Fluoro-Gold Protocol and Use Guide MainProtocol
1. Background
TheuseofFluoro-Goldisessentiallythesameasotherfluorescenttracers.ThemaindifferenceisthatFluoro-GoldismoreflexIBLeintermsofpost-injectionsurvivaltimes,concentrationrange,tissuetreatmentandcompatibilitywithotherhistochemicaltechniques.
2.StorageandShelfLife
DryFluoro-Goldshouldbekeptinalighttightclosedcontainerat4degreesCelsius.Storedproperly,Fluorogoldshouldhaveashelflifeexceedingoneyear.Thedyeinsolutionshouldalsobekeptinalighttightclosedcontainerat4degreesCelsiusandshouldremainstableforatleastsixmonths.
3.Vehicle
Fluoro-Goldcanbedissolvedindistilledwateror0.9%saline,orutilizedasasUSPension
in0.2Mneutralphosphatebuffer.
4.DyeConcentration
Fluoro-Goldhasbeensuccessfullyusedatconcentrationsrangingfrom1-10%.Initially,a4%concentrationisadvised.Ifundesirablenecrosisoccursattheinjectionsite,orlabelingistoointense,reducetheconcentrationtoa2%solution.Ifyouneedtousemoreprecisemeasurements,themolecularweightofFluoro-Goldis532.6daltons.
5.DyeAdmiNISTration A.PressureInjection -Thisisprobablythemostfrequentlyusedmodeofapplication.Volumesinjectedrangefrom.05-1 µl,typically.1-.2 µl. B.Iontophoresis -Discrete,smallinjectionsitesresultfrom4-10secondpulsediontophoretic(+5to+10ua/10min)application. C.Crystal -Acrystalofthetracercanbeadministeredfromthetipofamicro-Pipette.
6.Post-0perativeSurvivalPeriod
Goodretrogradelabelinghasbeenobservedwithperiodsrangingfromtwodaystotwomonths.Survivalperiodsofthreetofivedaysaretypical.Longsurvivalperiodsenhancefillingofdistalprocesseswithoutdiffusionofthedyefromthecell.
7.Fixation
Almostanyfixative,ornofixative,canbeused,Phosphateneutralbufferedsalinecontaining4%formaldehydeisfrequentlyemployed.Fixativescontaininghighconcentrationsofheavymetals(e.g.osmium,mercury)willquenchthefluorescence,whilehighconcentrations(over1%)ofglutaraldehydemayincreasebackgroundfluorescence
8.HistochemicalProcessing
TissuecontainingFluoro-Goldmaybeprocessedaccordingtovirtuallyanycommonhistologicaltechnique.Thisincludescryostatsectionsofunfixedtissue(10µm),frozensectionsoffixedtissue(20µm),andthinsectionscutfromtissueimbeddedineitherplastic(.2-4µm)orparaffin(3-10µm).Frozensectionsoffixedtissuearemostfrequentlyused.
9.CombinedMethods
Atthispointofprocessing,sectionsmaybefurtherprocessedforasecondMarkersuchasautorADIography,HRPhistochemistry,immunocytochemistry,asecondfluorescenttracer,fluorescentcounterstain,etc.
10.Mounting,ClearingandCoverslipping
Sectionsaretypicallymountedongelatin-coatedslides,air-dried,immersedinxylene,andcoverslippedwithnonfluorescentDPXplasticmountingmedia.Sectionsmaybedehydratedwithgradedalcohols,unlessthisisnotcompatiblewithasecondtracer.IfFluoro-Goldistobecombinedwithfluorescenceimmunocytochemistry,thensectionsareair-driedanddirectlycoverslippedwithneutralbufferedglycerine(1:2).
11.ExaminationandPhotography
Fluoro-Goldcanbevisualizedwithafluorescencemicroscopeusingawidebandultravioletexcitationfilter.AgoldcolorisemittedwhentissuehasbeenprocessedwithneutralpHbuffer,whereasabluecolorisemittedwhentissueisprocessedwithacidic(e.g.pH3.3)pHbuffer.Itcanbephotographeddigitallyorwithfilm(useEktachrome200-400ASAfilmforcolorprintsandcomparablespeedfilmforblackandwhiteprints,forexampleTri-X).Mostexposuretimesrangefrom10-60secondexposures,dependingontheobjectivemagnificationandtheintensityofthelabel.Thirty(30)secondexposuresareaboutaverage.MultipleexposuresmaybeexploitedtosimultaneouslyvisualizeFluoro-Goldandanothertracer.Thus,UVwouldbecombinedwithbrightfieldIlluminationtosimultaneouslylocateFluoro-GoldwithHRPorsilvergrainsinautoradiography.Similarly,bluelightexcitationcanbecombinedtoalsovisualizethegreenemissioncolorofFITC,whilegreenexcitationlightmaybeusedtosimultaneouslyobservetheredemissioncolorofpropidiumiodide,orethidiumbromide(afluorescentcounterstain).
