1.Dissectembryosinice-coldPBS.RemoveasmuchextraembryonicmembranesaspossIBLe.Puncturetheamnionandinolderembryos(>E10)puncturetheheadwithasyringeortungstenneedle.

2.Fixin4%paraformaldehyde(PFA)inPBSat4^oCovernight.

3.Washtwicefor5-10minutesinPBS.

4.Washwith50%MeOH/PBS,thentwicein100%MeOHfor5-15mineach.

Embryoscanbestoredat—20^oCatthispoint,foruptoafewmonths.

5.IncubateembryosinfreshlypreparedMeOH:DMSO:H₂O₂(4:1:1)atroomtemperaturefor5-10hours.

Atthispointembryoscanbetakeninto100%MeOHandstoredat—2⁰Catthispoint.

6.Rehydratetheembryosinaseriesof50%MeOH(20min)->PBS(2x15min)->freshlypreparedice-coldPBSMT(2x15min,1x1-2hour).

7.IncubatewithprimaryantibodyinPBMSTat4^oCovernight.

Theantibodyincubationcanbeextendedforseveralovernightswithoutcompromisingtheexperiment.

8.Washwithfreshlypreparedice-coldPBMST(2x15min,5x1hour).

KeepPBMSTonice,orinfridge,butperformthewashesatroomtemperature.

9.Incubatewithsecondaryantibody(FITCcoupledforgreen,orCy5,Cy3coupledforred)inPBSMTat4^oCovernight.

Theantibodyincubationcanbeextendedforseveralovernightswithoutcompromisingtheexperiment.

10.Washasinstep8.

11.RinseinPBT(2x10min).

12.Viewandphotographunderepifluorescenceoptics,forexamplefromBLS,Leica,orZeiss.