斑马鱼免疫荧光

Fixation：Fixtheembryosin4%paraformaldehydefor3hatroomtemperatureorovernightat4°C.20embryosineach1.5mLvolumemicrocentrifugetube.Afterfixation,replacethesolutionwithanequivalentvolumeofPBS(此步骤后胚胎可存放数周，但下次用时要wash5x5minwithPBS).Blocking:Dilutethegoatserumto10%(v/v)inPBTandincubate20-30embryosin0.5mLofthissolutionfor1hatroomtemperatureonarockingtable.Primaryantibodyincubation:Removetheblockingsolutionwithapipet.DilutetheprimaryantibodyinPBTplus1%goatserumtotheappropriateconcentration,asrecommendedbythesupplier.Applytheprimaryantibodyinaminimumvolumeof100rL.overnightat4°Conarockingtablewithgentleagitation.Washing:removeasmuchoftheprimaryantibodyaspossibleandreplacewithalargevolumeofwashingsolution(PBT).Changethewashthreetofivetimes,withatleast15minforeachwashwithgentlerockingatroomtemperature洗的时间长，背景会降低).Dilutethesecondaryantibody：DilutethesecondaryantibodyinPBTplus1%goatserumtotheappropriateconcentration,asrecommendedbythesupplier.overnightat4°Conarockingtablewithgentleagitation.keeptheembryosinthedarkfromthisstepontopreventbleaching.RemovethesecondaryantibodyandwashtheembryosinPBT.Changethewashthreetofivetimes,withatleast15minforeachwashwithgentlerockingatroomtemperature.FinallytransfertheembryostoPBSalonepriortothedetectionsteps.Atthispoint,iffluorescenceisused,cleartheembryosingradedglycerolsandmountin70%glycerolcontaininganantibleachingagentsuchasCitifluor(抗荧光衰退剂)(AgarScientificLtd).Examineuseconfocalmicroscope.4%多聚甲醛-0・1mol/L磷酸缓冲液（pH7.3）:配制方法：称取10g多聚甲醛，置于三角烧瓶中，加入125〜200ml0.1mol/L磷酸缓冲液（PhosphateBuffer以下简称PB），加热至60°C左右，持续搅拌（或磁力搅拌）使粉末完全溶解，通常需滴加少许1nNaOH才能使溶液清亮，最后补足0.1mol/L的PB于250ml，充分混匀。PBS:8.0gNaCl,0.2gKCl,1.44gNa2HPO4,0.24gKH2PO4in1LdH2O,pH7.4,PBT:PBSplus0.8%TritonX-100（Sigma）.goatserum（sigma）gradedglycerols:30%,50%,and70%glycerolinPBSCitifluor(抗荧光衰退剂)(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(抗荧光衰退-剂)(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