Cuticlepreparations

Thisprocedurewasadaptedfromonedescribedoverthephone(thanksP.G.).We"renotsureifitappearselsewhereinpublishedform.

-Collectembryosonapplejuice-agarplatesforsuitabletimeperiodfromawell-stockedcylinderorcage.Allowtoagefor24-36hrat25o.

-rinseunhatchedlarvaeintoanytexscreen.Dechorionatein3%bleachuntilembryosfloattothesurface(1-5min).Don"tworryaboutover-dechorionating.Rinsewithwater.

-removescreenanddipintoascintillationvialcontaining5mlPBS/5mlheptane.Theembryosshouldslideoff.Thosewithvitellinemembranesintactwillstayattheinterphase,whilehatchedlarvaewillsettletothebottom.Useapaintbrushtoremoveremaininglarvaefromthedechorionatingvesselanddipintoheptanelayertodislodgefrombrush.

-usinga1mlPipetteman,suckupvitellinecontaininglarvaefrominterphase.Ejectanyoftheloweraqueousphasesuckedupintheprocess.TransferembryosinupperheptanesolutiontoanEppendorftube.Adjustvolumetoapproximately0.5mlandaddanequalvolumeofmethanol.

-Closecapandshakevigorouslyfor15secondstodevitellenize.Themajorityofembryoswillsettletothebottom.Removemostofupperphasewithoutremovinganyembryos.Shakeonceagain.Nowalloftheembryosshouldsettletothebottom.Removeliquidandwash2-3xwithmethanol.

-larvaeatthebottomofthescintillationvialaregenerallywildtype.TheycanalsobedrawnupintheaqueoussolutioninaP-1000andtransferredtoaneppendorftube.Removethemajorityofliquidandaddaround0.5mlofmethanol.Larvaeshouldsettle.Rinse2Xmorewithmethanol.Larvaecanbepooledwiththoseaboveifdesired.

-againusingaP-1000,transferthelarvaetoacleanglassslide.Usetheexcessmethanoltodispersethelarvaeevenlybyaddingdropsoverclusteredlarvae.AllowthemethanoltoairdrybrieflyandthenaddadropofHoyer"s/lactate.Covercarefullywithasuitablysizedcoverslip,takingcaretoavoidbubbles.

-placeina65oovenovernighttoclearthelarvae.Oncecleared,flattenthelarvaeasfollows.Wrapinalayerofaluminumfoil.Placeonaflatsurface,coverwithasecondglassslideandplacealeadpigsuchasthosewhichcomewithrADIoactivesubstances.Withthisweight,larvaeshouldbesuitablyflattenedwithin1-4hr.Takecarenottomovethecoverslipwhileaddingorremovingtheweightandfoil,asthiswilldestroythecuticles.Uponremovingthefoil,excessHoyer"sshouldhavebeenexudedfromtheslide.Thiscanbecleanedawayusingfirstwaterandthenethanolsquirtedinastreamovertheslide.Thisavoidsphysicalcontactwhichmaymovethecoverslip.Allowtheslidetoairdryandsealtheedgeswithyourfavoritenailpolish.Oncedry,theslideshouldbepermanentandrelativelyresistanttophysicalabuse.

Hoyer"sMountant:Add30gofgumarABIcto50mldistilledwater,stirovernight.Whilestirring,add200gchloralhydrateinsmallquantities.Add20gglycerol.Centrifugeatleast3hrat12000gtoclear.Addlactatetoincreasecontrastanddecreaseclearingtime.Wefind1:4worksbest.

Wild-typefirstinstarlarvacuticlepreparation