1.Dechorionate26hrembryos(pharyngulastage)carefullywithtwofineforceps.

Transfertofixative(1%formaldehydeinPBS).Fixfor1hourrockingat4oC.

2.Washwith5ml0.1%BSAinPBSfor10minutes.Wash3Xwith5mlPBS,

10minuteseach.

3.Incubateovernightat4oC(coldroom)in0.5mlprimaryantibodyin0.2%saponininPBS.

Primaryantibodies:(A)znp-1@1/2000(primarymotoneurons)

(B)F6@1/500(somiteboundaries)

(C)noantibody(Control)

4.Washforaminimumof2hourswithseveralchangesof0.2%saponininPBS(canstoreincoldroomatthispoint).

5.Incubateovernightwith0.5mlsecondaryantibodyconjugatedtoperoxidase(HRP)(Goatanti-mouseIgG+IgM-HRP)@1/500at4oC.

6.Washforaminimumof2hourswithseveralchangesof0.2%saponininPBS(canstoreincoldroomatthispoint).

7.Washwith1mlHRPsubstratebuffer.

8.DevelopwithHRPsubstrate(metal-enhancedDAB).Monitorunderdissectingmicroscopeafter5minutes.WhenbrowncolorisclearlyvisIBLe,stopreactionbywashingwithPBS.

9.Clearembryobysoakingin1:1glycerol:CMFET,followedby1:4glycerol:CMFET.

Observewithdissectingmicroscope,andmountwithdepressionslidestoobservewithcompoundmicroscope.

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