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Millipore/MAB3424 | Anti-BrdU Antibody, clone AH4H7-1 / 131-14871/MAB3424/50 µg
  • Millipore/MAB3424 | Anti-BrdU Antibody, clone AH4H7-1 / 131-14871/MAB3424/50 µg

Millipore/MAB3424 | Anti-BrdU Antibody, clone AH4H7-1 / 131-14871/MAB3424/50 µg

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貨號(hào): MAB3424
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    • Description
      CatalogueNumberMAB3424
      ReplacesMAB1467
      BrandFamilyChemicon®
      TradeName
      • Chemicon
      DescriptionAnti-BrdUAntibody,cloneAH4H7-1/131-14871
      AlternateNames
      • BrdU
      BackgroundInformationBromodeoxyuridine(BrdU)isathymidineanalogandisspecificallyincor-poratedintoDNAduringDNAsynthesis.Anti-bromodeoxyuridinemonoclonalantibodyisusedtoidentifycellsthathaveincorporatedBrdU.ThisimmunologicaldetectionschemehasseveraladvantagesovertheuseofrADIoactivethymidineincorporationforidentifyingcellsunder-goingreplication.Labelinganddetectioncanbeperformedthesamedayinsteadofwaitingseveraldays,asrequiredforautoradiographyoftritium-labeledcells,andthenecessityofusingmultiplespecimensforobtainingtheoptimalexposuretimeiseliminated.Inaddition,anti-bromodeoxyuridinestainingwithflowcytometricanalysisallowsmultipleparameterstobeevaluatedsimultaneously.Anti-bromodeoxyuridinemonoclonalantibodyhasbeenusedforidenti-fyingproliferatingcellsinblood(Campanaetal.,1988),tissues(Schutteetal.,1987;Hayashi,etal.,1988),tumors(Hoshinoetal.,1986;Morstynetal.,1983),aswellasfordeterminingplasmacelllabelingindices(Greippetal.,1985).
      ProductInformation
      FormatPurified
      Control
      • AfterincorporationofBrdU,allDNAcontainingspecies
      PresentationLiquidin10mMPhosphatebuffer,pH7.4containing150mMNaCland0.1%sodiumazide
      StorageandShippingInformation
      StorageConditionsMaintainfor6monthsat-20°Cfromdateofshipment.Aliquottoavoidrepeatedfreezingandthawing.Formaximumrecoveryofproduct,centrifugetheoriginalvialafterthawingandpriortoremovingthecap.
      Applications
      ApplicationUseAnti-BrdUAntibody,cloneAH4H7-1/131-14871(MouseMonoclonalAntibody)validatedinFC,ICC,IHCtodetectBromodeoxyuridinealsoknownasBrdU.
      KeyApplications
      • FlowCytometry
      • Immunocytochemistry
      • Immunohistochemistry
      ApplicationNotesImmunohistochemistry:(6μg/ml)

      FlowCytometry:(0.2μg/100μl/10E6cells)Optimalworkingdilutionsmustbedeterminedbyenduser.

      APPLICATIONS

      Flowcytometry:ThemethodbelowisbasedonthatofM.Vanderlaanetal.(1986).Variationsofthismethodexistintheliterature,oneconsiderationbeingtheeffectvariousfixationprocedureshaveonthelight-scatteringpropertiesofdifferentcellpopulations.Procedure:

      1.Tolabelcells,pulsewith10μMbromodeoxyuridinefor30minutes.Harvestcellsfromculture.

      2.Fixcellsin70%ethanolat+2-8°Cforatleast30min.ExtracthistonesbyresUSPendingcellsin1mLchilled0.1MHCIcontaining0.5%TritonX-100;incubatethesuspensiononicefor10minutes.Diluteacidwith5mLdistilledwaterandcentrifugeat200xgfor10min.Resuspendcellsin2mLdistilledwater.

