AntibodystainingonDrosophilapost-emergencewings

(NicolasGompel,EvaAylaSchröder,2015)

ThisprotocolisforthepreparationofDrosophilawings,immediatelyaftertheemergenceoftheflyfrom the pupa. Upon dissection of the wings, separation of the dorsal and ventral surfaces andfixation,thesewingscanbeprocessedforantibodystaining.ThisprotocolcanbeadaptedforRNAinsituhybridization.

Material

HeptanglueUnroll 4 full rollsof tesa® fotostrip (15mmx10m)double-sidedtape.Removetheprotective linerand immersethetape into200mln-heptaneinaclosedbottle.Makesurethatthetapeiscoveredbyheptane.Thegluedissolvesin2to3daysatroomtemperature,resultinginagluesolution.Transferthisgluesolution(butnottheremainingtaperibbon)toanewbottle(figure1).

Afteralongstorage,thegluecanbecomeviscous,whichmakesitshandlingmorecomplicated.Addheptaneandmixwell tomake itmorefluid;alternatively,preparenewglue.

DissectionslideThedissectedwingswillbecollectedandtemporarilyarrangedona dissection slide, before their transfer to a finalmounting slide.The dissection slide is a microscope slide coated with a 3.5 cmpieceofdouble-sidedtape(withitsprotectivelinerontheotherside).Theprotectivelinerprovidesthe smooth surface, necessary tonotdamage thedissectedwings. For an illustration, see the lastpictureoffigure4orthefirstpictureoffigure5.

SlidesforfinalwingmountingThe slides, ontowhich the dissectedwingswill bemounted,must be coatedwith a thin layer ofheptaneglueintheircentralpart.

- Wipeanydustorglassdebrisofftheslidewithacleantissue(Kimwipes®).- Add a drop of heptane gluewith a Pasteur pipette and directly spread it evenlywith the

pipettetiptoanovalshapeofapproximately1x3cm(seefigure2).Spreadthegluequicklyto prevent the edges of the liquid from drying as you are spreading. This would result inunwantedridges.

- Let thegluedrywhile theslide isplacedona flat surface.Donotmove theslideuntil theheptaneisfullygone.

Figure1.Heptaneglue.Left,Tesafilmsoakinginheptane.Right,dissolvedtapegluetransferedtocleanbottle.(Photos:©AylaSchröder)

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- To flatten the elevated edges of the dried drop, stamp another clean slide onto the glue,ideallyturnedata90°angle.Avoidshearingmovementsasthesewillimpairthesmoothnessofthesurface.

Toseparatethedorsalandventralcell layersofthewings,twoslidesareneeded.Theschematicinfigure2referstothepositionsandshapesofthegluelayersoneachslide.

Fixationsolution5%Formaldehyde/PFAin1xPBT(PhosphateBufferSaline,0,1%TritonX-100).

Example: 75 ml 2x PBT + 75 ml 10% Roti®-Histofix (phosphate-buffered Formaldehydesolution;Roth)

Washbuffer1xPBT(PhosphateBufferSaline,0,1%TritonX-100).

Figure2.Preparingaslideforfinalwingmounting.Picturesontheleft:processingofasingleslide.Scematicontheright:shapesofthegluelayersthatneedtobeproduced.(Photos:©AylaSchröder)

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Methods

StageselectionforwingdissectionWithin 30 min of emergence from the pupal case, adult flies unfold their wings (figure 3). Thisprocess is generally blocked when the flies are anaesthetized with CO2 and does not resume.Therefore,donotanaesthetizefliesthathavejusthatchedwithCO2.

Upon unfolding, the wing surfaces look dull, likely because the cells of the wing blade are stillpresent.Thesecellsstarttodelaminateasthewingsunfold.Withintwohoursafteremergence,thedorsalandventralepitheliadissociateandthewingbladecellsmigratetothethorax.Theventralanddorsal cuticle surfaces become tightly bound, looking shiny and iridescent (figure 3; Kiger et al.,2006).

For antibody staining, or in situ hybridization on unfolded post-emergence wings, before celldelamination,thefliesmustbestagedasshowninfigure3(30’afteremergence).Additionallytothedull wings, flies of the right age remain overall lightly pigmented, in particular on the abdomen.Dorsally, thedark stripes at theposterior endof each segment are not yet distinct. Ventrally, thedarkspotontheleftsideoftheabdomenisstillwellmarked.

CollectionofflieswithunfoldedwingsAnalternativestagingmethodistoharvestfliesthathavejustemergedfromthepupalcase,withoutCO2anesthesia,andtoplacetheminacleanvialuntiltheyhaveunfoldedtheirwings.Theycanthenbeanaesthetizedifneeded.

- Flushfliesfromaculturewithlotsofemergingflies.- 30minuteslater,emptynewlyemergedfliesinacleandish.Mostshouldhavefoldedwings.- Gentlypushthenewlyhatchedfliesintoacleanvialwithabrush.- 30minutes later, the same flies should have unfolded their wings, and have reached the

appropriatestagefordissection.Thesefliescanbefrozenat-20°CinaPetridishcoatedwithWhatmanpaperforlaterdissection.

