Materials and Methods.docx
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MaterialsandMethods
MaterialsandMethods
CellCulture
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