Name: use of immunolabeling to analyze stable, dynamic, and nascent microtubules in the zebrafish embryo
Uploaded: 2017-09-20T13:00:00
Description: Watch this Scientific Journal Video about Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo at JoVE.com

Konfokal mikroskop blev købt med midler fra den amerikanske National Science Foundation (NSF), grant #DBI-0722569. Forskningen blev støttet af det amerikanske nationale institutter af sundhed/National Institute of General Medical Sciences (NIH/NIGMS) grant #GM085290 og amerikanske Institut for Defense (DOD) grant #W81XWH-16-1-0466 tildeles R.M. Brewster. E. Vital blev støttet af en bevilling til UMBC fra Howard Hughes Medical Institute gennem pre college og Undergraduate Science Education Program, yde #52008090. S.P. Brown blev støttet af en US Department af uddannelse GAANN Fellowship, en Meyerhoff Graduate stipendium finansieret af NIH/NIGMS tilskud, #GM055036, og en forskning undervisningsopholdet finansieret af den amerikanske DOD grant #W81XWH-16-1-0466.

etik erklæring: procedurerne beskrevet nedenfor følger University of Maryland Baltimore County dyrs pleje retningslinjer.
 1. analyse af stabile og dynamiske MTs ved hjælp af Immunolabeling (protokol 1) <ol><li>Manuel dechorionation af embryoer før fiksering <ol><li>få frisk opfostrede embryoner ved at hælde off overskydende system vand og derefter indsamle resterende embryoner i en plastik petriskål (Se Tabel of Materials).</li>
		<li>Fjerne snavs f...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+regulation+of+the+microtubule+and+actin+cytoskeleton+in+zebrafish+epiboly.">Dynamic regulation of the microtubule and actin cytoskeleton in zebrafish epiboly.</a> Biochem Biophys Res Commun. 452 (1), 1-7 (2014).</li><li>Bulinski, J. C., Gundersen, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilization+and+post-translational+modification+of+microtubules+during+cellular+morphogenesis.">Stabilization and post-translational modification of microtubules during cellular morphogenesis.</a> BioEssays. 13 (6), 285-293 (1991).</li><li>Magiera, M. M., Janke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+16+-+Investigating+Tubulin+Posttranslational+Modifications+with+Specific+Antibodies.">Chapter 16 - Investigating Tubulin Posttranslational Modifications with Specific Antibodies.</a> Methods Cell Biol. 115, 247-267 (2013).</li><li>Hong, E., Jayachandran, P., Brewster, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+polarity+protein+Pard3+is+required+for+centrosome+positioning+during+neurulation.">The polarity protein Pard3 is required for centrosome positioning during neurulation.</a> Dev Biol. 341 (2), 335-345 (2010).</li><li>Westermann, S., Weber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+modifications+regulate+microtubule+function.">Post-translational modifications regulate microtubule function.</a> Nat Rev Mol Cell Biol. 4 (12), 938-948 (2003).</li><li>Jayachandran, P., Olmo, V. N., 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=Microtubule-associated+protein+1b+is+required+for+shaping+the+neural+tube.">Microtubule-associated protein 1b is required for shaping the neural tube.</a> Neural Develop. 11, 1 (2016).</li><li>Nam, S. -. C. <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+Tau,+a+microtubule+associated+protein,+in+Drosophila+photoreceptor+morphogenesis.">Role of Tau, a microtubule associated protein, in Drosophila photoreceptor morphogenesis.</a> Genes N Y N 2000. 54 (11), 553-561 (2016).</li><li>Abal, M., Piel, M., Bouckson-Castaing, V., Mogensen, M., Sibarita, J. -. B., Bornens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+release+from+the+centrosome+in+migrating+cells.">Microtubule release from the centrosome in migrating cells.</a> J Cell Biol. 159 (5), 731-737 (2002).</li><li>Delgehyr, N., Sillibourne, J., Bornens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+nucleation+and+anchoring+at+the+centrosome+are+independent+processes+linked+by+ninein+function.">Microtubule nucleation and anchoring at the centrosome are independent processes linked by ninein function.</a> J Cell Sci. 118 (8), 1565-1575 (2005).</li><li>Manning, J. A., Lewis, M., Koblar, S. 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Der er i øjeblikket mange metoder til imaging MT dynamics i tidlige zebrafisk udvikling, lige fra live imaging af mærkede molekyler til immunolabeling af faste væv11,12,13,14. Selvom MTs i en enkelt celle kan findes i enten dynamiske eller stabile stater, er epithelialization en proces, hvor stabiliseret MTs gradvist over tid. Ved hjælp af markører for stabile og dynamiske MTs tilbyder en ...

