite+cytoskeleton+and+synergize+to+define+final+arbor+shape.">Knot/Collier and cut control different aspects of dendrite cytoskeleton and synergize to define final arbor shape.</a> Neuron. 56, 963-978 (2007).</li><li>Li, W., Gao, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+filament-stabilizing+protein+tropomyosin+regulates+the+size+of+dendritic+fields.">Actin filament-stabilizing protein tropomyosin regulates the size of dendritic fields.</a> J. 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>

För att förstå hur komplexa cell former uppnås är det viktigt att kunna korrekt analys mikrotubuli organisation. Här beskriver vi ett robust immunohistological märkning metod för analys mikrotubuli organisation av dendritiska sensoriska arborization neuron dendriter. Förutom färgning sensoriska nervceller, uppnår denna metod robusta immunohistological färgning av luftstrupe, muskler och andra kroppsvävnader vägg. Vi använder detta protokoll för att undersöka mikrotubuli orga...

<table border="1"><tbody><tr><th> Reagensets namn </th><th> Företag </th><th> Katalog Antalet </th><th> Kommentarer (Tillval) </th></tr><tr><td> Tång </td><td> Dumont </td><td> 11.251-20 </td><td></td></tr><tr><td> Microscissors </td><td> FST </td><td> 15.000-08 </td><td></td></tr><tr><td> Mus-anti-α-tubulin (Klon: DM1A) </td><td> Sigma </td><td> T9026 </td><td> Spädning 1 / 1000 </td></tr><tr><td> Mus-anti-Futsch (Klon: 22C10) supernatanten </td><td> Developmental Studier Hybridom Bank </td><td> 22C10 </td><td> Spädning 1 / 1000 </td></tr><tr><td> Rat anti-CD8 (Klon: 5H10) </td><td> Caltag </td><td> MCD0800 </td><td> Spädning 1 / 1000 </td></tr><tr><td> Alexa Fluor 488 anti-mus IgG </td><td> Invitrogen </td><td> A-11.001 </td><td> Spädning 1 / 500 </td></tr><tr><td> Cy3 anti-Rat IgG </td><td> Jackson Immunoresearch </td><td> 712-166-150 </td><td> Spädning 1 / 200 </td></tr></tbody></table>

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

För att förstå hur skillnader i komplexa cell former uppnås är det viktigt att noggrant följa mikrotubuli organisation. Det Drosophila larvkroppen vägg innehåller flera celltyper som är modeller för att studera celler och vävnader morfogenes. Till exempel tracheae används för att undersöka rör morfogenes 1 och dendritiska arborization (DA) sensoriska nervceller i Drosophila larven har blivit ett primärt system för belysning av allmänna och neuron-class-specifika mekanismer av dendritiska differentiering 2-5 och degeneration 6 . Formen på Dendrite grenar kan variera kraftigt mellan neuron klasser, och även mellan olika grenar av en enda neuron 7,8. Genetiska studier i Da nervceller tyder på att skillnaden cytoskelettala organisation kan ligga till grund för morfologiska skillnader i dendritiska gren form 4,9-11. Vi erbjuder en robust metod immunologiska märkning för att enssay in vivo mikrotubuli organisation i DA sensoriska neuron Dendrite berså (figur 1, 2, Movie 1). Detta protokoll illustrerar dissekering och immunfärgning första INSTAR larv, ett stadium då aktiv sensoriska neuron Dendrite utväxt och förgrenade organisation sker 12,13. Förutom färgning sensoriska nervceller, uppnår denna metod robusta märkning av mikrotubuli organisation i musklerna (Movies 2, 3), luftstrupe (Figur 3, Movie 3) och andra kroppsvävnader vägg. Det är värdefullt för utredare som vill analysera mikrotubuli organisation på plats i kroppen väggen vid utredning av mekanismer som styr vävnader och celler form.

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

För att förstå hur komplexa cell former som neuronala dendriter, uppnås under utveckling är det viktigt att kunna korrekt analys mikrotubuli organisation. Här beskriver vi ett robust immunohistological märkning metod för att undersöka mikrotubuli organisation av dendritiska sensoriska arborization neuron dendriter, luftstrupe, muskler och andra<em&gt; Drosophila</em&gt; Larv kropp vägg vävnader.

Immunohistological Märkning av mikrotubuli i sensoriska neuron dendriter, Tracheae och muskler i Drosophila Larva Body Wall

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

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Neuroscience

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

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

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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 ...

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

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

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

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Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...