MakingBoiledDonkey(orwhatever)Serum(BDS)forBlockingYouwantafinalworkingconcentrationof5%Serum.Ordertheserumfromthesamecompanyandorganismfromwhichyoubuysecondaryantibodies,JacksonImmunolabsfortheSalmonLab.Thereasonweusethisforallourincubationsisbecauseofoursecondaryantibodies.Weusedonkeysecondaryantibodiesandthusanysourceofnon-specificstainingorbackgroundwillbebestblockedwithdonkeyserum.Thatwayanythingyourantibodieswillbindinserumwillbindbeforeyouputonthesecondary.1)BoilnormaldonkeyserumintheportionofddH2Orequiredtomakea5%serumsolutioninPHEM(whichyou"lladdfromyour2xstockafterboiling)for10minutes.Note:usecautionwhenboilingasthistendstoeasilyboilover.Watchthesolutionboil.2)Allowtocool.3)Addappropriateamountof2xPHEMandsodiumazideto0.05%.4)Spinat18KinSorvallfor1hour.5)Filterthrough0.22umfilterandstoreinthefridge.

MakingHeat-InactivatedSerumforBlockingYouwantafinalworkingconcentrationof20%Serum.Ordertheserumfromthesamecompanyandorganismfromwhichyoubuysecondaryantibodies,JacksonImmunolabsfortheSalmonLab.Thereasonweusethisforallourincubationsisbecauseofoursecondaryantibodies.Weusedonkeysecondaryantibodiesandthusanysourceofnon-specificstainingorbackgroundwillbebestblockedwithdonkeyserum.Thatwayanythingyourantibodieswillbindinserumwillbindbeforeyouputonthesecondary.1)Heatnormaldonkeyserumto60-70oCintheportionofddH2Orequiredtomakea20%serumsolutioninPHEM(whichyou"lladdfromyour2xstockafterheating)for1hour.2)Allowtocool.3)Addappropriateamountof2xPHEMandsodiumazideto0.05%.4)Filterthrough0.22umfilterandstoreinthefridge.5)Youwillhavetodilutethisforblocking,doatestdilutionwithyourfavoriteantibody,likelythiswillbestworkata5-10%serumsolution.Thelastbatch,forexample,workedat6%,butthenextbatchworkedat3%.

Fresh4%FormaldehydeDOALLSTEPSINTHEHOODIn10mlsddH₂O,add0.8gparaformaldehydeBringto80oCin50mlconicaltubeinabeakerofdistilledwaterorusethesameflaskeachtime*NOTE:ifyou"reusingconicaltubes,itiscrucialtorememberthethingisboilingoryou"llmakeahugemessandbeveryembarrassed!Whensolutionbeginstoclear,removefromheatAdddropsof1NNaOHuntilcompletelyclearAdd10mls2xPHEMFilterthrough0.2umfiltertoeliminateanypolymerformedduringheating.Allowtocoolbeforeaddingglutaraldehyde(ifapplicable)Allowtocoolbeforefixingcells

MountingMedia20mMTrispH8.00.5%N-propylgallate90%GlycerolStoreat4oC

PHEM(500mls)2x18.14gPipes6.5gHepes3.8gEGTA0.99gMgSO₄pH7.0w/KOH

PBS(5xin500mls)20.45gNaCl0.465gKCl10.142gNa₂HPO₄*7H₂O0.545gKH₂PO₄pH7.2

F-Actin/MTFixationProtocol(5/19/98)updated4/2/01Note:Mycellshaven"tbeenstickingsoInowusea20secondquickfixtojustaffixtheoutsideofthecellstothecoverslippriortopermeablizing.1)Permeablizecellsin0.5%TritonX-100(freshlymade,sonicated,andpreheatedto37oC)inPHEMfor5minutesalone2)Slowlydribbleinabout6dropsoffix(freshlymade4%Formaldehyde/0.5%GlutaraldehydeinPHEM)intoPermeablizationbuffer.Letsitforanother2minutes.3)FixinFreshPHEMFixfor20minutes.4)Rinse3timesfor5minuteseachinPHEM.5)QuenchwithSodiumBorohydride(asmallpinch)inPHEMfor5minutes.Beverycarefulnottoletbigbubblesform,whichwilldamagefineactinstructures,byliftingoutevery10-20secondsorso.Thetimewillvary,sokeepacloseeyeonit.6)Quench2timesinSodiumBorohydride(asmallpinch)inPHEM(noTritonyet)for4minuteseachfollowingthesameextremecautionasabove.7)Rinse3timesfor5minuteseachinPHEM-T(PHEM+0.1%TritonX-100madefreshandheatedto37oC(sometimesIsonicateandsometimesIforget,butit"sprobablybettertobesafeandsonicatetobesurethedetergentisevenlydistributedinthebuffer).8)ALLBLOCKSANDINCUBATIONSSHOULDBEDONEWITHTHECOVERSLIPSFACINGUPONParafilmSOTHEFINESTRUCTURESDONOTGETDAMAGED.9)Blockat37oCinBoiledDonkeySerumforatleast45minutes,ideally60minutes.

Note:youcanleaveineitherblockorinprimaryAntibodyovernightat4oCanditstilllooksbeautiful.

10)PutonprimaryDM1A(mouse)at1:300inblock(thisconcentrationworksgreatformyrepeatedfrozen/thawedaliquot,butmaybeifit"safreshaliquotuseitat1:400)for30minutesat37oC.11)Rinse3timesfor5minuteseachrinseinPHEM-T.12)Putonsecondary(IuseDonkeyanti-mouse-RhodamineRedX)at1:100inblockfor30minutesat37oC.13)Rinse3timesfor5minuteseachrinseinPHEM-T.14)ForAlexa488-Phallodin,Iuse3uls/coverslipandIuseabout150ulsofblock/coversliptoapplyit.Thistimingissortofcrucial,onlyleaveitinfor20minutesatroomtemperature.15)Rinse1-2timesinPHEM-T16)IdoDAPIhere...about1ul/10mLsofPHEM-Tfor60-120seconds17)Rinse3timesinPHEM-Tfor5minuteseach.18)Rinse1timeinPHEMfor5minutestogetthebubblesoff,etc(Idon"treallyknowwhyIdothisstep,butitfeelsrighttoremovethedetergentformounting).19)Mountin6-7uls(Ihaveahardtimeavoidingbubblessoithelpstousethislargervolumeforme)of90%Glycerol+N-PropylGallate.