DyeFillingtoStainAmphidandPhasmidNeurons byMichaelKoelle,fromBethSawin 4/6/94 1.BuyDiOfromMolecularprobes,catalog#D-275.DiOfluorescescesgreen(useFITCfilters).Ifyouwantredfluorescence,DiIcanbesubstituted;stainexactlythesamewaybutusetheTexasredfiltersonthefluorescencescope.DiImaystainaslightlydifferentsetofcells. 2.Stocksolutionis2mg/mlindimethlyformamide,storedat-20deg.inafoilwrappedtube. 3.Dilutethestock1:200inM9.Somedyewillprecipitatewhenyoudothis;don"tworryaboutit. 4.Put150ulstaininamicrotiterwell,anduseawormpicktoputtransfersomewormsintothedye.Incubate2-3hoursatroomtemperature. 5.UseamouthPipettetotransferthewormstoafreshplate,andletthemcrawlonabacteriallawnforabout1hourtodestain.(Alternatively,washtheworms3XinM9). 6.Putwormsonagarpadswithsodiumazideandvisualizebyfluorescenceusingtheappropriatefilters. Thefollowingcellsstain(oneofeachbilaterallysymmetricpairisdiagrammedhere,andonlythecellbodiesareshown,butprocessesalsostain):