Ringraziamo il Bloomington Drosophila Stock Center per la fornitura di scorte mosca. Ringraziamo anche il Developmental Studies Ibridoma Bank e il dottor Lynn Cooley (Yale University) per la fornitura di anticorpi. Un ringraziamento particolare va a AhHyun Yoo per il suo aiuto sul manoscritto. Questo lavoro è stato sostenuto da sovvenzioni dal scienze naturali e ingegneria Research Council del Canada (Krieger), William e Ada Isabelle Steel Fund (Krieger), e la Canadian Institutes of Health Research (Harden).

1. Preparazione parete Corpo NOTA: Preparazione di terzo stadio larvale pareti corpo (per lo studio delle NMJs che innervano i muscoli della parete corpo) è stata eseguita come precedentemente descritto in Brent et al 10, o Ramachandran e Budnik 11,12, ma con alcune modifiche.. <ol><li> Dissezione <ol><li> Sollevare scorte mosca e croci a 25 ° C per 5-6 giorni usando procedure standard 13. </li><li> Scegli strisciando terzo instar larve di fiale o flaconi utilizzando una pinza sottile. </li><li> Lavare le larve in un piccolo piatto di Petri contenente tampone fosfato salino (PBS) per rimuovere eventuali particelle di cibo. </li><li> Posizionare un larva su un disco Sylgard e immergerlo in alcune gocce di ghiaccio freddo PBS. Utilizzando ghiacciata PBS aiuterà stordire la larva rendendo più facile da manipolare. Durante la dissezione, assicurarsi che la preparazione è sempre immerso in PBS per evitare che si asciughi. </li><li> Posizionare il larva con il suo lato dorsale rivolto verso l&#39;alto in modo che i due tratti tracheali sono visibili al microscopio da dissezione (Figura 2A). Utilizzando le pinze per afferrare un perno minutien, pin la larva giù alla fine posteriore vicino alle spiracoli (Figura 2b). Con un altro perno, penetrare attraverso la cuticola all&#39;estremità anteriore vicino i ganci bocca. Allungare delicatamente la larva fuori il lungo, poi pin basso (Figura 2B). </li><li> Utilizzando forbici microdissezione, pizzicare la fine posteriore vicino al pin per creare una piccola apertura. L&#39;incisione dovrebbe essere abbastanza superficiale passare solo attraverso la cuticola. </li><li> Posizionando la punta della lama inferiore delle forbici nell&#39;incisione, tagliare lungo l&#39;intera lunghezza della linea mediana dorsale tra i due tratti tracheali (Figura 2C). Puntare le lame a forbice leggermente verso l&#39;alto quando il taglio per evitare di danneggiare i muscoli della parete del corpo ventrali. </li><li> Fai una piccola incisione orizzontale leggermente anteriore al postpin eriorly-posto (Figura 2C). Fare un&#39;altra incisione simile poco posteriore al pin anteriormente classificata (Figura 2C). Le incisioni dovrebbero solo passare attraverso la cuticola. NOTA: Le tre incisioni di passaggi 1.1.7 e 1.1.8 combinati dovrebbe assomigliare ad un &quot; <img fo:content-width="0.09in" src="/files/ftp_upload/52139/52139_letter_I.jpg" width="15px" /> &quot;Una volta completato, cioè, un lembo sinistro e destro sul lato dorsale del corpo larvale dovrebbe essere prodotto. </li><li> Pulire