Nonfluorescent protein/RNA doublelabeling

1.Collectembryos,dechorionateandfixina(1:1)mixtureofheptane:fix.Fixis5%formaldehyde,0.05MEGTAin1XPBS.pH7.0.Devitellinizeembryosbyshakinginanequalmixtureofmethanol:heptane(embryoswillsinktothebottom)andwashtheembryoswellwithmethanol.Transfertheembryostoa0.5mleppendorftube.

2.Washtheembryoswith3%hydrogenperoxideinmethanolfor15min.Rinsewellinmethanol.

3.Wash3X30minuteswithPBTH(filtered1XPBS,0.1%Tween-20,50ug/mlheparin,and250ug/mltRNA).Youcanalsoinclude1%BSAprovidingyouknowitisultrapure/RNase-free.Withourreagents,wefindwegetsimilarresultswithoutit.

4.Incubatewiththeprimaryantibody(dilutedappropriatelyinPBTH)overnightat4C.ForextraRNaseprotection,include2-5UnitsofRNasin/250ulofantibodymixture.

5.WashatroomtemperaturewithseveralchangesofPBTHforatleast1-2hr.

6.Incubatewithbiotinylatedsecondaryantibody(dilutedinPBTH)for1.5hoursatroomtemperature(againaddRNaseinhibitor).

7.WashwithseveralchangesofPBTHfor60minutes.Fordetectionofthebiotinylatedsecondaryantibody,wegenerallyusetheVectorLabABCamplificationsystem.Mixthereagentspermanufacturer"sinstructions30min.priortouse.

8.Incubateembryoswiththepre-incubatedABCmixturefor30minutesatroomtemperature.

9.Washfor15-30min.with3-4changesofPBTH.Developthesignalwith20ug/mlDiaminobenzidine,0.03%hydrogenperoxideinPBTH.Thiswillproduceabrownsignal.StopthereactionwithseveralrinsesofPBTH.

InsituHYBRIDIZATION:

1.Fixtheembryosfor20minuteswith5%formaldehydeinPBT(PBT=filtered1XPBSwith0.1%Tween-20)atroomtemperature.

2.Wash3X5minuteswithPBT.

3.Incubatetheembryoswith50ug/mlofProteinaseKinPBTfor2-3minutes(timevarieswithdifferentProt.Kprepsandneedstobeoptimized).

4.StoptheProteinaseKreactionwithtwowashesof2mg/mlglycineinPBT.Eachwashshouldbe2-3minutes.ThenrinsetheembryostwicewithPBT.

5.Fixtheembryosagainfor20minuteswith5%formaldehydeinPBTatroomtemperature.

6.Washtheembryos5timeswithPBT.

7.RinsetheembryoswithPBT:Hybridizationbuffer(1:1),andthenwithhybridizationbufferalone.HybridizationbufferforDNAprobesis:5XSSC,100ug/mlsonicated,boiledsalmonspermDNA,100ug/mltRNA,50ug/mlheparin,and0.1%Tween-20(forRNAprobesandhybridizationconditions,seeforex.ourprotocolonFISHwhole-mountstaining).

8.Pre-hybridizetheembryosinhybridizationbufferforatleastonehourattheappropriatetemperature(48CforDNAprobes).Longerpre-hybridizationsofupto3hoursseemtoimprovethesignaltonoiseratio.

9.AddyourDigoxygenin-labeledprobetothehybridizationbufferandincubateovernight(12to16hours)attheappropriatetemperature.Useabout100ulhybridizationbufferper50ulsettledembryos.

10.Carryoutthefollowingpost-hybridizationwashesatthehybridizationtemperatureforatleast20minuteseach:

(i)hybridizationbufferalone

(ii)PBT:hybridizationbuffer(1:1)

(iii)PBTaloneforatleast5washes

11.Incubatewithanti-Digoxygeninantibody(BoerhingerMannheim)conjugatedtoalkalinephosphatase(diluted1:2000inPBT)for2hoursatroomtemperature.

12.WashwithPBT4timesfor20minuteseachwash.Rinsewithalkalinephosphatasestainingbuffer(100mMNaCl,50mMMgCl2,100mMTrispH9.5,1mMLevamisol,and0.1%Tween20).

13.DevelopthecolourreactionusingNBTandBCIPinthealkalinephosphatasestainingbufferasdescribedbythemanufacturer(BoehringerMannheim).

14.StopthereactionwithseveralwashesofPBTanddehydratewithanethanolseries(inPBT).

15.Rehydratetheembryosinareverseethanolseries(inPBT),andthenrinseseveraltimeswithPBT.

16.Mountin70%glyceroland30%0.1MTrispH8.0.