InSituHybridization1dayFixation1、dechorionate:in35mmdishadd20mg/mlpronase30ulto1mlsystemwater,roomtemperature10min,washtwicewithsystemwaterandtransferto1.5mlEP.2、fixembryosin4%paraformaldehyde(PFAinPBS)overnightat4℃(<14h).3、washPBST2×10min(RT).4、dehydratewith:methanol:PBST=1:210minmethanol:PBST=2:110min100%methanol2×10min.5、stockat-20℃.2dayHybridization1、rehydratetheembryosin:methanol:PBST=2:15minmethanol:PBST=1:25minPBST3×5min2、fixfor20minin4%PFA.3、washPBST2×5min(RT).4、treatwith10ug/mlproteinaseKinPBST(1:1000use),精确3min（5体节到24h）；24h10min；48h15min。5、用PBST轻洗，即加入后立刻吸出。6、PBST中静置5min。7、fixin4%PFA20min(RT).8、washPBST2×5min(RT).65℃(preheated)9、add400ul/tubeHYB-,5min.10、add400ul/tubeHYB+,4-6h.11、addpreheatedprobe2ug/ml,300ul/tube,overnight.3dayPosthybridizationwashs:现配现用，全部配好后放在65℃预热。1、wash5minin1ml50%formamide,5×SSC:2×SSC=1:1,0.1%Tween20.2、wash10minin1ml50%formamide,2×SSC,0.1%Tween20.3、wash30minin1ml50%formamide,2×SSC,0.1%Tween20.4、wash15minin1ml2×SSC,0.1%Tween20.5、wash2×30minin1ml0.2×SSC,0.1%Tween20.室温6、wash2×5mininMABT.Immunolocalizationofprobes1、addblockingsolution4℃overnight.4day2、incubatefor2hinanti-DIGFab-AP(1:5000inblockingsolution).3、washwith1ml25minMABTcontaining10%灭活山羊血清；25minMBAT；1hMBAT；25minMBAT。4、equilibrate3×5mininfreshmadedetectionbufferwith1mMtetramisole(pH9.5)5、indarkstainwithNBT/BCIP(5mMtetramisole)，一般30min可见强信号，2-3h至信号强度不增加背景开始出现。6、indarkwashPBST2×5min.7、fixembryosin4%PFAovernightat4℃.5day8、washPBST2×5min.9、transfertheembryosintoglycerol.Pronae:Roche10165921001Stock:20mg/mlinddH2O,stockat-20℃.Paraformaldehyde:上海西巴斯500ml:Heat400mlddH2Oto65℃,add20gPFA(加盖搅拌)，addafewdropsof5MNaOH，stopheating,cooltoRT,checkpH7.3andfilter(0.45um),50ml/tube,freezeat-20℃.PBST:1×PBS+0.1%Tween20proteinaseK:10ug/ulinddH2O,stockat-20℃.HYB-:50%formamide,5×SSC,0.1%Tween20.HYB+:50%formamide,5×SSC,500ug/mlyeastRNA,50ug/mlheparin,0.1%Tween20.MAB:0.1Mmaleicacid,0.15MNaCl,pH7.5.(一起加入再调pH，RT保存)MABT：MAB+0.1%tween20.Blocking