accuratamente fuori gli organi interni con le pinze. Aggiungendo qualche goccia forti di PBS aiuterà spostare gli organi dal corpo larvale, rendendo così più facile per rimuoverli. Evitare colpendo il corpo larvale in quanto causerà danni ai muscoli della parete del corpo. </li><li> Srotolare il corpo larvale aperto e appuntare gli angoli verso il basso (Figura 2D). Quando pinning, allungare la parete del corpo sia orizzontalmente che verticalmente per formare un rettangolo uniformemente-tensione (see Figura 2G per la forma), facendo attenzione a non strappare i muscoli della parete del corpo nel processo. </li><li> Finire rimuovendo eventuali organi interni rimanenti (Figura 2E). </li></ol></li><li> Fissazione e permeabilizzazione <ol><li> Immergere le pareti del corpo appuntato alcune gocce di soluzione di Bouin. Incubare per 15 minuti sul ghiaccio. In alternativa, utilizzare 4% paraformaldeide (PFA) come fissativo alternativo; incubare per 30 min. </li><li> Risciacquare tre volte con tampone fosfato Saline con Triton (PBT). </li><li> Utilizzando una pinza sottile, rimuovere con attenzione i perni e trasferire le pareti del corpo da parte dei loro angoli in un siliconato 0,65 ml provetta. </li><li> Conservare le pareti del corpo in PBT a 4 ° C fino al momento PLA. Per ottenere risultati ottimali, avviare immunocolorazione le pareti del corpo entro un giorno o due di dissezione. NOTA: Per risparmiare i reagenti e di garantire che tutte le pareti del corpo sono trattati allo stesso modo durante il test, diversi genotipi possono essere inseriti in un unicotube. I genotipi possono distinguere tagliando gli angoli delle pareti del corpo diverso (vedere Figura 2F per gli esempi). </li></ol></li></ol> 2. immunoistochimica NOTA: immunocolorazione di terzo stadio larvale pareti corpo è stata eseguita come precedentemente descritto in Brent et al 14 e Ramachandran e Budnik 11, ma con alcune modifiche 11,14.. NOTA: Eseguire tutte le operazioni a temperatura ambiente e con agitazione se non diversamente specificato. <ol><li> Blocco <ol><li> Lavare le pareti del corpo con PBT tre volte per 10 minuti ciascuno. </li><li> Blocco con 1% albumina sierica bovina (BSA) per 1 ora. </li></ol></li><li> Immunostaining <ol><li> Incubare le pareti del corpo con anticorpi primari di topo e di coniglio contro le due proteine ​​di interesse (diluito in 1% BSA) per 2 ore a temperatura ambiente, o per una notte a 4 ° C. In questo caso, utilizzare il mouse 01:10 anti-Dlg e 1: 250 coniglio unNTI-HtsM 15,16. Gli anticorpi contro i marcatori che non sono fatti nel topo o di coniglio possono essere inclusi - per esempio, utilizzare 1: 200 di capra anti-Hrp per delineare le membrane neuronali. </li><li> Lavare con PBT tre volte per 10 minuti ciascuno. </li><li> Incubare con anticorpi secondari coniugati fluoroforo per rilevare marcatori (diluito in 1% BSA) per 2 ore a temperatura ambiente, o per una notte a 4 ° C. Poiché il segnale PLA viene successivamente visualizzata con un fluoroforo rosso, un&#39;altra fluoroforo deve