Negativestainingisarapid,qualitativemethodforanalyzingmicrotubulestructureattheEMlevel.Becausenegativestaininginvolvesdepositionofheavyatomstains,structuralartifactssuchasflatteningofthecylindricalmicrotubuleandopeningupofmicrotubulesintoflatsheetsarecommon.Cryo-electronmicroscopy,wheremicrotubulesareflashfrozeninathinfilmofvitreousiceandimagedwithoutstaining,iscurrentlyregardedasthebestmethodtoviewnativemicrotubulestructureathighresolution.Nevertheless,negativestainingisveryusefulbecauseofitsease,rapidityandlackofrequirementforspecializedequipmentotherthanthatfoundinaregularEMfacility.

• I.Solutions&Supplies
• II.NegativeStainingProtocol

Backtoprotocols

I.SolutionsandSupplies0.5%(w/v)UranylAcetate(preparebydissolving50mgUAin10mlofddH₂O.AddwatertotubecontainingUA,covertubewithfoilandrotateincoldroomforseveralhourstillfullydissolved.Filterthrougha0.22µmfilterthathasbeprerinsedwellwithddH₂O.Filteredstainstoredat4¡Cinafoil-wrappedtubecanbeusedfor>1year.)

Filterstrips(preparedbycuttingWhatman#1filterpaperintosmallslivers)

Grids(200meshcoppergridsthathavebeenformvarcoated,carboncoated)

Rinse(ddH₂Owith5mMEGTAorasappropriate)

II.NegativeStainingProtocol1.Glowdischargeformvarandcarboncoatedgridsjustbeforeusetoincreasetheirhydrophylicity.

2.Placesampleonthegrid(1-3µl,sufficienttocoverthegridsurface).

3.~10seclaterslowlypipeton20µlofUAstainusingaP-20.Whilepipetingontothegrid,gentlyabsorbstainontheoppositesideusingafilterpapersliver.Thestainingprocedureshouldtake~30s-1".

4.AllowthegridtodryafterabsorbingasmuchstainaspossIBLewiththefilterpaperandexaminethegridassoonaspossible,preferablyonthesameday.Ifthereareproblemswithstainprecipitation,orwithgeneralstainbackgroundthenrinsespriortostainingmaybenecessary(seebelow).

WehavefoundthisstraightforwardnegativestainingproceduretoworkverywellwithstablemicrotubulessuchastaxolorGMPCPP-stABIlizedmicrotubules.WedonotrecommendusingUAdissolvedin50%methanol,sincewehadirreproducibleresultswiththisstainformulation.Ifthereisalotofsaltorsucrose/glycerolinthebuffer,thenwashingthesamplepriortostainingmaybenecessary.Ifdynamicmicrotubulesundergoingpolymerizationat37¡Cneedtobeexamined,thenarinsewithwarmBRB80maybenecessarytoremovethelargeamountofunpolymerizedtubulinbeforeapplyingstain.

Rinsing:Thereareseveralmethodsforrinsingthegridsurfacepriortoapplyingstain.Onemethodistoapplysampletothegrid,allowadsorptionfor~10sec,holdthegridtilteddownwardanddrop2-3largedropsofrinse(eitherddH₂O+5mMEGTAforremovinginterferingsalts/buffercomponents/sucrose/glycerolorwarmBRB80forremovingunpolymerizedtubulin)overitandthenapplythestain.Alternatively,rinsingcanbedonebyplacingalargedropoftherinsesolutiononParafilmandslowlydrawingthegrid,withthesample-coatedsurfacefacingtheparafilm,overthesurfaceoftherinsesolutiondrop.Thestaincanthenbeappliedasabove.

Dilutesamplescanbeconcentratedonthegridbyadsorptionforlongertimes(1"-3").Negativestainingisrelativelyquick,sotrybothdirectstainingandrinsingpriortostainingforthespecificreactionconditionsbeinganalyzed.