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.