Mikrotubuli (MTs) er polymerer af α - og β-tubulin, samles til lineære protofilaments, hvoraf flere kombinere til at danne et hult rør1,2. MTs er polariseret strukturer, med hurtigtvoksende plus ender og langsomt voksende minus ender, der er forankret på centrosome eller andre organisering af mikrotubulus center (MTOC)3. De novo MT dannelse er initieret af Nukleering på γ-tubulin ring kompleks (γ-TURC), som indeholder en skabelon til MT forsamling4. I en given celle, kan to populationer af MTs skelnes der tur til forskellige priser. Dynamisk MTs udforske deres cellulære miljø ved at skifte mellem faser af vækst og svind i en proces kendt som dynamiske ustabilitet5. I modsætning til dynamisk MTs, stabil MTs er ikke-voksende og har en længere halveringstid end dynamisk MTs6.

Årtiers forskning i cellebiologi har leveret en avanceret vifte af værktøjer til at studere MT struktur og funktion og resulterede i en omfattende viden om disse cytoskeletal elementer. For eksempel, MTs spiller en central rolle i etablering og vedligeholdelse af celle polaritet, som ikke kun kan tilskrives deres iboende polaritet, men også til en differentieret subcellulært fordeling af stabil versus dynamiske MTs7, 8. derimod langt mindre forstås om MT arkitektur og funktion i mere komplekse tredimensionale (3D) miljøer, såsom hvirveldyr embryoner, delvis på grund af udfordringen af imaging MT cytoskelettet med høj opløsning. Trods denne begrænsning, den seneste generation af normal god landbrugspraksis-udtrykker transgene linjer at mærke MTs eller forbigående udtryk af fluorescently markeret MT markører har øget vor forståelse af de dynamiske ændringer, som MTs gennemgå og deres cellulære og udviklingsmæssige rolle i zebrafisk embryo. Hele MT netværket kan være afbildet i transgene linjer i hvilke tubulin er enten direkte mærket9 eller tubulin polymerer er indirekte mærket ved hjælp af MT-associerede proteiner Doublecortin-lignende-kinase (Dclk) eller Ensconsin (EMTB)10, 11. Andre linjer (og konstruktioner) er blevet genereret, aktiverer vurdering af MT iboende polaritet ved specielt mærkning MT plus ender eller centrosome-forankrede minus ender11,12,13, 14. magt af disse værktøjer ligger i evnen til at studere MT dynamics i live, udvikle organismer. Sådanne undersøgelser har for eksempel afsløret den rumlige og dynamisk fordeling af MTs i specifikke cellepopulationer, orientering af mitotiske spindler i væv gennemgår morfogenese (en indikator af flyet af celledeling), polariteten af MT polymer da det drejer sig om processer såsom celle strækforlængelse og migration, og MT vækst bestemmes af comet hastighed9,13,15. Begrænsning af disse værktøjer er, at de ikke let diskriminerer mellem stabile og dynamiske MT populationer.

Tegning fra de rige celle biologi litteratur, er immunolabeling metoder til billede stabile og dynamiske MTs i zebrafisk embryo beskrevet her, der er komplementær til brugen af transgene linjer. Den udbredte brug af sådanne immunolabeling metoder i zebrafisk er blevet lidt hæmmet af vanskeligheden i at bevare MT integritet under proceduren fiksering. Protokol 1 beskriver en optimeret metode til immunolabeling samlet, dynamisk, og stabil MTs i cross dele af den tredje zebrafisk baghjernen. Derudover er en ligetil metode ved hjælp af kommercielt tilgængelige software beskrevet for at kvantificere disse MT populationer. Stabil MTs adskiller sig fra dynamiske MTs baseret på flere posttranslationelle modifikationer af α-tubulin, som acetylation og detyrosination, der akkumulerer på stabil MTs over tid16,17. I zebrafisk embryo opstår acetylation ciliaere og cytoskeletale MTs, men ikke stabilt interphase MTs18, begrænse nytten af denne markør til en delmængde af stabiliseret MTs. Derimod synes detyrosination at forekomme på alle stabile MTs i zebrafisk embryo18. Denne posttranslationel modifikation udsætter carboxy-terminal glutaminsyre α-tubulin (detyrosinated tubulin)18 og kan påvises ved hjælp af anti-Glu-tubulin19. Selv om detyrosination sker fortrinsvis på stabil MTs, angiver eksperimentelle bevismateriale, at denne posttranslationel modifikation er et resultat af, snarere end en årsag til MT stabilitet16. Den gensidige MT befolkning, sammensat af dynamiske MTs, udmærker sig ved hjælp af en antistof, anti-Tyr-tubulin, der specifikt genkender tyrosinated form af α-tubulin19. Efter immunolabeling med disse markører og konfokal imaging, kvantitativ analyse af MTs (længde, antal og relativ overflod) kan udføres i definerede regioner i udviklingslandene neuralrøret. En strømlinet metode er fastsat her til at udføre denne analyse ved hjælp af 3D-billedbehandling software. Denne metode kan anvendes til løsning af spørgsmål vedrørende morfogenese og etablering eller modning af celle polaritet20. Faktisk, udarbejdelse af polariseret arrays af stabil MTs ledsager mange udviklingsmæssige begivenheder, herunder fotoreceptor morfogenese21, epithelialization af celler i udviklingslandene neurale rør18 og axon dannelse8.

