石蜡切片和原位杂交protocol_from-Chongkang-植物所

共2页1RNA组织原位杂交实验记录（以小麦胚芽为例）ChongKang’sLab2011.07.08版本一、FIXATIONANDEMBEDDING1.固定:小麦胚芽浸入FAAfixative，抽气1小时,RT12小时(Overnight)2.脱水:50%,70%,80%,90%,95%,100%,100%,100%ethanol,eachfor30minRT3.透明:removetheethanolandreplacewiththemixture:25%xylene-75%ethanol,50%xylene-50%ethanol,75%xylene–25%ethanol,90%xylene-10%chloroform,90%xylene-10%chloroform,90%xylene-10%chloroform,30minforeachstep.氯仿的存在可使石蜡漂浮在上,这样可以保证让石蜡逐渐的增加浓度在。95％乙醇这一步可以加EosinY到0.1％，适当染色，可以overnight。目前市场有Histoclear代替二甲苯4.浸蜡:50%蜡-50%xylene,4-16hrs42℃,重复一次；移入60温箱℃,更换为100%石蜡,60保温℃2-6天,期间更换纯石蜡4次以上60过滤℃,勿超62℃5.包埋:固化前可以在60保温℃30分以除气泡。保包埋块4可以存℃1年以上二、SECTIONING直接购买涂有poly-Lys的载玻片。5—10μmsections,47粘片过夜（大于℃24h）,4保℃存。RNAwilllosewhentheribbonswerestoredformorethanonemonth三、SYNTHESISOFPROBE1.Transcription(20lsystem):xllinearizedtemplateDNA(1g)4l5transcriptionbuffer(containingrNTPmixture&DIG-UTPetc)20-(6+x)lDEPC-water2lRNApolymerase(T7orSP6orT3polymerase),42℃2hoursAccordingtheprocedureoftheNorthernKit可以T7、T3或SP6启动子序列合成引物，用PCR办法制备模板，省事省时。过去通过提取质粒线性化再回收很费劲!2.Degestionoftemplate:2lRNase-freeDnaseat37℃for15min,Add0.8l500mMEDTAtostopthereaction3.Precipitatingtheprobe(for20lreactionsystem):1l10mg/mltRNA(Itcanbeomitted)2.5l4MLiCl75l100%ethanol–20℃overnight.4℃13000rpmspindown,washing2timeswith70%ethanolandresuspendedin100lDEPC-water.Ifnecessary,incubatein60℃10mintogettheprobeintosolution.4.Hydrolysisofprobe(for50lprobesolution):20l200mMNaHCO3+30l200mMNa2CO3,hydrolyzedat60℃.Stopthereactionbyadding10lof1MpH4.7NaAcbuffer(or3l3MpH6.0sodiumacetateand5l10%glacialaceticacid)Precipitatetheprobe:1µl20mg/mlGlycogen(optional)+10µl4MLiCl+300µlEthanol.Incubateat-20°Covernight.4℃13000rpmspindown,washing2timeswith70%ethanolandresuspendedin100lDEPC-water.5.Semi-quantitativedeterminationofprobeaccordingtheprotocolofRNAlabelingKit:applya1ulprobesolutionofthedilutionseriestothenylonmembrane,fixthenucleicacidfor30minat120℃,incubateinmaleicacidbuffer2min,blockingfor30minatRTwithagitation,combindingwithantibodyinBlockingsolutionfor30minatRT,pourofftheantibodysolutionandwashwithwashingbufferfor2×15min,replacedthewashingbufferwithTNM50andincubatefor3min,coloringindetectionbuffer(NBT/BCIP)indarkforseveralminutes.水解时间:（Tmin）:T=(起始产度-昀终长度)÷(0.11×起始产度昀终长度×)，NaHCO3、Na2CO3用DEPC水临时配用10mg/mltRNA4MLiCl200mMNaHCO3:0.168gadd10mlDEPC-water200mMNa2CO3:0.212gadd10mlDEPC-waterBydotblotusedig-labeledactinRNAasstandard0.05~5.0ng/linworkingsolution（from4references）Maleicacidbuffer:0.1Mmaleicacid+0.15MnaClpH7.5RNAdilutionbuffer:0.5mlDEPCwater+0.3ml20×SSC+0.2mlformaldehydBlockingsolution:diluting10%blockreagentto1%bymaleiacidbufferWashingbuffer:maleicacidbuffer+0.3%Tween20Detectionbuffer：2%NBT/BCIPstocksolutioninTNM50四、PREHYBRIDIZATIONTREATMENT以下的操作要在无RNA酶的条件下进行容器，溶液和工具均要灭菌。。