Silver Enhancement of Colloidal Gold相关交流-蚂蚁淘在线

Generalremarks:Labeledsectionshavetobefixedtoavoidlossoflabelduringtheenhancementprocedureandthoroughlywashedwithbi-distilledwatertoremoveions(especiallychlorideions)whichcouldinterferewiththesilver-enhancementprocess.Enhancementiscarriedoutbyincubatingthegridsonadropof~100µloftheenhancersolution.Althoughmostoftheenhancerscanevenbeusedindaylight,werecommendcoveringthegridsorworkingunderredsafelightconditions,asthisdelaysself-nucleationofsilverions.Thereactionisstoppedbytransferingthegridstobi-distilledwater.Beforestainingwithuranylacetateandleadcitrate,componentslikegumarABIchavetobecompletelyremovedbythoroughlywashingthegrids.Ithastobenotedthathighertemperaturesandmovingofthegridsduringincubationontheenhancersolutionspeedupthedepositionofsilveronthegoldsurface.

Procedure:

Silverlactate,acidic(Danscher,1981): 0.6mlgumarabic(33%inbidistilledwater)plus0.1mlcitratebuffer(2.55gcitricacidplus2.35gtrisodiumcitratedihydrate,addbi-distilledwatertomake10ml,pH3.8)plus0.15mlhydroquinone(0.85gin15mlbidistilledwater)plus0.15mlsilverlactate(0.11gin15mlbi-distilledwater).Gumarabic,citratebuffer,andhydroquinonecanbepremixedandstoredinafreezer.Silverlactatehastobestoredseparatelyinafreezer.Incubationtimefor1-nmgoldisabout20min,forNanogoldabout25min(20-22°C).
Silverlactate(Lahetal.,1990): Asabove,replacecitratebufferby0.2MHEPESbuffer,pH6.8.Incubationtime~3min.for1-nmgold,forNanogold4to5min(20-22°C).ThefinalpHis4(seeBurry1995,p.220).Removeblockingbuffer,add25µloftheprimaryantibodysolutionpercoverslip(withafinalconcentrationintherangeof1-5µgspecificIgG/ml)andincubatefor30-60min.
Silveracetate,acidic(Hackeretal.,1988): Mixequalamountsof0.5%hydroquinonein0.5Mcitratebuffer(seeabove),and0.2%silveracetate.Finalconcentrationof16.5%gumarabicwaspreparedfromastocksolutionof33%gumarabic.Incubationtime~90minfor1-nmgold(20-22°C)(butonly~20minwithoutgumarabic).
HQSILVERª.(Nanoprobes): Equalamountsofthethreecomponentsinitiator,moderator,andactivatorweremixedbeforeuse(seeinstructionsofNanoprobes).Incubationtime~3minfor1-nmgoldand~5.5minforNanogold(20to22°C).HQSILVERcontainsaprotectivecolloid.

MostofthecommerciallyavailableenhancerslikeIntenSEM(Amersham),R-Gent(Aurion)andSEKL15(BritishBioCell)appeartobelessefficient.Theycanbeimprovedbyaddingtheprotectivecolloidgumarabic(fordetailsseeStierhofetal1991,1992,1995).

References:

DanscherG(1981)Histochemistry71,1-16..
LahJJ,HayesDM,andBurryRW(1990)JHistochemCytochem38,503-508.
BurryRW(1995)In:Immunogold-SilverStaining.Principles,Methods,andApplications.Ed.M.A.Hayat,CRCPress,BocaRaton,pp.217-230.
HackerGW,GrimeliusL,DanscherG,BernatzkyG,MussW,AdamH,andThurnerJ(1988)JHistotechnol11,213-221.
StierhofY-D,HumbelBM,andSchwarzH(1991)JElectronMicroscTech17,336-343.
StierhofY-D,HumbelBM,HermannR,OttenMT,andSchwarzH(1992)ScanningMicroscopy6,1009-1022.
StierhofY-D,HermannR,HumbelBM,andSchwarzH(1995)In:Immunogold-SilverStaining.Principles,Methods,andApplications.Ed.M.A.Hayat,CRCPress,BocaRaton,pp.97-118.

Reviews

seerelatedarticlesin:Immunogold-SilverStaining.Principles,Methods,andApplications.Ed.MAHayat,CRCPress,BocaRaton(1995).andin:ColloidalGold:Principles,Methods,andApplications.Vol1.Ed.MAHayat,AcademicPress,SanDiego(1989).

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Silver&nbsp;Enhancement&nbsp;of&nbsp;Colloidal&nbsp;Gold

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Silver Enhancement of Colloidal Gold