DescriptionCatalogueNumberMAB377BBrandFamilyChemicon®TradeNameChemiconDescriptionAnti-NeuNAntibody,cloneA60,biotinconjugatedAlternateNamesNeuron-SpecificNuclearProteinBackgroundInformationNeuNantibody(NEUronalNuclei;cloneA60)specificallyrecognizestheDNA-binding,neuron-specificproteinNeuN,whichispresentinmostCNSandPNSneuronalcelltypesofallvertebratestested.NeuNproteindistributionsareapparentlyrestrictedtoneuronalnuclei,perikaryaandsomeproximalneuronalprocessesinbothfetalandadultbrainalthough,someneuronsfailtoberecognizedbyNeuNatallages:INLretinalcells,Cajal-Retziuscells,Purkinjecells,inferiorolivaryanddentatenucleusneurons,andsympatheticganglioncellsareexamples(Mullenetal.,1992;Wolfetal.,1996).ImmunohistochemicallydetectableNeuNproteinfirstappearsatdevelopmentaltimepointsthatcorrespondwiththewithdrawaloftheneuronfromthecellcycleand/orwiththeinitiationofterminaldifferentiationoftheneuron(Mullenetal.,1992).ImmunoreactivityappearsaroundE9.5inthemouseneuraltubeandisextensivethroughoutthedevelopingnervoussystembyE12.5.Strongnuclearstainingsuggestsanuclearregulatoryproteinfunction;however,noevidencecurrentlyexistsastowhethertheNeuNproteinantigenhasafunctioninthedistalcytoplasmorwhetheritismerelysynthesizedtherebeforebeingtransportedbackintothenucleus.Nodifferencebetweenproteinisolatedfrompurifiednucleiandwholebrainextractonimmunoblotshasbeenfound(Mullenetal.,1992).ProductInformationFormatBiotinControlBraintissue,mostneuronalcelltypesthroughouttheadultnervoussystemPresentationPurifiedmousemonoclonalIgG1inbuffercontaining0.01MPBSpH7.1,0.1%sodiumazidewith15mg/mLBSAasastABIlizer.StorageandShippingInformationStorageConditionsStablefor1yearat2-8ºCfromdateofreceipt.ApplicationsApplicationThisAnti-NeuNAntibody,cloneA60,biotinconjugatedisvalidatedforuseinIC,IH,IH(P),WBforthedetectionofNeuN.KeyApplicationsImmunocytochemistryImmunohistochemistryImmunohistochemistry(Paraffin)WesternBlottingApplicationNotesImmunocytochemistryAnalysis:1:10-1:500dilutionofapreviouslotwasused.Neuronsincultureshouldbepermeablizedwith0.1%tritonX-100.Allprimaryantibodydilutionsshouldbeperformedwithsimplesolutionscontainingonlybufferandprimaryantibodywithoutexcessproteinblocksordetergents.Forduallabelingstudiesusingmousemonoclonals,antibodyincubationsshouldbesequentialwithMAB377Blast.Firstmousemonoclonalantibodyshouldbefirstdetectedwithanti-mousesecondarypriortoincubatingwithMAB377B.Excessanti-mouseIgGmaybeblockedbyincubatingwith1%mouseserumpriortoMAB377Bincubation.DetectionofbiotinylatedNeuNmonoclonalisviastreptavidin.Insomecasesinmaybenecessarytopretreatthetissuewithavidintoblockexcessbiotinpriortoimmunohistochemisty(WoodandWarnke,1981).Immunohistochemistry:1:200-1:2,000.Theantibodyworksbestonpolyesterwaxembeddedtissuebutalsoworksonparaffinembeddedtissueatalowerworkingdilution.Theantibodyworkswellwithformaldehyde-basedfixatives.Citricacidandmicrowavepretreatmenthasbeenusedsuccessfully(Sarnat,1998).WesternBlottingAnalysis:Apreviouslotofthisantibodywasusedonwesternblot.Recognizes2-3bandsinthe46-48kDarangeandpossIBLyanotherbandatapproximately66kDa.Optimalworkingdilutionsmustbedeterminedbyenduser.BIOLOGicalInformationImmunogenPurifiedcellnucleifrommousebrainCloneA60ConcentrationPleaserefertotheCertificateofAnalysisforthelot-specificconcentration.HostMouseSpecificityVertebrateneuron-specificnuclearproteincalledNeuN(NeuronalNuclei).MAB377Breactswithmostneuronalcelltypesthroughoutthenervoussystemofmiceincludingcerebellum,cerebralcortex,hippocampus,thalamus,spinalcordandneuronsintheperipheralnervoussystemincludingdorsalrootganglia,sympatheticchaingangliaandentericganglia.Theimmunohistochemicalstainingisprimarilyinthenucleusoftheneuronswithlighterstaininginthecytoplasm.ThefewcelltypesnotreactivewithMAB377BincludePurkinje,mitralandphotoreceptorcells.Developmentally,immunoreactivityisfirstobservedshortlyafterneuronshavebecomepostmitotic,nostaininghasbeenobservedinproliferativezones.TheantibodyisanexcellentMarkerforneuronsinprimaryculturesandinretinoicacid-stimulatedP19cells.Itisalsousefulforidentifyingneuronsintransplants.IsotypeIgG1SpeciesReactivityMouseRatHumanFerretChickenSalamanderSpeciesReactivityNoteRatandmouse.Itisexpectedthatthebiotinconjugatedantibodywillalsoreactwithhuman,ferret,chickandsalamander.AntibodyTypeMonoclonalAntibodyGeneSymbolNeuNA59PurificationMethodProteinAPurfiedMolecularWeight46-48kDaPhysicochemicalInformationDimensionsMaterialsInformationMaterialsInformation