1.Dewaxingandhydration：100%xylene20minatR.T.,100%xylene20minatR.T.,66%xylene2min,33%xylene2min,100%,100%,90%,70%,50%,30%,10%ethanolandH2O,H2O2minatR.Tforeachstep.Dipupanddownuntilthestreaksgoaway2.DigestionwithProteinaseK:37预热℃Kbuffer后加入蛋白酶K至1~5g/ml,37处理℃30分,无菌DEPC水洗三次,3.Acetylation:100mMpH8.0triethanolamine(2.68mlTriethanolamineper200mlEDPCwater,about0.8mlconcentratedHCltoadjustpH)5min.Stiringthechloroformtreatedstirbarhardandaddanhydridetofinalconcentrationof0.25%(500l/200ml),aftermixingfor5seconds,incubatetheslidesfor5minatRT.4.Dehydration:2×SSC,2×5min10%ethanol2minatR.T.30%ethanol2minatR.T.50%ethanol2minatR.T.70%ethanol2minatR.T.90%,2minatR.T.100%ethanol2minatR.T.100%ethanol2minatR.T.Drysectionsundervacuumatleast1huntilhybridization.Ifessentialpretreatedsectionscanbestoredat–20C100ml1MpH7.5Tris-HCl:80mlDEPC-H2O12.11gTrisbaseHCl6.5ml调pH7.5,定容，autoclave100ml500mMEDTApH7.5:80mlDEPC-H2O18.61gEDTANa2.2H2O约2gNaOH调pH7.5,定容，autoclaveKbuffer200ml:20ml1MpH7.5Tris-HCl20ml500mMEDTApH7.5160mlDEPC-H2O10×SSC1000ml:800mlDEPC-H2O87.65gNaCl44.1gNaCit10NNaOHadjustpHto7.0addDEPC-H2Oto1000mlautoclaveDipupanddownuntilthestreaksgoway共2页2五、HYBRIDIZATIONPrehybridizationisnotessentialandcanbeomitted.1.Prehybridization:45℃1-2小时注意往往会产生气泡1mlprehybridizationsolution:1)500lformamide2)200lDEPC-H2O3)100l10×Blockreagent4)100l3MNaCl5)73lDEPC-H2O6)15l10mg/mltRNA7)10l1MTris.HClpH7.58)2l500mMEDTApH7.5150lhybridizationsolution:mix115.8lAwith34.2lB100lhybridizationsolution:mix77.2lAwith22.8lBB34.2l:1)3.75l20g/lpolyA2)2.25l10mg/mltRNA3)28.2lprobe&DEPC-H2OB22.8l:1)2.5l20g/lpolyA2)1.5l10mg/mltRNA3)18.8lprobe&DEPC-H2O2.hybridization:42℃overnight湿室中垫有用0.3MNaCl－50％甲酰胺饱和的吸水纸A7.72ml分装备用:1)5000lformamide2)1000l50%dextransulfate3)1000l10×blockreagent4)600l5MNaCl5)100l1MTris.HClpH7.56)20l500mMEDTApH7.5HeatBat80℃5minandputoniceimmediately六、WASHING1.washing:40ml4xSSCRT,5-10min40ml4xSSCRT,5-10min40ml4xSSCRT,5-10min40ml4xSSCRT,5-10min2.RNase处理:预热37℃RNasebuffer(500MmNaCl-1mMEDTA-10mMTris.HClpH7.5),addRNaseAtofinalconcentration25g/ml,放入切片保温30min3.RNasebufferresine15minX3timesat37℃1000mlRNasebuffer:29.22gNaCl0.37gEDTANa2.2H2O1.21gTrisadjustpHto7.5withHClRNasesolution:2.5ul10mg/mlRnase+1mlRNasebuffer20×SSC1000ml:175.3gNaCl88.2gCitNa800mldH2OandadjustpHt