essere utilizzato per rilevare il marcatore - esempio, utilizzare 1: 200 FITC-coniugato anti-capra per rilevare l&#39;anticorpo anti-Hrp capra. Grazie all&#39;uso di reagenti sensibili alla luce, mantenere i tubi al buio da questo punto in poi. NOTA: Il kit utilizzato permette di PLA da eseguire tra anticorpi primari sollevate nel topo e coniglio, con il segnale visualizzato sul canale rosso al microscopio confocale. Se lo si desidera, altri kit sono disponibili che consentono il dosaggio di essere fatto con primaanticorpi Ry sollevate in altre specie, e il segnale visualizzati su altri canali. </li></ol></li></ol> 3. prossimità legatura Assay NOTA: Eseguire tutte le operazioni a temperatura ambiente e con agitazione se non diversamente specificato. <ol><li> PLA Sonde <ol><li> Lavare le pareti del corpo con PBT tre volte per 10 minuti ciascuno. </li><li> Incubare con sonde PLA (1: 5 di diluizione ciascuno in 1% BSA) per 2 ore a 37 ° C. In questo caso, utilizzare 40 ml di PLA sonda anti-topo MENO, 40 ml di sonda PLA PLUS anti-coniglio e 120 ml di 1% BSA per garantire il corretto immersione e miscelazione di 5-10 pareti del corpo. Fino ad una diluizione 1:25 sonde PLA ancora può risultare in un rapporto adeguato segnale-rumore (cioè, per questo esperimento). </li></ol></li><li> Legatura <ol><li> Lavare le pareti del corpo con tampone di lavaggio A due volte per 5 minuti ciascuno. </li><li> Incubare con la soluzione di legatura (1:40 diluizione della Ligase in tampone legatura) per 1 ora at 37 ° C. In questo caso, usare 5 ml di Ligase, 40 ml di tampone 5 legatura e 155 ml di acqua ad elevata purezza per garantire il corretto immersione e miscelazione di 5-10 pareti del corpo. </li></ol></li><li> Amplificazione <ol><li> Lavare le pareti del corpo con tampone di lavaggio A due volte per 2 minuti ciascuna. </li><li> Incubare con la soluzione Amplification (1:80 diluizione della polimerasi in tampone Amplificazione) per 2 ore a 37 ° C. In questo caso, utilizzare 2,5 ml di polimerasi, 40 ml di 5 buffer di amplificazione e 157,5 ml di acqua ad elevata purezza per garantire il corretto immersione e miscelazione di 5-10 pareti del corpo. </li></ol></li><li> Preparazione Imaging <ol><li> Lavare le pareti del corpo con tampone di lavaggio B due volte per 10 minuti ciascuno. </li><li> Lavare con 0,01X tampone di lavaggio B una volta per 1 min. </li><li> Equilibrare in poche gocce di soluzione di montaggio per almeno 30 minuti prima del montaggio, o conservare una notte a 4 ° C. </li><li> Utilizzando una pinza sottile, trasferire accuratamente le pareti del corpo su una piattaforma scorrevole wesimo loro cuticole rivolto verso il basso. Posizionare le pareti del corpo in righe e nello stesso orientamento in una o due gocce di montante. Posizionare un 22 mm x 40 millimetri coprioggetto sulla preparazione facendo attenzione a non creare bolle d&#39;aria, quindi sigillare il vetrino con smalto trasparente. </li><li> Conservare i vetrini al buio a -20 ° C fino al momento per l&#39;imaging confocale. </li></ol></li></ol>

