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Millipore/AG325 | Fluoro-Jade? C/AG325/50 mg

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貨號: AG325

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Description
CatalogueNumber	AG325
BrandFamily	Chemicon®
TradeName	Fluoro-Jade Chemicon
Description	Fluoro-Jade?C
Overview	Fluoro-Jade?C,likeitspredecessors,Fluoro-Jade?andFluoro-Jade?B,werefoundtostainalldegeneratingneurons,regardlessofspecificinsultormechanismofcelldeath.Therefore,thepatternsofneuronaldegenerationseenfollowingexposuretoeithertheglutamateagoNIST,kainicacid,ortheinhibitorofmitochondrialrespiration,3-NPA,werethesameforalloftheFluoro-Jadeadyes.However,therewasaqualitativedifferenceinthestainingcharacteristicsofthethreeFluorochromes.Specifically,Fluoro-Jade?Cexhibitedthegreatestsignaltobackgroundratio,aswellasthehighestresolution.Thistranslatestoastainofmaximalcontrastandaffinityfordegeneratingneurons.Thismakesitidealforlocalizingnotonlydegeneratingnervecellbodies,butalsodistaldendrites,axonsandterminals.ThedyeishighlyresistanttofADIngandiscompatIBLewithvirtuallyallhistologicalprocessingandstainingprotocols.TriplelabelingcanbeaccomplishedbystainingdegeneratingneuronswithFluoro-Jade?C,cellnucleiwithDAPIandactivatedastrocyteswithGFAPimmunofluorescence. APPEARANCE:Coffeebrowntobrickredpowder. MOLECULARWEIGHT:823 EXCITATIONPEAK:485nm EMISSIONPEAK:525nm FILTERSYSTEM:Fluorescein/FITC SOLUBILITY:Highlysolubleinwaterandbases,moderatelysolubleinalcoholandweakacids. TOXICITY:Althoughthecompoundappearstobeoflowtoxicity,ithasnotbeenextensivelyevaluatedandthereforeroutinelaboratorycautionshouldbeexercised.Notintendedforhumanconsumption.
BackgroundInformation	Fluoro-Jade?C,likeitspredecessors,Fluoro-Jade?andFluoro-Jade?B,werefoundtostainalldegeneratingneurons,regardlessofspecificinsultormechanismofcelldeath.Therefore,thepatternsofneuronaldegenerationseenfollowingexposuretoeithertheglutamateagonist,kainicacid,ortheinhibitorofmitochondrialrespiration,3-NPA,werethesameforalloftheFluoro-Jade?dyes.However,therewasaqualitativedifferenceinthestainingcharacteristicsofthethreefluorochromes.Specifically,Fluoro-Jade?Cexhibitedthegreatestsignaltobackgroundratio,aswellasthehighestresolution.Thistranslatestoastainofmaximalcontrastandaffinityfordegeneratingneurons.Thismakesitidealforlocalizingnotonlydegeneratingnervecellbodies,butalsodistaldendrites,axonsandterminals.Thedyeishighlyresistanttofadingandiscompatiblewithvirtuallyallhistologicalprocessingandstainingprotocols.TriplelabelingcanbeaccomplishedbystainingdegeneratingneuronswithFluoro-Jade?C,cellnucleiwithDAPIandactivatedastrocyteswithGFAPimmunofluorescence.Fluoro-Jade?isaregisteredtrademarkofHisto-Chem,Inc.

ProductInformation

StorageandShippingInformation
StorageConditions	Thelyophilizedpowdershouldbestoredwellsealedatroomtemperature,preferableinadesiccator(duetoitshygroscopicnature)foruptooneyearfromdateofreceipt.Theliquidstocksolution(0.01%)indistilledwatercanbestoredat2-8°Cforupto3months.The.0002-.0001%workingsolutionin0.1%aceticacidshouldbeusedwithin4hrsofpreparation.

