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1、BromodeoxyuridineImmunohistochemistrySharonMorgenbesser,10/15/93RevisedbyNanetteJ.Liégeois7/1/96Introduction:Thismethodforthedetectionofcellularproliferationincludesseveralmodificationsofapreviouslypublishedprotocol(Hayashi,etal.,1988,J.Histochem.Cytochem.36:511-514).Thismethodutilizesformalin-fixa
2、tion,paraffin-embedding,andananti-BrDUmAbthatdetectsBrDUinssDNAafterdenaturationandneutralization.Becauseithasbeenoptimizedformidgestationalmurinelensanalysis,itmayrequirefurtheradjustmentforothertissues.Abbreviations:e(embryonicday),RT(roomtemp.),EtOH(ethanol),O/N(overnight)Method:A.BrDUTreatment,
3、Fixation,andSectioning:1.PrepareBromodeoxyuridine:10mg/mlin1XPBS.MakeupBrDUinalight-protectedconicalandsolubilizeat37°C.EstimatetheamountofBrDUatpreparebyassumingthateachmouseis40g.2.InjectBrDUintoapregnantmouseintraperitoneallyat100µgBrDU/gbodyweight(MillerandNowakowski,1988,BrainRes.457:44-52)usi
4、nga26gaugeneedleanda1ccsyringe.3.Embryosareremovedfromthemotherandplacedquicklyinadishofice-cold1XPBS.4.Fixembryoheadsfor2hrRT.Tissuesamplesareplacedinascintillationvialcontainingapx.20ml10%bufferedformalin.Fore14.5andolderembryos,theheadshouldberemovedandfixedseparatelyfromthebody.Placeonanorbital
5、shakersuchthatthesamplemovesslightly.Thesamplemustsettletothebottom.5.Rinsewith1XPBSGentlypouroffthefixativeintobiohazardcontainer.Donotletthesamplesdrybetweenthefollowingsteps,anduseapx.20mlforeachreagent(step3-7).6.30%,50%,70%,95%,100%x2,RT,30min.each,withslightagitation.Thisstepfunctionstodehydr
6、atethetissuesthroughanincreasingethanolseries:Samplesin70%,95%,or100%maybestoredO/N,4°Ciftimedoesnotpermitcompletionthroughatleastoneparaffinembeddingstep(step6).7.EtOH/xylene(1:1),RT;xylene,RT;andxylene/paraffin(1:1),58°C;30min.eachwithslightagitation.xylenesshouldbehandledinachemicalfumehoodwithg
7、loves,anddisposedofinabiohazardcontainer.8.3paraffinchanges,58°C,atleastoneisO/Nandtheothersareatleast3hrs.;manuallyshakeoccasionally.9.Orienttheheadinmeltedparaffin,andletsolidifyforonehouronice.Placesecti