      3.DenaturecellularDNAbysubmergingthecellsuspensionintoaboilingwaterbathfor10min.Afterwards,quicklycoolbyplacingthecellsuspensioninaniceslurryforseveralminutes.WashcellsinPBSthatcontains0.5%TritonX-100.

      4.Resuspendthecells(1-2x106cells)in100μLofsolutioncontainingapproximately2μg/mLanti-bromodeoxyuridineantibodydilutedinPBScontaining0.1%BSA(0.2μg/test).Incubatefor30minatroomtemperature.WashcellswithPBS.

      5.Resuspendcellsin100μLofdilutedgoatanti-mouseIgG-FlTCWashcellswithPBS.

      APPLICATIONS(Cont.)

      Immunohistochemistry:BelowisaprocedureforstainingcellsthathavebeenlabeledwithBrdUinvivoorinvitro.TheprocedureisbasedonthemethodsofB.Schutteetal.(1987)andD.Campanaetal.(1988).

      Preparationoftissue:

      Injectanimalwith50mgBrdU/kgbodyweight.Sacrificeanimalonehourlaterandremoveorganortissueunderstudy.EmbedtissueinOCTmediumandsnap-freezebyimmersionintoliquidnitrogen.Cut4mmfrozensectionswithacryostat.Placesectionsoneitheralbumin-orgelatin-coatedslides.

      Preparationofcells:

      Pulsecellswith10mMBrdUfor60min.Cellsgrownoncoverslips,orcytocentrifugepreparationsmadefromcellsgrowninsuspension,canbeusedforanti-bromodeoxyuridinestainingaccordingtotheprocedurebelow.

      Procedure

      1.Fixtissuesectionsorcells(onslideorcoverglass)byimmersinginabsolutemethanolfor10minutesat+2-8°C.Airdryafterremovingfromfixative.Theslidescanbestoredat-20°Cinasealedbox,orrehydratedtopreparefortheassayprocedure.Torehydrate,immerseinPBSfor3min.

      2.DenatureDNAbyincubatingtheslidesin2NHCIfor60minat+37°C.

      3.Neutralizetheacidbyimmersingtheslidesin0.1Mboratebuffer,pH8.5.Changethebuffertwiceovera10minperiod.

      4.WashslideswithPBS,changingthesolutionthreetimesovera10minperiod.

      5.Placeslidesinahumidifiedchamber(e.g.,asealedplasticboxlayeredwithwetpapertowels)andcovercellswith150-300μLofsolutioncontainingapproximately6μg/mLanti-bromodeoxyuridineantibodydilutedinPBSwith0.1%BSA.Incubatefor60minatroomtemperature.

      6.WashslideswithPBS,changingthesolutionthreetimesovera10minperiod.

      7.Applyoptimaldilutionofasecondantibodyconjugate(e.g.,anti-mouseIgG-peroxidase),incubate,wash,andperformdetectionwithasubstratethatproducesaninsolubleproduct.Afterdetection,counterstainwithHarris-modifiedhematoxylinifdesired.Slidescanthenbedehydratedandmounted.
      BIOLOGicalInformation
      ImmunogenBromodeoxyuridine-bovineserumalbuminconcentrate
      CloneAH4H7-1/131-14871
      ConcentrationPleaserefertotheCertificateofAnalysisforthelot-specificconcentration.
      HostMouse
      SpecificityBindstobromodeoxyuridineandcrossreactswithiodouridine(10%).Anti-bromodeoxyuridinedoesnotcrossreactwithfluorodeoxyuridine,norwithanyendogenouscellularcomponentssuchasthymidineoruridine.
      IsotypeIgG1
      SpeciesReactivity
      • All
      AntibodyTypeMonoclonalAntibody
      PurificationMethodProteinAPurfied
      MolecularWeightdependentuponthemolecularweightofthebromodeoxyuridineincorporatedproteinbeingdetected
      PhysicochemicalInformation
      Dimensions
      MaterialsInformation
      MaterialsInformation
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