Protokol 2 beskriver en i vivo tilpasning af en celle biologi analyse til at analysere MTs under deres forsamling fase (Nukleering/anchorage og vækst)22,23. Spirende MTs er nukleeret på centrosome og efterfølgende forankret til subdistal vedhæng af mor centriole23. En metode til at analysere spirende MT genvækst efter depolymerization er beskrevet. Denne protokol indeholder oplysninger om nocodazole behandlingen af depolymerize MTs, drug udvaskning procedure og efterbehandling tilbagebetalingsperioden. MT re-vækst overvåges med regelmæssige mellemrums indlæg udvaskning af immunolabeling med markører for samlede MTs (anti-β-tubulin) sammen med markører for centrosome (anti-γ-tubulin) og kerne (4', 6-diamidino-2-phenylindole (DAPI)), efter de generelle procedurer beskrevet i protokol 1. MT depolymerization trin i denne protokol er afgørende, da det giver mulighed for vurdering af de novo MT vækst og ikke forlængelse af allerede eksisterende MTs. Denne teknik er derfor adskiller sig fra andre offentliggjorte procedurer til at måle MT vækstrater (i mangel af depolymerization) ved hjælp af en plus spids markør såsom end bindende protein 3 sammenvoksede til grøn fluorescerende proteiner (EB3-NGL), som vist i Tran et al., 201211. Desuden, denne analyse er især nyttig til at analysere embryoner defekt i de novo MT forsamling, som de tidligere rapporterede NEDD1 mutanter, rekruttering af γ-tubulin til centrosome er nedsat, hvilket resulterer i ufuldstændig neuralrøret dannelse og neuronal defekter24.

<table border="0" cellpadding="0" cellspacing="0" width="916">
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	<tbody>
		<tr height="140">
			<td height="140" style="height:140px;width:207px;">Low Melting Point Agarose</td>
			<td style="width:177px;">IBI scientific</td>
			<td style="width:159px;">IB70050</td>
			<td style="width:373px;">Only used for embedding. 
			 