AdditionalInformationConcerningtheUseofFluoro-Gold
Vehicle
Forpressureinjectionsthroughamicrosyringeormicropipette,Fluoro-Goldshouldbedissolvedindistilledwateror.9%saline.Fluoro-Goldmayalsobeutilizedasasuspensionin.2Mneutralphosphatebuffer,however,thesuspendedparticlesmayclogafinemicropipettetipsodistilledwateror.9%salineisthepreferredvehicle.Foriontophoresis,a1%Fluoro-Goldsolutionismadeupin.1Macetatebuffer(pH=3.3).Well-cleaned(95%ETOH,water)glassmicropipettesshouldhavetipsof10-20µm.Optimaliontophoresisparametersare+1to+5uampsdeliveredwithpulsedcurrent(4-10secondson,4-10secondsoff)overa10-20minuteperiod.
InjectionSites
VirtuallyanycentralorperipheralnervoussystemstructurecanbeinjectedwithFluoro-Goldforanalysisofretrogradetransport.Intheperipheralnervoussystem,gangliaandperipheraltargetscanbestudied.Forstudiesofperipheralnerve,thenerveshouldbecutordamagedandeitherdippedin,orinjectedwith,aq5%solutionofFluoro-Gold.SinceFluoro-Goldisnotsignificantlytakenupbyintactfibersofpassage,thefibersmustbecutorseverelydamagedforuptakeofthedyetooccur.
TransportandSurvivalTime
Fluoro-Goldisusedasaretrogradeaxonaltracer,althoughorthogradeaxonaltransportdoesoccur.Thesurvivaltimeshouldbevaried(especiallytoveryshortsurvivaltimesof12hours-2days)tomaximizeorthogradetransportinthespecificneuronalsystemunderstudy.Forretrogradetransport,thesurvivaltimesshouldbevariedfrom4daysto14days.Sevento10daysworksformostsystems,althoughlongpathways(e.g.,spinalcordtobrainstem)andpathwaysinlargemammals(e.g.,cats,monkeys)mayrequirelongersurvivaltimes(e.g.,14days).Inaddition,sinceFluoro-Goldremainsfastwithinretrogradelylabeledneurons,survivaltimesofseveralmonthswillalsoproduceexcellentresults.Foriontophoresis,a2-5daysurvivaltimeisrecommended.Itisestimatedthattransportoccursatabout2cmperdayformammals;itisslowerforcold-bloodedanimals.
TissueProcessing
Tissueprocessingiscoveredindetailintheuseguideandintheoriginalpublication(SchmuedandFallon,1986,BrainResearch377:147-154).SinceFluoro-Goldisstableinmanysolventsandremainsfastwithinretrogradelylabeledneurons,it"suseiscompatiblewithmanyhistochemicaltechniques.Itcanbeusedwithotherretrogradetracers,immunofluorescence,PAPandABCimmunocytochemistry,HRPhistochemistry,autoradiography,counterstains(ethidiumbromideisthepreferredfluorescentcounterstain),paraffinembeddingandplasticembedding.However,iftissueisunfixed,additionalprocessingoftissueinaqueoussolutionsforoveranhourortwowillresultinlossofFluoro-Goldfluorescencefromlabeledneurons.Fluoro-Goldmaybeusefulinelectronmicroscopy.Fluoro-Goldcanbeusedinabrainwhichhasbeensectionedandtransferredtophosphatebuffer.Sectionsaretypicallymountedongelatin-coatedslides,airdried,immersedinxyleneandcoverslippedwithDPXplasticmountingmedia(FLUKAChemicalCorp.,255OserAvenue,Hauppauge,NewYork,11788,Catalog#44581).Tissuemayalsobeviewedonslideswithoutfurtherprocessing,canberunthroughgradedalcoholsfordehydration,or,forimmunocytochemistry,thesectionscanbeairdriedanddirectlycoverslippedwithneutralbufferedglycerine(1:2).
ExaminationandPhotography
Fluoro-Goldisvisualizedwithafluorescencemicroscopeusingawidebandultraviolet(UV)excitationfilter.UsethesamefilterpackyouwouldforotherfluorescentretrogradetracersexcitedunderwidebandUV(e.g.,TrueBlue,FastBlue,NuclearYellow),suchastheLeitzPloemfiltersystemA(WideBandUV,ExcitationfilterBP340-380),MirrorRKP400,BarrierFilterLP430).Objectivesshouldbemadeespeciallyforfluorescencemicroscopy(suchasthatmadebyZeiss)glycerine,orwater.SinceplasticdoesabsorbUVlight,itisnotadvisedtoviewthroughplasticpetridishes,etc.RecommendedfilmsareT-Max(Kodak,black&white)andEktachrome200(Kodak,colorslides).Exposuretimesusuallyvaryfrom20secondsto1.5minutes.
ChemicalAnalysis |
美国Fluorochorome公司于1985年成立于美国科罗拉多州丹佛市，Fluorochorome一直致力于荧光染料的研究与开发，其专利产品Fluoro-Gold（荧光金）自1985年来在全球范围内被广泛应用，并有大量的参考文献。同时，Fluorochrome公司特别开发了高灵敏度的Fluoro-GoldAntibody、以及Fluoro-Ruby（红色荧光金）等产品，Fluorochrome公司坚持以优质的产品和热情的服务赢得了良好的知名度和口碑。
