FixationandpermeABIlizationprotocol5/30/91MikeFinney

1)Fixation:

-Washanimalsfromanunstarvedplatewithbuffer.Washthemfreeofbacteriawithwater,leavingtheminwaterinamicrofugetubeonice.

-Addcold2XMRWBtoafinalconcentrationof1Xand20%formaldehydetoafinalconcentrationof1-4%(totalvolumeusually1ml).Mixwell,freezeindryice/ethanol,meltonice,andincubateonicewithoccasionalagitationfor0.5hrtoovernight.Frozensamplesmaybeprocessedimmediatelyorstoredindefinitely.

Notes:Methanolprecipitatesproteins,reducingdiffusionbeforecrosslinking.Spermidineandformaldehydetogethercrosslinkproteins.Formaldehydeconcentrationandfixationtimeareimportantandshouldbeoptimized;agoodplacetostartis1%for0.5hr;morefixationwillstabilizesomeantigensbutdestroyothers(likeunc-86).Freezingcrackseggshells,lettingthefixativesin.

2)Reduction:

-WashwormstwicewithTrisTritonbuffer,thenincubateinTrisTritonbuffer+1%BME,1-2hoursat37degreeswithmildagitation.Afterthispointthewormsarefragileandshouldn"tbespunhard(5000rpminamicrofugeisOK).Note:SteveSalserreportsthatspinningthewormsisessentialfortheprotocoltowork;lettingthemsettleoutapparentlydoesnotprovideenoughstresstoopenthecuticle.

-Washwormsoncein10-15vols.1xBO3buffer,thenincubatein1xBO3buffer+10mMDTT,15minwithagitation.

Notes:BMEandDTTreducethedisulfidelinkagesthathelpholdthecuticletogether;Tritonkeepsthewormsfromstickingtoeachother.Thedisulfidereductioniscompleteinminutes;theextendedincubationat37degreesistokillwormenzymeslikeDNases,proteasesandperoxidases(whichwreakhavokintheoxidationstep).

3)Oxidation:

-Washwormsoncein10-15vols.1xBO3buffer,thenincubatein1xBO3buffer+0.3%H2O2,15minatroomtemperature.AgitategentlybutkeeptubesuprightbecausethecapmaypopopenfromtheO2pressure.

-Washoncewith1xBO3bufferandonceforatleast15minwithantibodybufferB.Storewormsat4degreesinantibodybufferA.Notes:H2O2oxidizesthe-SHgroupsto-SO3.Donotover-wash,sodisulfidesdonotreform.BO3bufferprovidesthebasicpHneededforthereaction.Cysteinesandmethioninesinotherproteinswillbeoxidizedaswell,possIBLyaffectingsomeepitopes.MetbutnotCyscanberestoredbyasecondDTTtreatment.

4)Staining:

DoantibodyincubationsinbufferAandwashesinbufferB.

Buffers:

2XModifiedRuvkun"switchesbrew(MRWB)160mMKCl40mMNaCl20mMNa2EGTA10mMSpermidineHCl30mMNaPIPESpH7.450%methanol

TrisTritonbuffer100mMTrisClpH7.41%TritonX-100orNP-401mMEDTA

40xBO3buffer(pH~9.2at25mM)1MH3BO30.5MNaOH

Antibodybuffers:A1XPBS1%BSA0.5%TritonX-100orNP-400.05%NaAzide1mMEDTA

BSameasAexcept0.1%BSA

20%formaldehydeWeighsomewhatmoredryparaformaldehydethanyouneed(<300mg)andputitinamicrofugetube.Multiplytheweightinmgby4.5andaddthatvolumeinmicrolitersof5mMNaOH.Placein65degreebathfor30minuteswithoccasionalmixing.Spinfor1min.topelletundissolvedparaformaldehyde.Usethesupernatantimmediately.