CAMASSAYShell-lessembryocultureFertilizedwhiteleghornchickeneggs(SPAFASInc.,Norwich,CT)werereceivedatday0andincubatedfor3daysat37°Cwithconstanthumidity.Onday3,eggswererinsedwith70%ethanolandopenedinto100mm2tissueculturecoatedPetridishesunderasepticconditions.Theembryoswerethenreturnedtoahumidified38°Cincubator2for7-9additionaldays.MeshassaySubstratesVitrogen(CollagenBiomaterials,PaloAlto,CA)wasdilutedtoaconcentrationof1.46mg/mlwithanequalvolumeofDMEM(HEPESbuffer,nophenolred,GibcoBRL,Gaithersberg,MD).This1:1mixturemadeuphalfofthetotalvolumetobePipettedontothemesh(finalconcentration=0.73mg/ml).Matrigel(BectonDickinson,Bedford,MA)wasdilutedtoafinalconcentrationof10mg/mlwithDMEManddirectlypipettedontomeshes.Nylonmeshwith250μm2openingswerecutinto4mmx4mmsquaresandautoclaved.Inpreparationforpolymerization,mesheswereplaced,underasepticconditions,ontoanonbindingsurface(i.e.,bacteriologicalPetridish).Thepolymerizationconditionsforeachsubstratewereidentical;aftermixingthegrowthfactorsand/orcompounds,40μlwerepipettedontoeachmeshinabacteriologicalPetridishasdescribedabove.ThePetridishwasplacedinahumidified37°Cincubatorwith5%CO2for30minutestoallowpolymerizationfollowedbyanincubationat4°Cfor2hours.GrowthFactorsVPF/VEGF165(PeproTech,RockyHill,NJ)wasresUSPendedat100ng/μlinHBSS(Sigma,St.Louis,MO).ForeachmeshcontainingVPF/VEGF,250μgwasused.PlacementofmeshesInatissuecultureenclosure,mesheswereplacedontotheperipheryoftheCAMofaday12-14embryo,excludingareascontainingmajorvessels.Theembryoswerethenreturnedtothehumidified38°Cincubatorwith3%CO2for24to48additionalhours.VisualizationofvesselsEmbryoswereremovedfromtheincubatorandmesheswereviewedunderadissectingmicroscopeforgrossevaluation.Forinjection,borosilicateglasscapillaries(OD1.0mm,ID0.75mm;SutterInstrumentCompany,Novato,CA)werepreparedwithP-87micropipettepuller(SutterInstrumentCompany).NeedleswereconnectedwithTygontubing(ID1/32",OD3/32",wall1/32")toa3ccsyringewitha20-gaugeneedle.Thesyringewasattachedtoaninfusionpump(HarvardApparatus,SouthNatick,MA).Injectionof400μlFITCdextran,MW2,000,000(Sigma,St.Louis,MO),intotheumbilicalveinwasperformedatarateof200μlperminute.TheFITCdextranwasallowedtocirculatefor5minutesand3.7%formaldehydeinPBSwasapplieddirectlyoneachmesh.Theembryoswerethenincubatedat4°Cfor5minutesandthemeshesweredissectedofftheCAMandfixedin3.7%formaldehydefor10minutestoovernight.QuantificationofvesselsAfterfixation,meshesweremountedonslideswith90%glycerolin1XPBSandvisualizedonaninvertedfluorescencemicroscope.ANikonDiaphotwithaSonyDXC-151Acameraattachedtothesideportwasusedforcaptureofimages,whichweretransferredtoaPowerMacintosh7100/66AV.NIHImage1.61(publicdomainprogramavailableontheInternetathttp://rsb.info.nih.gov/nih-image/)wasusedtocaptureandanalyzeimages.Foreachmesh,5randomstaggeredimages(approximately600μmeach)werecapturedusing"CaptureFrames,"followedby"MakeMontage"inordertodisplayalloftheframesatonce.Theareasofhighintensitywerehighlightedusing"DensitySlice"with(LUTselectedat105)andmeasuredusingthe"Measure"function.Whendensityslicingisusedinanimage,the"Measure"functioncalculatestheareasofhighlightedpixels.

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