Applications
KeyApplications	Immunohistochemistry
ApplicationNotes	SUGGESTEDPROTOCOLFORUSINGFLUORO-JADE?C Processing:Halfofeachgroupofbrainswereparaffinembeddedandcutonarotarymicrotomewhiletheremainderwerecutonafreezingslidingmicrotome.Paraffinsectionswere10uminthicknesswhilefrozensectionswerecutatathicknessof25um.Priortostaining,sectionsweremountedfromdistilledwaterontogelledslides.Gelatincoatedslideswerepreparedbyimmersionina60degreeCsolutionof1%pigskingelatin(Sigma;typeA,300Bloom)andthenovendriedovernightatthesametemperature.Thesectionsweremountedontotheslidesfromdistilledwaterandthenairdriedforatleast30minonaslidewarmerat50degreesC.Slidesbearingfrozencuttissuesectionswerefirstimmersedinabasicalcoholsolutionconsistingof1%sodiumhydroxidein80%ethanolfor5min.Theywerethenrinsedfor2minin70%ethanol,for2minindistilledwater,andthenincubatedin0.06%potassiumpermanganatesolutionfor10min.Followinga1-2minwaterrinse,theslideswerethentransferredfor10mintoa0.0001%solutionofFluoro-Jade?Cdissolvedin0.1%aceticacidvehicle.Theproperdilutionwasaccomplishedbyfirstmakinga0.01%stocksolutionofthedyeindistilledwaterandthenadding1mLofthestocksolutionto99mLof0.1%aceticacidvehicle.Theworkingsolutionwasusedwithin2hofpreparation.Thestocksolution,whenrefrigerated,canbekeptforlongperiodsbutshouldbediscardedifthesolutionbecomescloudy.Theslideswerethenrinsedthroughthreechangesofdistilledwaterfor1minperchange.Excesswaterwasdrainedontoapapertowel,andtheslideswerethenairdriedonaslidewarmerat50degreesCforatleast5min.Theairdriedslideswerethenclearedinxyleneforatleast1minandthencoverslippedwithDPX(FlukaorSigma)nonfluorescentmountingmedia.Polarcoverslippingmedia,suchasthosethatcontainwater,alcoholorglycerolwereneverused.Forcomparativepurposes,someslideswerestainedwithFluoro-Jade?Baccordingtothepreviouslydescribedprocedure.Whenworkingwithparaffinprocessedtissue,thesectionsarefirstdeparaffinizedthroughtwo10minchangesofxyleneandthenthesectionsarerehydratedthroughagraduatedalcoholseries,omittingthebasicalcoholsolution.Onceindistilledwater,thesectionsaretransferredtothepotassiumpermanganatesolutionatwhichpointthestainingprocedureisidenticaltothatdescribedforfrozensections.Multiplelabeling:Fluoro-Jade?CcanreadilybecombinedwithotherfluorescentMarkers.Multiplelabelingwasachievedusinganti-glialfibrillaryacidicprotein(GFAP)immunocytochemistrytolabelactivatedastrocyteswhileusingDAPItolabelnuclearDNA.Incorporating4",6-diamidino-2-phenylindole(DAPI;Sigma,St.LouisMO)asafluorescentnuclearstainisaccomplishedbysimplyincorporating0.0001%intotheFluoro-Jade?Cstainingsolution.Thisisaccomplishedbytheadditionof1mlof0.01%DAPIstocksolutionto99mlof0.1%aceticacid.Fluoro-Jade?CwasalsocombinedwithimmunofluorescentlabelingofGFAPaccordingtothefollowingprocedure.Loosefrozentissuesectionswereincubatedinapredilutedsolutionofanti-GFAP(ChemiconhasanumberofdifferentantibodiestoGFAP)atabout5degreesCintherefrigeratorfor1-3days.Itshouldbementionedthatalthoughinthisstudyallimmunocytochemistrywasperformedonfrozensections,themethodsarefullycompatiblewithparaffinprocessedtissueaswell.Sectionswererinsedintwochangesofbufferedsalinefor10mineachandthentransferredtoatetramethylrhodamineisothiocynate(TRITC)labeledsecondaryantibody(ChemiconhasanumberofdifferentTRITClabeledsecondaryantibodies),diluted1:100inbufferedsaline,for1hatroomtemperature.Sectionswererinsedintwochangesofbufferedsalinefor10mineachandthenthesectionsweremountedontogelledslidesfromdistilledwaterandairdriedonaslidewarmerat50degreesCfor30min.TocombinewithFluoro-Jade?C,theslidemountedsectionswererehydratedfor2minindistilledwaterandthentransferredtothe0.06%potassiumpermanganatesolutionfor10min.Itisworthmentioningthattheincubationtimeinpotassiumpermanganatemayneedtobereducedwhenco-localizingthoseantigenicepitopessusceptibletochemicaloxidation.Theslideswerethenrinsedfor2minindistilledwater,transferredtotheFluoro-Jade?Cworkingsolutionfor10minandthenrinsed,airdehydrated,xyleneclearedandcoverslippedwithDPX,aspreviouslydescribed.ThebluenuclearlabelconferredbyDAPIisvisualizedviaultravioletlightexcitation,whiletheredTRITClabeledantibodyisvisualizedbygreenlightexcitation.