<ol>
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Questo rapporto dimostra come PLA può essere applicata alla Drosophila larvale NMJ. Il test viene eseguito su preparazioni muscolari parete corpo larvali al fine di rilevare le interazioni proteina-proteina endogeni presenti al NMJ. Con questa tecnica, Dlg e HTS vengono mostrati per essere in stretta vicinanza l&#39;uno all&#39;altro, e pertanto esistere in un complesso, in particolare in corrispondenza della zona 27 postsinaptico. A sostegno di questo risultato, un precedente studio ha fornito prov...

Drosophila dischi di grandi dimensioni (Dlg) è un membro conservato della guanilato famiglia chinasi associata alla membrana di ponteggi proteine ​​che aiutano a orchestrare l&#39;assemblaggio di grandi complessi proteici in siti specifici della membrana plasmatica. Originariamente identificato come una proteina soppressore del tumore, Dlg serve come un importante determinante di epiteliale 1,2,3 apicobasal polarità. Dlg serve anche come un importante modulo di ponteggio a livello della giunzione neuromuscolare (NMJ) dei motoneuroni glutamatergici durante lo sviluppo larvale 4. Dlg svolge ruoli diversi al larvale NMJ, e la sua pleiotropismo si basa sulla sua capacità di associare con più proteine ​​5,6. Una di queste proteine ​​è Hu-li tai shao (Hts), un omologo alle adducins mammiferi che sono stati descritti principalmente per quanto riguarda i loro ruoli nella regolazione della actina-spettrina citoscheletro 7. È stato precedentemente dimostrato che Dlg e Hts possono formare un complesso con l&#39;altro sulla base in vitro </em&gt; esperimenti di co-immunoprecipitazione coinvolgono tutta adulti di mosca lisati 8. Una carenza di questi risultati, tuttavia, è che non indicano dove Questo forme complesse. Con l&#39;uso di immunoistochimica, le distribuzioni di Dlg e HTS vengono osservate per sovrapporsi alla membrana postsinaptica del NMJs larvali, ma sono in un complesso in questa regione 8? Come recentemente dimostrato e dettagliato ulteriormente qui, di prossimità legatura Assay (PLA) è utilizzato per cercare un in situ associazione tra Dlg e Hts specificamente al larvale NMJ 27. PLA è una relativamente nuova tecnica utilizzata principalmente in cellule e tessuti cultura in grado di rilevare le interazioni proteina-proteina in situ 9. In questo saggio, gli anticorpi primari contro le due proteine ​​di interesse vengono rilevati con un paio di anticorpi secondari specie-specifici, definito sonde PLA, che sono coniugati ad oligonucleotidi (Figura 1A, B). Se le due proteines sono in stretta vicinanza l&#39;uno all&#39;altro (cioè entro poche decine di nanometri), la distanza tra le sonde PLA allegate può essere colmato attraverso ibridazione di oligonucleotidi due connettori aggiuntivi (Figura 1C). In questa conformazione, le estremità libere delle oligonucleotidi connettore sono abbastanza vicino per fare contatto con l&#39;altro, e una molecola di DNA circolare chiuso possono essere formati su di legatura in situ (Figura 1D). La molecola di DNA circolare serve come modello per in situ cerchio di rotolamento di amplificazione, che è innescato da uno degli oligonucleotidi coniugati alle sonde PLA (Figura 1E). Sequenze all&#39;interno amplificato, prodotto DNA concatemeric risultante possono poi essere visualizzati con, sonde oligonucleotidiche complementari fluorescenza marcata (Figura 1F). Poiché il DNA amplificato rimane attaccato ad una delle sonde PLA, la localizzazione subcellulare della proteina-proteina interazione withina tessuto può essere prontamente accertata. Diversi metodi sono comunemente usati per rilevare le interazioni proteina-proteina anche in tecniche in vitro, come co-immunoprecipitazione, pull-down test e lievito di screening doppio ibrido, e in tecniche vivo come il trasferimento Förster Resonance Energy (FRET) e bimolecolari complementazione fluorescenza ( BiFC). Una trappola delle tecniche in vitro è che essi non identificano in cui l&#39;interazione è endogeno in corso, mentre le tecniche vivo il citate in coinvolgono l&#39;espressione artificiale di proteine ​​di fusione che non può riflettere il comportamento nativo dei loro omologhi endogeni. Uno dei principali vantaggi di PLA è che è in grado di determinare all&#39;interno di un tessuto la localizzazione subcellulare di interattori proteici endogeni che sono in stretta vicinanza l&#39;uno all&#39;altro e probabilmente formano un complesso, con il grado di vicinanza necessaria per generare un segnale è paragonabile alla FRET e BiFC. PLA può rilevare interazioni con alta specificità e sensibilità a causa dell&#39;accoppiamento del riconoscimento anticorpale e amplificazione del DNA. Così, il dosaggio può generare segnali luminosi discreti, in forma di puncta che rivelano la posizione esatta dell&#39;interazione. Inoltre, gli antigeni difficilmente visibili possono essere rilevati. Infine, PLA è una tecnica relativamente semplice da eseguire, e non richiede una procedura di immunoistochimica standard per completare. Pertanto, PLA offre un vantaggio tecnico rispetto agli altri saggi di interazione proteina-proteina che sono spesso afflitto con tempi lunghi di preparazione e ampie risoluzione dei problemi. Questo protocollo illustra come PLA può essere applicato al Drosophila larvale NMJ al fine di rilevare le interazioni proteina-proteina endogena in situ. Qui, PLA viene eseguita su preparazioni muscolari parete corpo larvali dove Dlg e Hts sono mostrati di esistere davvero in un complesso in zona post-sinaptica di NMJs. PLA non è stata precedentemente utilizzata per studiare la larvale NMJ, e ci sono al momento solo una manciata di articoli pubblicati che hanno utilizzato questo test nel tessuto Drosophila. Si spera che ulteriore esposizione del PLA per la comunità Drosophila si tradurrà in un maggiore ricorso come uno strumento aggiuntivo per integrare altri, più comunemente usati proteina-proteina saggi di interazione.

