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Wholemount in situ hybridisation
Wholemountinsituhybridisation BasedonWilkinsonprotocol,modifiedbyMurrayHargrave(m.hargrave@cmcb.uq.edu.au),Koopmanlab A.SYNTHESISOFPROBE 1Mixinthefollowingorderatroomtemperature: 2Incubateat37°Cfor1hour,thenaddanother20UofRNApolymerase. 3Incubateforafurtherhourat37°C. 4Removea1µLaliquotandrunona1%agarose/TAEgeltoestimatetheamountsynthesised.AnRNAbandofapproximately10foldgreaterintensitythantheplasmidbandindicatesthat~10µgofprobehasbeensynthesised. 5TherearetwosuccessfulmethodsforpurificationoftheRNAprobe: A)forthelazy(thisworksfine): B)fortheparanoid: 6RedissolvepelletinDEPCmilliQH2Oat~0.1µg/µLandstoreat-20°C. B.PRETREATMENTOFEMBRYOS Day0 1Dissectembryosinice-coldPBS.TrytoremoveasmuchoftheextraembryonicmembranesaspossIBLe.Besuretoremoveoratleastpuncturetheamnionandinembryosof10dpcorolder,puncturetheheadwithasyringeneedletoavoidtrappingofreagentsinthelumen. 2Fixin4%paraformaldehyde(PFA)inPBSat4°Covernight. Varyingthefixationtimefrom3htoovernighthasnoeffectonsignalorbackground. 3WashtwicewithPBTXfor10minuteseachat4°C. 4Washwith50%,MeOH/PBTX,thentwicewith100%MeOHfor10minuteseach. Canstoretheembyrosat4°Cor-20°Catthispoint,foruptoafewmonths,orpreferablyinpre-hyb(seestep12). Day1 5RehydratebytakingtheembryosbackthroughaMeOH/PBTX(75%MeOH->50%MeOH->25%MeOH->PBTX)seriesinreverse 6WashtwicewithPBTXfor10minuteseach. 7Treatwith10µg/mLProteinaseKinPBTXfor5-20minutesatroomtemperature. ThelengthofthistreatmentdependsonthesizeofthesampleandthebatchofproteinaseK.Eachbatchshouldideallybetested.Asaroughguide,use5minforE7.5,7minforE8.5,9minforE9.5,11minforE10.5,12-14minforE11.5. 8WashtwicewithPBTXfor5minuteseach. Becareful-theembryosarefragile! 9Refixwithfresh0.2%glutaraldehyde/4%PFAinPBTXfor20minutes. 10WashtwicewithPBTXfor10minuteseach. 11Placeina2mlscrewcapEppendorftubeandfillwithprehybridisationmix,andallowtheembryostosink,replaceprehybsoln. 12Incubateat65°Cfor2h. Thisstepcanalsobeperformedovernight.Alternativelytheembryoscanbestoredinthissolutionat-20°C. 13Removeprehybandaddhybridisationmixincluding1.0µg/mLDIGlabelledRNAprobe. Ifhighbackgroundisseen,probeconcentrationcanbedecreasedto0.5µg/mL. Thetubeneedstobefullsothatprobedoesnotdryontothesample. 14Incubateat65°Covernight. Iftheprobeisshortorheterologous,55°Ccanbeusedforpre-hyb,hybandstringencywashes. Day2 C.POST-HYBRIDISATIONWASHES FromthispointonRNase-freeconditionsarenolongernecessary. 1Washwiththefollowingfor5minuteseachat65°C(or55°C) Duringthesewashes,startpreabsorbingtheantibodyasdescribedbelow. 2Washwith2xSSC,0.1%CHAPStwicefor30minuteseachat65°C(or55°C). 3Washwith0.2xSSC,0.1%CHAPStwicefor30minuteseachat65°C(or55°C). 4WashwithTBTX,twicefor10minuteseachatroomtemperature. 5Preblocktheembryoswith10%sheepserum,2%BSAinTBTXfor2-3hoursatroomtemperature. 6Removethe10%sheepserum,2%BSAfromtheembryosandreplacewiththepreabsorbedantibody(seebelow).Rockovernightat4°C. D.PREABSORPTIONOFANTIBODY 1Duringthewashingoftheembryos(step1above),weighout3mgofembryopowderintoamicrotube,add0.5mLof10%sheepserum,2%BSAinTBTXand1µLanti-DIG-APFabfragment(Boehringer1093274). Embryopowdershouldmatchthespeciesbeingstudied. 2Rockgentlyat4°Cfor3hoursorlonger. 3Spininamicrofugefor10minutesat4°C. 4Dilutethesupernatantto2mLusing10%sheepserum,2%BSAinTBTX. 5Storeat4°Cuntiluse. E.POST-ANTIBODYWASHESANDHISTOCHEMISTRY Day3 1WashatleastfivetimeswithTBTXcontaining0.1%BSAfor1houreachatroomtemp. Theantibodysolutioncanbekeptat4°Candreusedupto4times. 2Washovernightat4°CwithTBTXcontaining0.1%BSA. Thiswashisoptionalbutusuallyconvenient. Day4 3WashtwicewithTBTXfor15minuteseach. 4WashthreetimeswithNTMTfor10minuteseach. 5IncubatewithNTMTincluding4.5µLNBTand3.5µLBCIP(X-phosphate)permL.Rockforthefirst20minutesthentransfertheembryostoaglassembryodishorscintillationvial. Avoidusingaplasticpetridishascrystalstendtoform. Keepinthedarkasmuchaspossibleandallowthecolourreactiontoproceeduntilsignalisstrongestwithoutproducingbackgroundstaining. Itisbesttoslightlyoverstain,assubsequentwashingswilltendtodestainsamples.Ifsamplesaretobesectioned,overstainingisrecommended.YoucanstopthecolourreactionbywashinginNTMT,thenTBTXovernight,thenre-startthecolourreactionthenextmorning. 6Whenthecolourhasdevelopedtothedesiredextent,washwithNTMTthenwithPBTX. 7WashseveraltimesinPBSwith1%TritonX-100. Thiswillbluethestainanddecreasebackgroundandsignal.Someobservationandjudgementisrequiredhere.Forweaksignal,thisstepcanbeshortenedoromitted.Ifsignalisstrongandbackgroundisweak,thenatotalofafewhoursisrecommended.Overstainedorhighbackgroundsamplescanbewashedforuptoseveraldays. 8Fixthestainbyincubatingtheembryosin4%PFAinPBTXovernightat4°C. 9Photographembryosassoonaspossible. Thesignalcanfadeortheenitreembryocanturnblueuponstorage.Positionembryos,immersedinPBS,ingroovescutinalayerofagaroseinapetridish.Adjustlightingtooptimizethetranslucencyofthesample. 10Iftheembryosaretobestoredforextendedperiods,usePBScontainingsodiumazide,ortakethemthroughaPBTX/glycerolseriesinto100%glycerol. SOLUTIONS ThismustbemadefreshonthedayofuseaspHdecreasesduringstorageduetotheabsorptionofCO2. Itisbesttoletthesereagentswarmtoroomtemperaturebeforeuseasitmaydecreasetheamountofcrystalformationinthecolourreaction. REFERENCE Wilkinson,D.G.(1992)Wholemountinsituhybridizationofvertebrateembryos,inInsituhybridization:APracticalApproach(D.G.Wilkinson,ed)pp75-83,IRLPress,Oxford.NBT:75mg/mLindimethylformamide(storeat-20°C)
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