Day1:PrehybridizationandHybridization

1. Rehydratesamplesina75%,50%,25%methanol/PBTseries.WashtwiceinPBTfor5minutesatroomtemperature.
2. leachembryos(ifnecessary)with6%hydrogenperoxideinPBTfor1houratroomtemperaturewithgentlerocking.(Note:bleachingtimecanbereducedorextendedifdesired.)Embryosmusthavebeenpreviouslydehydratedinmethanolifyouaregoingtoincludethebleachingstep!!
3. Wash3timeswithPBTfor5minuteseach.
4. ProteinaseKtreatment:Chickembryos:Foryoungerembryos(<st10),treatwith1to3g/mlproteinaseKinPBTfor15minutesatroomtemperature.Asimilartreatmentcanbeusedfordetectingectodermalgeneexpression.Forolderembryos,(>st10)aharshertreatmentwithproteinaseKisnecessary.Twoparameterscanbemanipulated:theconcentrationofproteinaseKand/orthelengthoftreatment.Embryosyoungerthanstage18areusuallytreatedwith10mg/mlproteinaseKforabout15minutesatroomtemperature.Forembryosbetweenstages18and24,proteinaseKtreatment(10mg/ml)canbeextendedfor20to25minutesatroomtemperature.Forstages26to29,enzymetreatmentwith10mg/mlproteinaseKcanlastforupto40minutesatroomtemperature.Alternatively,forembryosatstage26andolder,theconcentrationofproteinaseKcanbeincreasedwhilekeepingtheincubationtime(15minutesatroomtemperature)constant:stage:concentrationofproteinaseK26-27:20mg/ml28-29:30mg/ml30-31:40mg/ml32andolder:50mg/mlMouseembryos:incubatein10mg/mlproteinaseKinPBTfor5-10minutes,dependingonembryostage.Usethefollowinginformationasaguideline:embryoage:durationofincubation6.5dpc:4min7.5dpc:4-5min8.5dpc:6min9.5dpc:10min10.5dpc:15min
5. Wash10minutesin2mg/mlglycineinPBT(makefresh).
6. Washtwotimesfor5minuteseachwithPBT.
7. Postfixwith4%paraformaldehydeand0.2%glutaraldehyde(0.2mlof25%stockper25ml)inPBTfor20minutesatroomtemperature.
8. Wash2timesfor5minuteseachinPBT.Formouseembryos,aslowequilibrationinhybridizationsolutionissuggested.Thisapproachhasnotbeenroutinelyusedforchickembryos,butitcouldprobablybeusedsuccessfully.
9. Wash10minutesina1:1mixtureofhybridizationsolution/PBT
10. Wash10minutesinhybridizationsolution.
11. Incubateat70°Cinhybridizationsolutionforatleast1hour.
12. Replacehybridizationsolutionwithfresh,addRNAprobe(typicallyanywherefromone-fiftiethtoone-tenthofatranscriptionreaction)andincubateovernightat70°C.

1. PrewarmsolutionIto70°C.Washembryos3timesfor30minuteseachat70°CwithprewarmedsolutionI.
2. PrewarmsolutionIIIto65°C.Washembryos3timesfor30minuteseachat65°CwithprewarmedsolutionIII.
3. Wash3timeswithfreshTBSTfor5minuteseachatroomtemperature.
4. Blockingstep:
   Forchickembryos,blockwith10%heat-inactivatedsheepseruminTBSTforatleastonehouratroomtemperature.
   Formouseembryos,blockembryosbyincubatingatroomtemperaturefor60-90minutesinblockingsolution(10%heat-inactivatedsheepserumand0.1%BoerhingerMannheimblockingreagentinTBST).
5. Preabsorbtheantibody,ifdesired.(Note:Anumberofpeopledonotpreabsorbtheantibodyforchickembryosandstillgetgoodresults,usingadilutionof1:5000to1:10,000).Forchickembryos,putroughly3mgofchickembryopowderintoamicrotubewith0.5mlTBST.Heatat70°Cfor30minutes,vortexfor10minutes.Coolonice,addmlsheepserumand1mlanti-digAPantibody.Shakegentlyat4°Cfor1hr.Spininmicrofugefor10minutesat4°C.Collectsupernatantintoa15mltube.Dilutewith1%sheepserum/TBSTtoavolumeof2to8mlspervialofembryos,dependingonthedesiredantibodydilution.Formouseembryos,followtheaboveprocedurebutuseroughly3mgofmouseembryopowderand0.5mlTBSTplusBoerhingerblockingreagent.

6. Removeblockingsolutionfromembryos.Addantibodyandincubateovernightat4°C.

Day3:Post-antibodywashes

1. Wash3timesfor5minuteseachwithTBSTatroomtemperature.
2. Wash5timesfor1to1.5hoursinTBSTatroomtemperature.
3. WashovernightinTBSTat4°Cwithgentlerocking.

Day4:Colordevelopment

1. Wash3timesinNTMTforatleast10minuteseachatroomtemperature.
2. RemoveNTMT,addreactionmix(125mg/mlBCIPand250mg/mlNBTinNTMT)andallowthereactionstodevelopatroomtemperaturewithgentlerocking.Throughoutthedevelopmentreaction,keepsamplescoveredwithaluminumfoiltoprotectthemfromlightandcheckthemperiodicallytomonitortheprogressofthereaction.
3. Whenthereactionisjudgedcomplete,washwithPBSorPBTandpost-fixin4%paraformaledehye/0.1%glutaraldehyde.
4. WashtwotothreetimesinPBS.

Solutions

Allsolutionsshouldbemadewithhigh-qualityreagentgradewaterthatisRNase-free.(Note:WeroutinelyusewaterdirectlyfromourpurificationsystemwithoutDEPCtreatment.)Inaddition,allsolutionscontainingTween-20shouldbefilteredandkeptsterile.

10XPBS(1liter):

80gNaCl
2gKCl
14.4gNa₂PO₄
2.4gKH₂PO₄
(adjusttopH7.4withHClandbringto1literwithwater)

PBT:

1XPBSplus0.1%or1%*Tween
*NOTE:Formouseembryos,itisrecommendedthatallsolutionswithTweencontain1%,ratherthan0.1%.
Tweenisroutinelyusedat0.1%inthechickprotocol.

Hybridizationsolution:

50%formamide
5XSSC,pH4.5(usecitricacidtopH)
1%SDS
50mg/mlyeasttRNA
50mg/mlheparin

SolutionI:

50%formamide
5XSSC,pH4.5
1%SDS

SolutionIII:

50%formamide
2XSSC,pH4.5
0.1%Tween(NOTE:thisisonlyincludedfor
youngchickembryos,approx.st4-st9)

Sheepserum:

inactivatebyheatingto70°Cfor30minutes,storeinaliquotsat-20°C.

10XTBS(for1liter):

80gNaCl
2gKCl
250ml1MTris-HCl,pH7.5
(bringto1literwithwater)

TBST:

1XTBSplus0.1/1%Tween
2mM(0.5mg/ml)Levamisole
(NOTE:mostpeoplenolongeraddlevamisoletoTBST)

NTMT:

100mMNaCl
100mMTris-HCl,pH9.5
50mMMgCl₂
0.1/1%Tween-20
2mM(0.5mg/ml)Levamisole

200XNBT:

50mg/mlNBTin70%DMF

200XBCIP:

25mg/mlBCIPinwater