Doubledyefilling(usingDiOanddi-4ANEPPS)(KristaWilliams)

DyeFilling

1. Label15mlcentrifuge(c/f)tubeswithstrainname.Squirt~1-2mlM9ontoplatewithaPasteurpipet.RinseplatewithM9usingapasteurpipetandtransfersolutiontoc/ftube.
2. Spinpulseonhighinaclinicalc/fandcarefullyremovesupernatant(s/n).Wormpelletwillbeveryloose.
3. WashoncewithM9(optional).
4. Add2mlM9.
5. AddDiO(2mg/mlinDMF)at5l/mlofM9.
6. Adddi-4ANEPPS(1mg/mlinDMF)at8l/mlofM9andcoverthec/ftubeinfoiltoprotectfromlight
7. Rockthetubefor~1hourat20°Conalabquakerocker.
8. Afterrocking,spinpulseonhighandremoves/nwithPasteurpipet.
9. WashoncewithM9
10. Transferwormpellettolabeledplate(s).Useenoughplatesthatwormshavesufficientfood.
11. Allowtheplatestositat20°Cfor~20minutesorsufficienttimetopassthedyeoutofthedigestivetract.NOTE:Ifwormsarelefttoolong,thedi-4ANEPPSwillspreadtoallmembranesinwormandmakeaverystrongredbackgroundinwholebody.

1. Makepadsusing5%agarwith10mMNaN₃(10lof1MNaN₃/mlof5%agar--ourtesttubescontain4mlof5%agar).
2. Apply4-6lofM9tooneendofthepad.
3. Transfer25-50wormstotheM9,addingmoreM9ifitstartstoevaporate.
4. Putcoversliponandviewwithuprightandfluorescence.Phasmidneuronsshouldstaingreen(DiO)andphasmidsocketcellsshouldstainred(di-4ANEPPS).BothstructuresshouldbevisIBLethroughsamefilter.Limitexposureofwormstolight,asdi-4ANEPPSisinactivatedbylight.

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