<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.

In wild-type terzo stadio larvale NMJs, Dlg è prevalentemente trova a livello della membrana post-sinaptica di tipo I boutons glutamatergiche, con livelli di immunoreattività Dlg essere più pronunciato in tipo IB boutons di tipo è boutons (figura 3A) 4. Hts è presente in tutto il muscolo, ma si concentra in zona post-sinaptica con Hts livelli immunoreattività appare uguale in entrambi di tipo I boutons, ed è anche trovato presinapticamente (figura 3A &#39;) 8,17.<...

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

Questo protocollo dimostra come Proximity legatura test può essere utilizzato per rilevare in situ interazioni proteina-proteina in Drosophila larvale neuromuscolare giunzione. Con questa tecnica, dischi grandi e Hu-li Shao tai sono presenti per formare un complesso in corrispondenza della zona postsinaptico, un&#39;associazione precedentemente identificato mediante co-immunoprecipitazione.

Rilevamento di<em&gt; In Situ</em&gt; Complessi proteina-proteina del<em&gt; Drosophila</em&gt; Larvale neuromuscolare Junction Utilizzando prossimità legatura 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 ...

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

Research

JoVE Journal

Neuroscience

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

13.0K Views.  Simon Fraser University.  Questo protocollo dimostra come Proximity legatura test può essere utilizzato per rilevare in situ interazioni proteina-proteina in Drosophila larvale neuromuscolare giunzione. Con questa tecnica, dischi grandi e Hu-li Shao tai sono presenti per formare un complesso in corrispondenza della zona postsinaptico, un'associazione precedentemente identificato mediante co-immunoprecipitazione. 

Video: 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.

Visualizzazione dei nervi larvali segmentale in 3 Rd Instar Drosophila I preparativi larvali

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

Ricerca

Neuroscienze

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.

Nella visualizzazione in vivo delle vescicole sinaptiche In Drosophila assoni larvali segmentale

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.

Dissezione e immagini di zone attive nel Drosophila Giunzione neuromuscolare

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.

Sequential Photo-sbiancante per Delineare cellule di Schwann singoli sulla giunzione neuromuscolare

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.

Perché Quantificazione Matters: Caratterizzazione dei fenotipi al<em&gt; Drosophila</em&gt; Larvale Giunzione neuromuscolare

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.

Funzionalità di misurazione della giunzione neuromuscolare

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).

Due Algoritmi per high-throughput e multi-parametrica Quantificazione degli<em&gt; Drosophila</em&gt; Giunzione neuromuscolare Morfologia

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.

Macropatch focale registrazioni delle correnti sinaptiche da giunzione neuromuscolare larvale della drosofila

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.

Dissezione e immunoistochimica della gamba adulta di Drosophila per rilevare i cambiamenti alla giunzione neuromuscolare per un motoneurone identificato

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.

Rilevazione dell'espressione del recettore accoppiato alla proteina G nei neuroni afferenti vagali di topo utilizzando l'ibridazione multiplex in situ

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