Materials and MethodsWord格式.docx

Materials and MethodsWord格式.docx

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ToassesstheeffectsofAngIIonneuraldifferentiation,weusedinvitrofetalmouseneurospheresandratneuralcellspreparedasdescribedpreviously.1AT2receptor-deficientmouse（Agtr2-;

basedonC57BL/6Jstrainbredinourlaboratory）andwild-typemouse（Agtr2+）orSprague-Dawleyratfetalbraintissuewasdissectedintocells.Forneurospherepreparation,cellswereplatedat105cells/mlinneuralstemcellbasalmedium（NeuroCult,StemCellTechnologiesInc.,Vancouver,BC）,withdifferentiationsupplements（NeuroCultDifferentiationSupplements,StemCellTechnologiesInc.）andbasicfibroblastgrowthfactor（b-FGF,20ng/ml）.Neurospheresformedafter7-10daysinculture.Countingneurosphereswasperformedintheviewat×

20magnificationwithanAxioskopmicroscope（CarlZeiss）.Theywerepassagedevery10-14daysbymechanicaldissociationintosinglecells.Forneuralcellpreparation,cellswereplatedat105cells/mlonpoly-D-lysine-coateddisheswithserum-freeneurobasalmediumsupplementedwith2%B-27supplement（Invitrogen,Carlsbad,CA）,1mmol/Lglutamine,100units/mlpenicillinand100μg/mlstreptomycinsulfateat37oCina5%CO2atmosphere.

Immunoblotanalysis

Totalproteinwaspreparedfromneurospheres,andimmunoblottingwasperformedaspreviouslydescribed2usinganti-MAP2（CHEMICONInternationalInc.,Temecula,CA）,anti-βIIItubulin,anti-GFAPantibodies（PromegaCorporation,Madison,WI）andanti-MMS2（SantaCruzInc.,CA）antibodies.Thecelllysate（20μg）wasrunon10%SDS-PAGEandtheseparatedproteinswereelectrophoreticallytransferredtoanitrocellulosemembrane（HybondECL;

GEHealthcare,Piscataway,NJ）.Blotswereincubatedwithspecificantibodiesasindicated.ThebandswerevisualizedwithanECLsystem（GEHealthcare）.Densitometricanalysiswasperformedusinganimagescanner（EPSONGT-8000,RicohSystemKaihatsuCompanyLtd.,Tokyo,Japan）andNationalInstitutesofHealthimagingsoftware.

Immunofluorescence

Neurosphereswerefixedwithanethanolandmethanolsolution（1:

1mixed）priortoincubationwithrabbitpolyclonalanti-mouseAT1,AT2receptorantibody（SantaCruzInc.,CA）andmousemonoclonalanti-MMS2antibody（ZymedLaboratoriesInc.,SouthSanFrancisco,CA）.Thesecondaryantibodywasgoatanti-rabbitoranti-mouseCy3-conjugatedantibody（JacksonImmunoResearch,WestGrove,PA）.NegativecontrolstainingwasperformedwithmouseIgG（Sigma-AldrichCorp.,St.Louis,MO）tomeasurebackgroundstainingduetononspecificbindingorcross-reactivity.CellswerecounterstainedwithDAPIforthedetectionofnucleus.Imageswereviewedat×

40magnificationwithanAxioskopmicroscope（CarlZeiss,Oberkochen,Germany）,usingimageanalysissoftware（AxioVison,CarlZeiss）.

MMS2-siRNAAssay

ForsmallinterferingRNA（siRNA）assay,neuralstemcellsisolatedfromthemousefetalcortexandneurospheresweretransientlytransfectedwithnegativecontrolsiRNA,orMMS2-specificsiRNA,acocktailofthreesiRNAsdesignedbyB-Bridge（Sunnyvale,CA）,usingLipofectaminePLUS（Invitrogen）.Thirty-sixhoursaftertransfection,cellswereobservedandtreated.KnockdownofMMS2-genewasevaluatedbyimmunoblotusinganti-MMS2antibody（ZymedLaboratoriesInc.）.

ImmunohistochemicalStudy

Frozen,enzymaticallyintact,10-μm-thicksectionswerepreparedfrommousebrain24hoursafterMCAocclusionandfixedwith10%formaldehydesolutionpriortoincubationwithmousepolyclonalanti-MMS2antibody（ZymedLaboratoriesInc.）.Afterincubation,sectionswerestainedbyDABstainingusinganABCstainingkit（DakoCytomation,Glostrup,Denmark）.NegativecontrolstainingwasalsoperformedwithmouseIgG（Sigma-AldrichCorp.）tomeasurebackgroundstainingduetononspecificbindingorcross-reactivity.

AnimalsandTreatment

ThisstudywasperformedinaccordancewiththeNationalInstitutesofHealthguidelinesfortheuseofexperimentalanimals.AllanimalstudieswerereviewedandapprovedbytheAnimalStudiesCommitteeofEhimeUniversity.AdultmaleAT2receptor-deficientmice（Agtr2-）andwild-typemice（Agtr2+）（8to12weeksofage;

bodyweight,25to30g,purchasedfromNihonClea,Tokyo,Japan）wereusedinthisstudy.Theanimalswerehousedinaroomwherelightingwascontrolled（12hourson,12hoursoff）andthetemperaturewaskeptat25°

C.Theyweregivenastandarddiet（MF,OrientalYeastCo.Ltd.）andwateradlibitum.AnAT1receptorselectiveblocker（ARB）,valsartan（providedbyNovartisPharmaAG）,wasadministeredatadoseof3mg/kg/dayintraperitoneallyviaanosmoticmini-pump（Alzetmodel1002,DURECTCorporation,Cupertino,CA）implantedintraperitoneally10daysbeforeMCAocclusion.Bloodpressurewasmeasuredbythetail-cuffmethod（MK-1030,MuromachiCo.Ltd.,Tokyo,Japan）.

MCAOcclusion

Focalcerebralischemiawasinducedbyocclusionofth