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LacZcell Staining
LacZ-cellStaining Materials: 1)0.5%glutaraldehydeinPBS2)20mMK4Fe(CN)6o3H2O0.44ginto50mlPBS3)20mMK3Fe(CN)60.33ginto50mlPBS4)X-gal2%inDMF5)PBScontaining1mMMgCl26)Stainingsolution Procedures: 1)Washthecellstwicewith2mlPBS(I"massumingyou"reworkingwitha35mmdish,otherwise,adjustyourvolumeaccordingly).2)Fixthecellsfor10minutesatroomtemperaturein2mlof0.5%glutaraldehydedilutedinPBS.3)Washthecells3timeswith2mlPBScontaining1mMMgCl2.4)Coverthecellswithstainingsolutionandincubateat37¡Cuntilthedesiredstainingisachieved.5)WashcellswithPBS. β-galactosidaseEmbryoStainingProtocol 1.Preparefixasfollows:weighout0.5gofsolidparaformaldehydeintoa50mlconicaltube.Tothistubeadd: 5ml0.5MPipesbuffer(pH6.9)50µl1MMgC12125µl0.2MEGTA(pH8.0) Heatinmicrowavefor5secondintervalsuntilPFAdissolvescompletely.Beverycarefulnottooverheatthetube(ITWILLEXPLODE)andalwaysreleasepressurebyunscrewingthetubeinbetweenheatings. 2.Embryosarefirstdissectedawayfromtheplacentalmaterialandtheamnionisremoved.Whendissectingmanyembryosatonce,theinitialembryoscanbestoredin1XPBSwhileotherembryosaredissected.Priortofixation,theembryosareverydelicatesoexercisecareinhandling.AcutoffP1000pipettipworkswellformovingembryosaround. 3.Oncealltheembryosaredissected,washthem1or2timesin1XPBS.ThenfixtheembryosbyplacingtheminthePFAsolutionfor15minutes.Althoughfixationtendstohardentheembryos,oneshouldstillexercisecarewhenmanipulatingtheembryos. 4.Afterfixingtheembryos,washthem3times@5minutesperwashin1XPBS/1mMMgCl2. 5.Preparestainingsolutionasfollows,mix: 250µl20mMK3Fe(CN)6/PBS250µl20mMK4FE(CN)6o3H20/PBS1µl1MMgCl21µl10%sodiumdesoxycholate2.2µl9%NP4050µl2%Xgal andbringuptoafinalvolumeof1.05mlswith1XPBS.Onecanalsoadddetergentstothestainingsolutionifdesired,addThismayhelptoincreasethepermeABIlityoftheembryoduringstaining. 6.Placeembryosinthestainingsolutionandincubateat30-37ûCovernight. 7.Afterdesireddegreeofstainingisachieved,washembryosin1XPBS.Embryosmaythenbepost-fixedorstoreddirectlyin1XPBS.10XPBS(pH7.2)1L_NaC180.06g1.37MKC12.01g27mMNa2HPO411.35g80mMKH2PO43.53g26mM20mMK4Fe(CN)6o3H20Mix0.44221gsolid5ml10XPBSBringvolumeupto50mlwithwater.20mMK3Fe(CN)6Mix0.32926gsolid5ml10XPBSBringvolumeupto50mlwithwaterX-galismade2mg/mlor2%inDMF.
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