			Prepare 4% LMP agarose 
			by heating a solution of&#160; 4 grams LMP agarose per 100 ml 1X TBS in a microwave 
			until polymerized. Keep warm on a 50 &#730;C hot plate with the cap secured</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:207px;">Agarose</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">Used to treat petridishes.&#160;&#160; Prepare 1% agarose 
			by heating a solution of&#160; 1 gram agarose per 100 ml 1X embryo medium in a microwave 
			until polymerized.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">Nocodazole</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">487928-10MG</td>
			<td style="width:373px;">Make stock by dissolving entire bottle of powder in distilled water and aliquoting into 500ul aliquots.&#160; Store at -20 &#730;C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">8% Formaldehyde</td>
			<td style="width:177px;">Electron Microscopy Sciences</td>
			<td style="width:159px;">157-8-100</td>
			<td style="width:373px;">Aliquot and store at -20 &#730;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Kpipes</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">P7643</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">NaCl</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">S7653</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Tris-HCl</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">T3253-500G</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">KCl</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">P9333-500G</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">CaCl2&#183;2H2O</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">C5080</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">NP-40</td>
			<td style="width:177px;">American Bioanalyticals</td>
			<td style="width:159px;">AB01424</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">EGTA</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">E3889-25G</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">MgCl2</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">M2670-500G</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Normal Goat Serum</td>
			<td style="width:177px;">Millipore</td>
			<td style="width:159px;">S26-100ml</td>
			<td style="width:373px;">Aliquot and store at -20 &#730;C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Bovine serum albumin (BSA)</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">BP1605</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Triton-x</td>
			<td style="width:177px;">American Bioanalyticals</td>
			<td style="width:159px;">AB02025</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">Pronase E (non-specific Protease from Streptomyces griseus)</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">P5147-1G</td>
			<td style="width:373px;">make stock solution by diluting 10mg per ml of ddH2O. Aliquot in 1ml aliquots and store at -20 &#730;C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Anti-Fade mounting medium</td>
			<td style="width:177px;">Invitrogen</td>
			<td style="width:159px;">P10144</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:207px;">DAPI</td>
			<td style="width:177px;">Invitrogen</td>
			<td style="width:159px;">D1306</td>
			<td style="width:373px;">Prepare 50 ml DAPI solution by first combining 1 &#181;l DAPI stock&#160; with 1 ml sterile water and then adding the DAPI/water mix to 49 ml 1X TBS; store in foil at 4 &#730;C for months.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:207px;">Mouse anti-&#946;-tubulin</td>
			<td style="width:177px;">Developmental studies Hybridoma Bank</td>
			<td style="width:159px;">E7</td>
			<td style="width:373px;">1/200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Rabbit anti-&#947;-tubulin</td>
			<td style="width:177px;">Genetex</td>
			<td style="width:159px;">GTX113286</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Rabbit anti-&#945;-tubulin</td>
			<td style="width:177px;">Genetex</td>
			<td style="width:159px;">GTX108784</td>
			<td style="width:373px;">1/1000*</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Rabbit anti-detyrosinated-tubulin</td>
			<td style="width:177px;">Millipore</td>
			<td style="width:159px;">AB3201</td>
			<td style="width:373px;">1/200-1/1000*&#160; Titrate antibody with first use of new lot.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Rabbit anti-tyrosinated-tubulin</td>
			<td style="width:177px;">Millipore</td>
			<td style="width:159px;">ABT171</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Mouse anti-centrin</td>
			<td style="width:177px;">Millipore</td>
			<td style="width:159px;">04-1624</td>
			<td style="width:373px;">1/1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Goat 488 anti-rabbit</td>
			<td style="width:177px;">Thermofisher</td>
			<td style="width:159px;">A11008</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Goat 594 anti-rabbit</td>
			<td style="width:177px;">Thermofisher</td>
			<td style="width:159px;">A11012</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Goat 594 anti-mouse</td>
			<td style="width:177px;">Thermofisher</td>
			<td style="width:159px;">A11005</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Goat 488 anti-mouse</td>
			<td style="width:177px;">Thermofisher</td>
			<td style="width:159px;">A11001</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Vibratome</td>
			<td style="width:177px;">Vibratome</td>
			<td style="width:159px;">1500</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Forceps</td>
			<td style="width:177px;">World Precision Instruments</td>
			<td style="width:159px;">555227F</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">100 mm petri dish</td>
			<td style="width:177px;">Cell treat</td>
			<td style="width:159px;">229693</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">35 mm petri dish</td>
			<td style="width:177px;">Cell treat</td>
			<td style="width:159px;">229638</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">50 ml falcon tube</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">14-432-22</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Woven nylon mesh 70 um</td>
			<td style="width:177px;">Amazon.com</td>
			<td style="width:159px;">B0043D1SZG&#160;</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Micropipette</td>
			<td style="width:177px;">Gilson</td>
			<td style="width:159px;">F123602</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Glass pipette</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">NC-999363-9</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Aquarium sealant</td>
			<td style="width:177px;">Amazon.com, by MarineLand</td>
			<td style="width:159px;">Silicone Sealer 1 oz (Tube)&#160;</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Ring stand</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">14-675BO</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Microbore PTFE Tubing, 0.022&#34;ID</td>
			<td style="width:177px;">Cole-Parmer</td>
			<td style="width:159px;">WU-06417-21</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Modeling clay</td>
			<td style="width:177px;">Amazon.com</td>
			<td style="width:159px;">Sargent Art 22-4000</td>
			<td style="width:373px;">Any wax or oil based non-toxic modeling clay will suffice</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Clamp</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">02-215-466</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">60ml syringe</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">14-820-11</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;width:207px;">Embryo medium (E3)</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">34.8 g NaCl 
			1.6 g KCl 
			5.8 g CaCl2&#183;2H2O 
			9.78 g MgCl2&#183;6H2O 
			 
