RUVKUNAntibodyStainingProtocolrevised10/29/90

2.Fixin5mlofFRESHTHATDAY1%paraformaldehyde:

0.05gparaformaldehyde80mMKCl20mMNaCl2mMEGTA0.5mMspermidineHCl0.2mMspermine0.5%BME15mMPIPESpH7.4

towhichadd3mlheptaneorH2O(heptanenotnecessary)and1ml90%methanol;0.25MEGTApH8.0

3.Wormsputonicefor10minutesinordertostoppumpingpriortofixation.Wormsplusaboveemulsionshakenforcefullyfor10minutesatroomtemp..Frozenindryice/ethanol.Immediatelydefrostedunderwarmtapwaterorstoredat-70Cforlateruse.

4.Afterwarming,emulsionshakenforanother60minutesatRT.Specimenspelletedinclinicalcentrifuge,don\"tspinhard.

5.Washwormsonce(inmicrofugetube)withTrisTritonbuffer:

100mMTrisClpH7.41%TritonX-1001mMEDTA

REDUCINGDISULFIDESTO-SH:6.IncubateinTrisTritonbuffer+1%BME,37C,morethan2hourswithagitation.(SomethinkBMEstepisn\"tnecessary.)Afterthispointthewormsarefragileandshouldn\"tbespunhard:approximately1000rpm.Washwormsoncein1XBO3buffer.100XBO3buffer(pH9.2):

1MH3BO30.5MNaOH

7.IncubateinBO3buffer+10mMDTT,15minutes,37Cwithagitation.Washoncein1XBO3buffer.

OXIDIZE-SHGROUPSTO-SO3:8.IncubateinBO3buffer+1%H2O2,1hour,RT.AgitategentlybutkeeptubesuprightbecausecapcanpopopenfromO2pressure.WashoncewithBO3bufferandoncefor15min.ormorewithAntibodyBufferB:

0.1%BSA1XPBS0.5%TritonX-1000.05%NaAzide1mMEDTA

9.StorewormsinAntibodyBufferA:

1%BSA1XPBS0.5%TritonX-1000.05%NaAzide1mMEDTA

10.Incubateasmallaliquotofwormsin10ug/mlRNAseA,1hour,37C.StainRNAsedandunRNAsedaliquots(smallamts!,don\"tuseupsample)with1%toluidineblue.RinseseveraltimesinbufferB.Comparetwosamplesat50Xunderdissectingscope.Ifanimalsare\"open\", + RNAse animals will have very little staining of internal tissues.

ANTIBODYINCUBATIONS:

11.CanpreincubateinBufferA(1%BSA)atRTforafewhours.

12.Spindownwormsgentlyandincubatewith1inBufferA(30ulaffinitypurifiedanti-lin-14antibody-preadsorbedwith292vectorprotein-straightordiluted1:1or1:2)for24hoursatRT.

13.Washfor4-24hoursatRTinBufferB(usuallydo5quickwashesandthen2-3hourslaterdo1-2more).

14.Incubatewith2inBufferA,4-24hours,RT(1:200FITC-coupledgoatanti-rabbitantibodyforlin-14).(2shouldbepreincubatedatRTfor4-24hourswithfixedC.eleganstoremovecross-reactingantibodies.)

15.Washfor4-24hoursatRTinBufferB(usuallyalotofwashesforfinalstep(about8-10X).StoreinBufferB.

PERMANENTSPRINGTIMEMOUNTING:

16.MakeapadonaslideofPermanentSpringtimeAgarose:

50mMTrisClpH9.55mMMgCl2%agarose

17.Puta3-5uldroponthepadofNPGsolution(keeps1-2monthsat4C):

-dissolve2mgn-propylgallatein70ulglycerol(65C)-add30ul100mMTrisClpH9.5-add0.2ul1mg/mlDAPIorHoechststain(weargloves,CARCINOGENS)AddanequalvolumeofstainedwormsandmixwithPipettetip.

18.Coverwithacoverslipandexamineusingepiflourescence.AfteranhourortwotheDAPIstainfadestoyellowand\"bleeds through\" to FITC channel, so don\"t mount too many samples at once. Store stained worms in aluminum foil covered Eppendorfsat4C.Ifneedtokeepslides,dothesame.

PHOTOGRAPHY:

19.KodakTriX400blackandwhitefilmorCOLOR:KodakTMY5053orFuji400colorfilmbothpushed2f-stopsindevelopment.

NOTES:

-usenailpolishtosealslidesifneedtokeep-slidescanbekeptabout2daysat4Cwithoutsealant-n-propylgallateistokeepfADIngdownandisbestforFITCconjugated2s(Ithink).Anotherantifaderisp-phenylnendiaminewhichisacarcinogen(seeANTIBODYMANUAL).-don\"tuseglasstubesorpipettessincewormssticktothem