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.
