DescriptionCatalogueNumberMAB345BrandFamilyChemicon®TradeNameChemiconDescriptionAnti-O4Antibody,clone81AlternateNamesSulfatideBackgroundInformationOligodendrocytesandastrocytesarederivedfromcommonprecursorcells,glioblasts(Sommer,1981).O-Antigensaresulfatides,whichfunctionasdifferentiationMarkersonthesurfaceofoligodendrocytesofthecentralnervoussystem.O4isformedpostnatally(Schachner,1981)andisamarkerforcellbodiesandprocessesofoligodendrocytestypesIandII(hairyeyeballtype).Duringoligodendrocytedifferentiation,O4occursinpro-oligodendrocytes,butnotinO-2A-Progenitorcells.O4occursfromday3onwardsincellculturesofembryonicmousebrain.Anti-O4canbeusedinmyelination,demyelinationandremyelinationstudiesandinregenerationexperiments.ProductInformationFormatPurifiedControlRatcorticalstemcellsorday3cellculturesofbrainsfrommouseembryosPresentationPurifiedimmunoglobulinin0.05MPotassiumphosphatebuffer,pH8.0with0.3MNaCland0.05%sodiumazide.StorageandShippingInformationStorageConditionsMaintainfor1yearat2–8°Cfromdateofshipment.Aliquottoavoidrepeatedfreezingandthawing.Formaximumrecoveryofproduct,centrifugetheoriginalvialafterthawingandpriortoremovingthecap.ApplicationsApplicationAnti-O4Antibody,clone81isanantibodyagainstO4foruseinIC,IHwithmorethan50productcitations.KeyApplicationsImmunocytochemistryImmunohistochemistryApplicationsNotRecommendedImmunoprecipitationWesternBlottingApplicationNotesImmunohistochemistry:10-20μg/mLonunfixed,shockfrozentissue.Immunocytochemistry:10-20μg/mLoncellsfixedwith4%paraformaldehyde.Note:O4isasulfatide,whichcanbedissolvedoutofthemembranebyorganicsolvents;acetoneandmethanolshouldnotbeusedforfixation.Optimalworkingdilutionsmustbedeterminedbytheenduser.Immunohistochemistryprotocol1.Preparesectionsfromunfixed,shockfrozentissue.Thesectionsshouldbe4-5μmthick.Placethesectionsonmicroscopeslides.2.Washtheslidethreetimesfor5min.eachinPBSatroomtemperature.3.Blockthenon-specificbindingsitesbyincubatingthesectionsinahumidchamberwith5%FCSatroomtemperaturefor30minutes.4.Washtheslidesasdescribedinstep2.5.CoverthesectionswithasufficientamountofMAB345(10-20μg/mLinPBS)andincubateinahumidchamberat37°Cforonehour.6.WashtheslidesbrieflythreetimeswithPBS.Carefullydryaroundtheareatobestained.7.Coverthesectionswithasufficientamountofanti-mouseIgM-fluorescein*solutionandincubateinahumidchamberat37°Cforonehour.8.Washtheslidesasdescribedinstep6.9.Coverthesectionswithasuitableembeddingmedium,coverwithacoverslip,andexaminebyfluorescencemicroscopy.*HRPorABCcanalsobeused.OptimalresultscanbeobtainedbytitratingtheprimaryandsecondaryantibodiesImmunocytochemistry1.Fixthepreparationswith4%paraformaldehyde(inPBS)atroomtemperaturefor10minutes.O4isasulfatidewhichcanbedissolvedoutofthemembranebyorganicsolvents;acetoneandmethanolshouldnotbeusedforfixation.2.Washtheslidethreetimesfor5min.eachinPBSatroomtemperature.3.Blockthenon-specificbindingsitesbyincubatingthesectionsinahumanchamberwith5%FCSatroomtemperaturefor30minutes.4.Washtheslidesasdescribedinstep2.5.CoverthesectionswithasufficientamountofMAB345(10-20μg/mLinPBS)andincubateinahumidchamberat37°Cforonehour.6.WashtheslidesbrieflythreetimeswithPBS.Carefullydryaroundtheareatobestained.7.Coverthesectionswithasufficientamountofanti-mouseIgM-fluoresceinsolutionandincubateinahumidchamberat37°Cforonehour.8.Washtheslidesasdescribedinstep6.9.Coverthesectionswithasuitableembeddingmedium,coverwithacoverslip,andexaminebyfluorescencemicroscopy.Note:Donotallowthepreparationstodryoutduringstaining.BIOLOGicalInformationImmunogenHomogenateofwhitematterofcorpuscallosumfrombovinebrain.Clone81(alsoreferredtointheliteratureasmABO4)ConcentrationVaries,seelotspecificCoAHostMouseSpecificityRecognizesOligodendrocytemarkerO4.Alsoreactswithcertaingalactolipidsinsperm(seeAdditionalInformationlibraryforlist).IsotypeIgMSpeciesReactivityChickenHumanMouseRatAntibodyTypeMonoclonalAntibodyPurificationMethodAmmoniumSulfatePrecipitationPhysicochemicalInformationDimensionsMaterialsInformationMaterialsInformation