- Continuetocollectevery30minutes.

Figure3.Stagingwings.(Photos:©NicolasGompel&AylaSchröder)

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DissectionThegoalofthedissectionistocarefullycutorpluckthewingsatthestagedescribedabove.Weuse2pairsofforceps(5-Dumoxel-H,Dumont),butothertools(e.g.,mountedneedles)mayworktoo.

- Put 1-10 flies of the correct stage on a CO2pad. They usually lie on their side, facilitatingwingdissection. [Orusefliesaccumulatedat-20°C].

- Hold a fly with one pair of forceps by gentlypressingitsthorax(figure4).

- Pinchonewingatitshingewiththeotherpairof forceps. It is important to onlymanipulatethewingbyitshinge,tonotdamagethefragilewingblade.

- Inagentlebutfirmmotion,detachedthewingfromthethorax.

- Transferpluckedwingstothedissectionslide;arrange them in the same orientation and inrows onto the liner of the double-sided tape,as illustrated above. This arrangementfacilitateslaterimaging(figure5).

- Avoidpressingontothewingsurfaces,tolimitlesions.

- Thewingsareloose,butwillbefirmlyattachedto the glue of the mounting slide aftertransfer. Protect the loose wings from airmovementssuchasslammingdoors.

Wingtransfertothefinalslide- Apply a slide for final wing mounting, glue-

coatedsidedown,ontothewings,alignedonthe dissection slide. Gently press for a shorttime. Avoid sliding movements that wouldshearthewings.

- Thewingsshouldnowbeattachedtothefinalslide(figure5).

Figure4.Dissectingwings.(Photos:©CarolinBleese)

Figure5.Transferingwings.(Photos:©CarolinBleese)

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Wingsurfaceseparation- Place the slide with the transferred wings on a

softpad(e.g.,acomputermousepad)- Applya secondglue-coatedslideonto thewings,

ata90°angle(figure6).Thetwogluedropsmustbepositionedaboveeachother.

- Shortlypressonthetopslidewithyourthumbsoralightobject(e.g.,packofmicroscopeslides).Thewingsbecomevisiblyattachedtotheupperslide.Itmaybenecessary to apply pressuredirectly intheregionoftheslideoverlap.

- Separate the slides in a steady movement,withoutshearing.Thisisbestdonebyholdingthebottom slide with one hand at both ends andlifting the upper slide at one endwith the otherhand.Thewings shouldopen inhalvesalong thewingmargin,likeawallet.

Fromnowon, it is crucial that thewingsdonotdryout.Transfer the slides directly after opening the wings intothestainingjarwithfixationsolution(figure7).

Fixation- Transfertheslidesintoaverticalstainingjarwith

fixationsolution(underthefumehood).- Incubatefor30minutesatroomtemperature.- Washtheslides3x15minuteswith fresh1xPBT

inanotherstainingjar.

Figure6.Separatingwingsurfaces.(Photos:©CarolinBleese)

Figure7.Jarforwingfixationandwashes.(Photos:©CarolinBleese)

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AntibodystainingPrepare a humid chamber by placing wettissues at the bottom of a slide box (or asimilarkindofbox)(figure8).Allowenoughspace between the tissues and the upperend of the spacers to prevent contactbetween the edge of the slide and thetissue. This would result liquid drain fromtheslide.

- Take the slide with specimens outofthestainingjarandplaceitovertwo spacer of the humid chamber(figure8).Donot let thewingsdryout, proceed quickly to the nextstep.

- Dispense 200 µl of primaryantibody solution onto the wings.Fullycovertheslidetopreventtheliquid from escaping thehydrophobicgluesurface.

- Incubateovernightat4°C.- WashwithPBT:3xbriefly,then3x10’- Applysecondaryantibody,incubate1houratRTinthehumidchamber.- WashwithPBT:3xbriefly,then3x10’

Figure8.Antibodyincubation.(Photos:©CarolinBleese)

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Mounting- Cutthinstripsofdouble-sidedtape(approximately3cmx2mm)thatwill laterbeusedas

spacersbetweentheslideandthecoverslip.- Remove the liquid from the long edges of the slide with the fixed, open wings, using a

Kimwipes®tissue.Thisallowsthespacertosticktotheslide.- Placeaspacerstriponeachsideoftheslide;useforcepsoradissectingneedletoposition

thestrips;removetheprotectiveliner.- ImmediatelyapplyadropofVectashield®onthewings(theymustnotdryout).- Slowlyloweracoverslipontothewingsatanangletoavoidairbubbles(figure9).

BibliographyKiger,J.A.,Natzle,J.E.,Kimbrell,D.A.,Paddy,M.R.,Kleinhesselink,K.,&Green,M.M.(2007).TissueremodelingduringmaturationoftheDrosophilawing.DevelopmentalBiology,301(1),178–191.

Figure9.Finalmountingofstainedwings.(Photos:©AylaSchröder&CarolinBleese)