I.Solutions&Supplies

BRB80(1X):80mMPIPES,1mMMgCl₂,1mMEGTA,pH6.8withKOH(generallymadeasa5Xstockandstoredat4¡C)

Fix:1%glutaraldehydeinBRB80(preparefroma50%glutaraldehydestockjustpriortouse)

Cushion:BRB80+10%(v/v)glycerol

Spindowntubeswith12mmcoverslips(Evansetal.J.CellBiol.,1985)

HB-4/HB-6orequivalentrotor

Acommonproblemwithspindownsisguessingtherightvolumetosedimentontoa12mmdiametercoverslip.Thiswillrequiresomeamountofpractice/troubleshooting.Withstablemicrotubules(taxol-orGMPCPP-stABIlized),usesquashestoguesswhatvolumetospindown.Mostofthetime,peopleerronthesideofexcess--e.g.,itonlytakes~0.2µlof1µMCPPmicrotubulestogetagooddensityofsinglemicrotubulesona12mmdiametercirclecoverslip.

II.SpindownProtocol1.Setupspindowntubeswithchocks(plexiglassinserts)andcoverslipsandadd5mlBRB80.ThenuseaverycutoffbluetiptounderlaytheBRB80with~2mlofCushion(BRB80+10%glycerol).Makesurethetubesarebalancedandstoreinanicebucket.

2.Fixthereactioncontainingmicrotubulesbyadding10volumesofFixandmixinggentlywithacutofftip.IncubateatRTfor3".

3.Dilutefixedreactionwith20-30volumesofBRB80,mixbyinversionandstoreoniceuntilallsampleshavebeenprocessed.

4.PipettheappropriateamountoffixedanddilutedreactionontopoftheBRB80inthespindowntubes.Spinat12,500rpminanHB-4/6rotorat20¡Cfor1-1.5hours(startwithrotorandcentrifugeat4¡C;thecentrifugewillheatupto20¡Cveryrapidlyandthiswillsavesomecentrifugewear&tear)

5.Aspirateandpostfixin-20¡Cmethanolfor5".Rehydrateandeithermount(ifmicrotubulesarefluorescent)orperformindirectimmunofluorescence.

Thesameprocedurecanbeusedwithmicrotubulesnucleatedoffaxonemesorcentrosomes.Forpelletingtheselargerstructures,usea5ml30%(v/v)glycerolcushionandspinat10Kfor15"-20"at20¡C.Washthesamplecushioninterfacewellpriortoaspiratingandprocessingthecoverslips.