			To prepare a 60X stock, dissolve the ingredients in H2O, to a final volume of 2 L. Adjust the pH to 7.2 with NaOH. Autoclave. To prepare 1X medium, dilute 16.5 mL of the 60X stock to 1 L.&#160;</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:207px;">Blocking Solution</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">50 ml TBS-NP-40 
			2.5 ml normal goat serum 
			1 g BSA 
			625 &#181;l Triton-X</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">TBS-NP-40 (pH 7.6)</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">155 mM NaCl 
			10 mM Tris HCl 
			0.1% NP-40</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">2x MAB (pH6.4)</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">160 mM KPIPES 
			10 mM EGTA 
			2 mM MgCl2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Commercial 3-D Image processing Software</td>
			<td style="width:177px;">PerkinElmer</td>
			<td style="width:159px;">Volocity (V 6.2)</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Dry block heater&#160;</td>
			<td style="width:177px;">VWR</td>
			<td style="width:159px;">12621-108</td>
			<td style="width:373px;">Used as a hot plate to melt agarose in Protocol 1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Dissecting Microscope</td>
			<td style="width:177px;">Leica</td>
			<td style="width:159px;">MZ12</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Confocal Microscope</td>
			<td style="width:177px;">Leica</td>
			<td style="width:159px;">SP5</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Flat embedding mold</td>
			<td style="width:177px;">emsdiasum.com</td>
			<td style="width:159px;">BEEM 70904-01</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Public domain image processing software</td>
			<td style="width:177px;">NIH</td>
			<td style="width:159px;">ImageJ (V 1.5)</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td style="width:532px;">* Success varies by lot number</td>
		</tr>
	</tbody>
</table>

use of immunolabeling to analyze stable, dynamic, and nascent microtubules in the zebrafish embryo

Mikrotubuli (MTs) er dynamisk og skrøbelige strukturer, der er udfordrende at billedet i vivo, navnlig med hvirveldyr embryoner. Immunolabeling metoder er beskrevet her til at analysere forskellige populationer af MTs i zebrafisk embryo udvikling neuralrøret. Mens der er fokus på neurale væv, er denne metode generelt gældende for andre væv. Procedurerne, der er optimeret for tidligt til midten af somitogenesis fase embryoner (1 somite til 12 somites), men de kan tilpasses til en række andre faser med relativt mindre justeringer. Den første protokol indeholder en metode til at vurdere den geografiske fordeling af stabile og dynamiske MTs og udføre en kvantitativ analyse af disse befolkninger med billedbehandling software. Denne tilgang supplerer eksisterende værktøjer til image mikrotubulus dynamik og distribution i realtid, ved hjælp af transgene linjer eller forbigående udtryk af mærket konstruktioner. Ja, sådanne værktøjer er meget nyttige, men de ikke let skelner mellem dynamisk og stabil MTs. Evnen til at billede og analysere disse særskilte mikrotubulus befolkninger har stor betydning for forståelse underliggende celle polarisering og morfogenese mekanismer. Den anden protokol beskriver en teknik til at analysere spirende MTs specifikt. Dette opnås ved at indfange de novo vækstegenskaber af MTs over tid, efter mikrotubulus depolymerization med stoffet nocodazole og en restitutionsperiode efter narkotika udvaskning. Denne teknik endnu ikke er anvendt til undersøgelse af MTs i zebrafisk embryoner, men er en værdifuld analyse for at undersøge in vivo funktion af proteiner involveret i mikrotubulus forsamling.

Analyse af stabile og dynamiske MTs ved hjælp af immunolabeling I protokol 1, er fordelingen af MT delpopulationer i begyndelsen (neurale køl) og slutningen (neurale stang) stadier af neuralrøret udvikling afsløret, bruger Glu-tubulin og Tyr-tubulin som markører for stabile og dynamiske MTs, henholdsvis. Dynamisk MTs dominerer i baghjernen på stadiet neurale køl (4-5 somites) (figur 2A-D). Som køl ...

Watch this Scientific Journal Video about Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo at JoVE.com

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Immunolabeling metoder til at analysere forskellige populationer af mikrotubuli i udviklingslandene zebrafisk hjernen er beskrevet her, som anvendes bredt til andre væv. Den første protokol skitserer en optimeret metode for immunolabeling stabile og dynamiske mikrotubuli. Den anden protokol indeholder en metode til at billede og kvantificere spirende mikrotubuli specifikt.

Brug af Immunolabeling til at analysere stabil, dynamisk og vordende mikrotubuli i zebrafisk Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

Research

JoVE Journal

Developmental Biology

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Produktion og anvendelse af Lentivirus til selektivt at transducere Primary oligodendrocyt precursorceller for<em&gt; In vitro</em&gt; myelinering Analyser

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Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

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The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Microbead Implantation in the Zebrafish Embryo

Mikroperle Implantation i Zebrafisk Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Analyse af zebrafisk Larver Skeletmuskel Integritet med Evans blå farvestof

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

Imaging Subcellulære Strukturer i Living Zebrafisk Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Observation Mitotisk Division og Dynamics i en Live zebrafisk Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

AFM og Microrheology i cellen zebrafisk Embryo æggeblomme

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

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Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

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During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...