DescriptionCatalogueNumberAG105TradeNameBLACK-GOLDDescriptionBlackGoldIIMyelinStainingKitOverviewBlack-GoldIIisanaurohalophosphatecomplexwhichstainsspecificallyformyelinwithinthecentralnervoussystem.Black-GoldIIstainingprovideshighresolution,highcontrast,ashorthistochemicalprocessingtime,andhighreproducibility.Black-GoldIIisanewandimprovedversionofitspredecessor,Black-Gold(Cat#AG390).TheadvantagesofMillipore’sBlack-GoldIIMyelinStainingKitoverBlackGoldIarethatitismorereADIlysoluble,canbeutilizedatahigherconcentration,producesamoreuniformandconsistentstaining,takeslesstimetostain,anddoesnotrequirepost-stainingintensificationprocedures.TheuseofBlack-GoldIIistailoredtostudiesusingformalin-fixed,non-solventprocessedtissue.Thetechniquestainslargemyelinatedtractsdarkred-brown,whiletheindividualmyelinatedfibersappearblack.Thisnoveltracercanbeusedtolocalizebothnormalandpathologicalmyelin.Black-GoldIIcandemonstrateandcharacterizespecificmyelinchangesassociatedwithexposuretodiverseneurotoxicantsincludingkainicacid,methamphetamine,acrylamide,domoicacid,3-nitropropionicacid,Fluoro-Goldandisoniazid.TheBlack-GoldIIMyelinStainingKitalsocomeswithsodiumthiosulfateandcresylviolet.ThesodiumthiosulfatesolutionisrequiredforextractingexcessBlack-GoldIIstain.WhereasthecresylvioletisaNisslstainthatcolorscellbodiesabrilliantbluishhueandisusedasacounterstainwithBlack-GoldII.BlackGoldIIPurity:Nodetectableamountofuncomplexedgoldwasfound.BlackGoldIIIllumination:Brightfieldordarkfield.BlackGoldIISolubility:Freelysolubleinwater,saline,ordiluteacids.ProductInformationComponentsBlack-GoldII:150mgdrypowder,yellowincolor.CresylVioletStain:150mgdrypowder,purplish-blackincolor.5%SodiumThiosulfate:10mlPresentationSeeComponentsStorageandShippingInformationStorageConditionsThekitcanbestoredatroomtemperature.Ideally,theBlack-GoldIIpowdershouldbekeptinadesiccator,becauseofitshygroscopicnature.Thedrypowder,ifunopenedandstoredproperly,isuseableforuptooneyearfromdateofreceipt.OnceresUSPendedtheBlack-GoldIIstainingsolutionshouldbestoredinthedarkat4°Candisgoodforupto3months.TOXICITY:Althoughthecompoundappearstobeoflowtoxicity,ithasnotbeenextensivelyevaluatedandthereforeroutinelaboratorycautionshouldbeexercised.Notintendedforhumanconsumption.ApplicationsKeyApplicationsImmunohistochemistryApplicationNotesTissueFixation:Tissuecanbefixedwitheitherformalinor4%PFA.Typicallyanimalsareperfusedwithneutralphosphatebuffered10%formalin(or4%formaldehyde)viatheascendingaorta.Thebrainsarethenremovedandfixedovernightinthesamefixativesolution.Longerfixationtimesaregenerallynotrecommended.Forcryosections,thebrainsmaybeprotectedthroughtreatmentwithsucrosesolution(20-30percent).Subsequentlyfrozenbraintissueiscutonafreezingslidingmicrotome.Note:Black-GoldIIwillnotstainparaffinembeddedorunfixedtissue.Sectioning:Eitherfrozenorfixed,non-frozenvibratomesectionscanbeusedandshouldbecutatathicknessof20-40μm.Thesectionsarethentypicallymountedonpositivelychargedslides.Thesectionscanalsobestainedloose,althoughthesectionsareeasiertohandlewhenmountedonslides.Solutions:Black-GoldIIpowdershouldberesuspendedwithsalinesolution(0.9%NaCl)toafinalconcentrationof0.3%.Simplyaddthe150mgofBlack-GoldIIpowderinto50mlsof0.9%NaClsolutionandmixtoresuspend.OnceresuspendedtheBlack-GoldIIstainingsolutionshouldbestoredinthedarkat4°Candisgoodforupto3months.CresylVioletpowdershouldberesuspendedtoafinalconcentrationof0.1%usingamixtureofMilliQwaterandglacialaceticacid(3µlofacidpermlofwater).Onceresuspendedthecresylvioletsolutionshouldbestoredat4°Candisgoodforupto6months.Thesodiumthiosulfateshouldbedilutedto1%usingMilliQwaterpriortouse.Staining:Pre-heat0.3%Black-GoldIIand1%sodiumthiosulfatesolutionsto60°C.RehydratetissuesectionsinMilliQwaterfor2minutesthentransfertopre-warmedBlack-GoldIIsolution.IncubateslidesinBlack-GoldIIsolutionat60°Cfor12minutes.Atthispointtheslidesshouldbemonitoredat2-3minuteintervalstodeterminetheextentoflabeling.Stainingiscompletewhenthefinestmyelinatedfibersarestainingdarkredtoblack.Theappearanceofalavenderbackgroundcolorindicatesoverstainingandtheprocessshouldbestopped.Note:Theexactstainingtimewillvarydependingonsectionthickness,fixingconditions,typeoftissue,andageofstainsolution.Thesectionsarethenrinsed2XinMilliQwaterfor2minuteseach.Sodiumthiosulfatesolution(1%)isthenaddedandincubatedfor3minutesat60°C.Thesectionsarethenrinsed3XwithMilliQwaterfor2minuteseach.Cresylvioletsolutionisthenaddedandincubatedfor3minutesatroomtemperature.Thesectionsarethenrinsed3XwithMilliQwaterfor2minuteseach.Post-stainingsteps:Finally,dehydratetheslidesusingaseriesofgradatedalcohols.Thecresylstainwillleachfromthetissueatthistimeandtheextentofstainingcanbecontrolledbyhowlongtheseincubationslast.Thedehydratedsectionsarethenclearedinhistoclearorxylenefor2minutesandthencoverslippedwithmountingmedia.FrequentlyAskedQuestions(FAQs):1)ItappearsthatMyelinimpregnationisincompletewithBlack-GoldII?Answer:Returnslidesto60°CBlack-GoldIIsolutionandmonitorevery2-3minutesundermicroscopeuntilfineparallelfibersoflayer1cortexarevisIBLe.2)Backgroundhaslavendercolor?Answer:Sectionhasbeenoverstainedandstainingtimeshouldbereduced.3)Backgroundstainingoccursbeforeimpregnationofallfinemyelinatedfibers?Answer:Tissuewasprobablyover-fixed.Trytoavoidleavingbraininfixative,orsectionsinbuffer,forlongerthanonemonth.4)WhataretheadvantagesoverothermyelinstainslikeLuxolfastblue,orSudanBlack?Answer:Betterresolutionandcontrast.Faster.5)DoothercounterstainsworkwithBlack-GoldII?Answer:Yes,mostconventionalNisslstains(includingblue,red,andgreen)canbeusedasacounterstain.Additionally,thecounterstaincanbeomittedifonlymyelinstainingisdesired.BIOLOGicalInformationPhysicochemicalInformationDimensionsMaterialsInformationMaterialsInformation