斑馬魚免疫熒光

Fixation：Fixtheembryosin4%paraformaldehydefor3hatroomtemperatureorovernightat4°C.20embryosineach1.5mLvolumemicrocentrifugetube.Afterfixation,replacethesolutionwithanequivalentvolumeofPBS(此步驟后胚胎可存放數(shù)周，但下次用時(shí)要wash5x5minwithPBS).Blocking:Dilutethegoatserumto10%(v/v)inPBTandincubate20-30embryosin0.5mLofthissolutionfor1hatroomtemperatureonarockingtable.Primaryantibodyincubation:Removetheblockingsolutionwithapipet.DilutetheprimaryantibodyinPBTplus1%goatserumtotheappropriateconcentration,asrecommendedbythesupplier.Applytheprimaryantibodyinaminimumvolumeof100rL.overnightat4°Conarockingtablewithgentleagitation.Washing:removeasmuchoftheprimaryantibodyaspossibleandreplacewithalargevolumeofwashingsolution(PBT).Changethewashthreetofivetimes,withatleast15minforeachwashwithgentlerockingatroomtemperature洗的時(shí)間長，背景會(huì)降低).Dilutethesecondaryantibody：DilutethesecondaryantibodyinPBTplus1%goatserumtotheappropriateconcentration,asrecommendedbythesupplier.overnightat4°Conarockingtablewithgentleagitation.keeptheembryosinthedarkfromthisstepontopreventbleaching.RemovethesecondaryantibodyandwashtheembryosinPBT.Changethewashthreetofivetimes,withatleast15minforeachwashwithgentlerockingatroomtemperature.FinallytransfertheembryostoPBSalonepriortothedetectionsteps.Atthispoint,iffluorescenceisused,cleartheembryosingradedglycerolsandmountin70%glycerolcontaininganantibleachingagentsuchasCitifluor(抗熒光衰退劑)(AgarScientificLtd).Examineuseconfocalmicroscope.4%多聚甲醛-0?1mol/L磷酸緩沖液（pH7.3）:配制方法：稱取10g多聚甲醛，置于三角燒瓶中，加入125?200ml0.1mol/L磷酸緩沖液（PhosphateBuffer以下簡(jiǎn)稱PB），加熱至60°C左右，持續(xù)攪拌（或磁力攪拌）使粉末完全溶解，通常需滴加少許1nNaOH才能使溶液清亮，最后補(bǔ)足0.1mol/L的PB于250ml，充分混勻。PBS:8.0gNaCl,0.2gKCl,1.44gNa2HPO4,0.24gKH2PO4in1LdH2O,pH7.4,PBT:PBSplus0.8%TritonX-100（Sigma）.goatserum（sigma）gradedglycerols:30%,50%,and70%glycerolinPBSCitifluor(抗熒光衰退劑)(AgarScientificLtd)RearlarvaeinPTUdilutedinrearingmediumtopreventpigmentformation.Attherequiredstage,washlarvaeinrearingmediumwithoutPTU,anesthetizein0.03%MS222,washinPBSbriefly,thenfixin2%TCAfor3hatroomtemperature.Fixationshouldnotbelongerthan4h.Afterfixation,washthetissueinPBSfor3x5min,andstoreatthisstageat4°Cuntilrequired.Note:Careshouldbetakenwhenhandlingthetissueduringthislabelingtechnique,asthelarvaearefragileafterfixation.Priortopermeabilization,washtissuetwiceinPBTatroomtemperature,thenchillonice.ReplacethePBTwith200-300rLofchilledtrypsinsolution(0.25%inPBT)andincubateoniceusingthefollowingtimesasguidelines:48-72hold:4min72-96hold:5min120h-6dold:6min或者washindH2Ofor5min,soakfor7minin100%acetoneat-20°C,rinseindH2Oonce,thentwiceinPBT.Blocking:Dilutethegoatserumto10%(v/v)inPBTandincubate20-30embryosin0.5mLofthissolutionfor1hatroomtemperatureonarockingtable.Primaryantibodyincubation:Removetheblockingsolutionwithapipet.DilutetheprimaryantibodyinPBTplus1%goatserumtotheappropriateconcentration,asrecommendedbythesupplier.Applytheprimaryantibodyinaminimumvolumeof100rL.overnightat4°Conarockingtablewithgentleagitation.Witholderstages,betterresultscanbeobtainedbyincubatingforlongerperiodsuptoseveraldaysandthisshouldbedeterminedforeachantibody.RemovetheantibodyandwashoverseveralhourswithPBT,changingthebufferatleastfivetimes.Finally,washwithPBS.Dilutethesecondaryantibody：DilutethesecondaryantibodyinPBTplus1%goatserumtotheappropriateconcentration,asrecommendedbythesupplier.overnightat4°Conarockingtablewithgentleagitation.keeptheembryosinthedarkfromthisstepontopreventbleaching.RemovethesecondaryantibodyandwashtheembryosinPBT.Changethewashthreetofivetimes,withatleast15minforeachwashwithgentlerockingatroomtemperature.FinallytransfertheembryostoPBSalonepriortothedetectionsteps.Atthispoint,iffluorescenceisused,cleartheembryosingradedglycerolsandmountin70%glycerolcontaininganantibleachingagentsuchasCitifluor(抗熒光衰退-劑)(AgarScientificLtd).AsTCA-fixedtissueisfragile,itisnecessarytopostfixin4%PFAat4°Covernight,asthismakesiteasiertohandlethelabeledtissue.ThetissueiswashedinPBSandclearedto70%glycerolfordissectionandmounting.Examineuseconfocalmicroscope.PTU:PTU(0.2mMphenylthiocarbamide;Sigma,Poole,UK):makea10Xstock(dissolve30mgin100mLofembryo-rearingmedium;ref.9)andstoreat4°C,dilutewhenrequired.Caution:PTUisextremelytoxicandmustbeweighedinafumecupboardandprotectiveclothingworn.0.03%MS222:Anaesthetic—0.03%MS222(3-aminobenzoicacidethylester;Sigma):Makea10Xstock(dissolve300mgofpowderin