Applications

KeyApplications

Immunohistochemistry

ApplicationNotes

SUGGESTEDPROTOCOLFORUSINGFLUORO-JADE?C

Processing:Halfofeachgroupofbrainswereparaffinembeddedandcutonarotarymicrotomewhiletheremainderwerecutonafreezingslidingmicrotome.Paraffinsectionswere10uminthicknesswhilefrozensectionswerecutatathicknessof25um.Priortostaining,sectionsweremountedfromdistilledwaterontogelledslides.Gelatincoatedslideswerepreparedbyimmersionina60degreeCsolutionof1%pigskingelatin(Sigma;typeA,300Bloom)andthenovendriedovernightatthesametemperature.Thesectionsweremountedontotheslidesfromdistilledwaterandthenairdriedforatleast30minonaslidewarmerat50degreesC.Slidesbearingfrozencuttissuesectionswerefirstimmersedinabasicalcoholsolutionconsistingof1%sodiumhydroxidein80%ethanolfor5min.Theywerethenrinsedfor2minin70%ethanol,for2minindistilledwater,andthenincubatedin0.06%potassiumpermanganatesolutionfor10min.Followinga1-2minwaterrinse,theslideswerethentransferredfor10mintoa0.0001%solutionofFluoro-Jade?Cdissolvedin0.1%aceticacidvehicle.Theproperdilutionwasaccomplishedbyfirstmakinga0.01%stocksolutionofthedyeindistilledwaterandthenadding1mLofthestocksolutionto99mLof0.1%aceticacidvehicle.Theworkingsolutionwasusedwithin2hofpreparation.Thestocksolution,whenrefrigerated,canbekeptforlongperiodsbutshouldbediscardedifthesolutionbecomescloudy.Theslideswerethenrinsedthroughthreechangesofdistilledwaterfor1minperchange.Excesswaterwasdrainedontoapapertowel,andtheslideswerethenairdriedonaslidewarmerat50degreesCforatleast5min.Theairdriedslideswerethenclearedinxyleneforatleast1minandthencoverslippedwithDPX(FlukaorSigma)nonfluorescentmountingmedia.Polarcoverslippingmedia,suchasthosethatcontainwater,alcoholorglycerolwereneverused.Forcomparativepurposes,someslideswerestainedwithFluoro-Jade?Baccordingtothepreviouslydescribedprocedure.Whenworkingwithparaffinprocessedtissue,thesectionsarefirstdeparaffinizedthroughtwo10minchangesofxyleneandthenthesectionsarerehydratedthroughagraduatedalcoholseries,omittingthebasicalcoholsolution.Onceindistilledwater,thesectionsaretransferredtothepotassiumpermanganatesolutionatwhichpointthestainingprocedureisidenticaltothatdescribedforfrozensections.Multiplelabeling:Fluoro-Jade?CcanreadilybecombinedwithotherfluorescentMarkers.Multiplelabelingwasachievedusinganti-glialfibrillaryacidicprotein(GFAP)immunocytochemistrytolabelactivatedastrocyteswhileusingDAPItolabelnuclearDNA.Incorporating4",6-diamidino-2-phenylindole(DAPI;Sigma,St.LouisMO)asafluorescentnuclearstainisaccomplishedbysimplyincorporating0.0001%intotheFluoro-Jade?Cstainingsolution.Thisisaccomplishedbytheadditionof1mlof0.01%DAPIstocksolutionto99mlof0.1%aceticacid.Fluoro-Jade?CwasalsocombinedwithimmunofluorescentlabelingofGFAPaccordingtothefollowingprocedure.Loosefrozentissuesectionswereincubatedinapredilutedsolutionofanti-GFAP(ChemiconhasanumberofdifferentantibodiestoGFAP)atabout5degreesCintherefrigeratorfor1-3days.Itshouldbementionedthatalthoughinthisstudyallimmunocytochemistrywasperformedonfrozensections,themethodsarefullycompatiblewithparaffinprocessedtissueaswell.Sectionswererinsedintwochangesofbufferedsalinefor10mineachandthentransferredtoatetramethylrhodamineisothiocynate(TRITC)labeledsecondaryantibody(ChemiconhasanumberofdifferentTRITClabeledsecondaryantibodies),diluted1:100inbufferedsaline,for1hatroomtemperature.Sectionswererinsedintwochangesofbufferedsalinefor10mineachandthenthesectionsweremountedontogelledslidesfromdistilledwaterandairdriedonaslidewarmerat50degreesCfor30min.TocombinewithFluoro-Jade?C,theslidemountedsectionswererehydratedfor2minindistilledwaterandthentransferredtothe0.06%potassiumpermanganatesolutionfor10min.Itisworthmentioningthattheincubationtimeinpotassiumpermanganatemayneedtobereducedwhenco-localizingthoseantigenicepitopessusceptibletochemicaloxidation.Theslideswerethenrinsedfor2minindistilledwater,transferredtotheFluoro-Jade?Cworkingsolutionfor10minandthenrinsed,airdehydrated,xyleneclearedandcoverslippedwithDPX,aspreviouslydescribed.ThebluenuclearlabelconferredbyDAPIisvisualizedviaultravioletlightexcitation,whiletheredTRITClabeledantibodyisvisualizedbygreenlightexcitation.

BIOLOGicalInformation
Purity	SilicaTLC(acetanitrile/water,6/4)revealedthepresenceoftwofluorescentspots,presumablycorrespondingtothedisulphatehomologues.Thepresenceofprecursorsorfreefluoresceinwasnotdetected.

PhysicochemicalInformation

Dimensions

MaterialsInformation

MaterialsInformation

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Millipore/AG325 | Fluoro-Jade? C/AG325/50 mg

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