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Rapidlythawandimmediatelyplaceoniceonealiquoteachofaxonemes,GolgiorERmembranes,45uMtubulin,ratlivercytosol,and20xenergyregenerationsystem.DilutetheaxonemeswithPMBuffertotheproperdilution(determinedafterpreparation,seesupportprotocol2below)andprepare6xmembranes(determinedafterpreparation,seesupportprotocol4below)bydilutingorganelleswithPMbuffercontaining1mMGTP.Prepareandplaceonicea30ulMembraneMix:5ul6xGolgiorERmembranes1.5ulof20xenergyregenerationsystem10ulof45uMtubulin1ulof15mMMgGTP12.5ulofcytosolPerfuseaxonemesintoasimpleperfusionchamberbyslowlypipeting~10ulofproperlydilutedaxonemesagainstoneopenendofthechamberandallowingthechambertofill.Becarefultoavoidintroducinglargebubblesintothechamber.Placetheperfusionchamberintothehumidchamberandincubateatroomtemperaturefor10mintoallowtheaxonemestoadheretotheglass.Washoutunadheredaxonemes.SlowlyPipette10ulofPMbufferagainstoneendoftheperfusionchamber,whilesimultaneouslywickingexcessbufferfromtheoppositesideofthechamberwiththetipofasquareoffilterpaper.Repeatthis2moretimes.Dilute5ulof45uMtubulinwith10ulofPMcontaining1mMMgGTP.Perfusethedilutedtubulinintothechambercontainingthewashedaxonemes,placeadropofimmersionoilonthetopandbottomoftheslide,andtransferittothemicroscopestage.Focusontheaxonemeswiththe100xobjectivelens.ItisdifficulttofocusonaxonemesonthesurfaceofthecoverslipbecauseoftheirverysmallsizeandtheverybrightIlluminationneededforVE-DIC.Aligntheslideonthestagesothatanedgeofthedoublesticktapeformingtheperfusionchamberperfectlybisectstheareailluminatedbythemicroscopecondenserlens.Immersethe100xobjectivelensinoil,andfocusontheedgeofthetape.Whenyouhavebroughttheedgeofthetapeintoview,backofffinefocusuntiltheveryedgeofthetapejustbeginstogooutoffocus.Ifyoumovetheslidesothelensiswithintheareacoatedwithaxonemes,youshouldnowbequiteclosetofocusandtheaxonemesshouldbefoundwithlittledifficulty.OptimizetheimageforvisualizationofindividualMTsbyaligningthemicroscopeforKoehlerillumination(seeunitbyE.D.Salmon),andusetherealtimeimageprocessortoperformbackgroundsubtraction,contrastenhancement,andframeaveraging(seeSalmonandTran,1998).ObserveandrecordontoS-VHSvideotapeimagesofpolymerizationdynamicsofindividualMTsasthearenucleatedoffofaxonemes.Notethedifferencebetweentheplus(longer,fastergrowingMTs)andminus(shorter,slowergrowingMTs)endsoftheaxonemes.DuringtheobservationofMTdynamics,allowthemembranemixtowarmtoroomtemperature.Perfuse12ulofmembranemixintothesimpleperfusionchamberonthemicroscopestage.Sealthechamberedgesonbothedgeswithadropofmeltedvalap.ObserveandrecordthedynamicinteractionsbetweentheorganellesandMTs.Notethatoftenittakesupto45minformotilitytodevelop.Thistimeperiodisproportionaltoroomtemperature
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