GUS-foolproof 染色

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1、E:file-serverfs_tmpdownload80068e7646d61e7ed906d3a1a020e9b8.doc15March2007FrankslabGUSstaining-15March2007GUSProtocol(fool-proofversion)KirstenBomblies,adaptedfromFrançoisParcy'sprotocolWithalterationsspecificforfrankslab13March2007Harvesttissueandplaceincold90%Acetoneonice.Thisshould

2、stayoniceuntilallsamplesareharvested.Forsamplecontainers,eppendorftubesandglassscintillationvialsworkwell.Microtiterplatesareusefulforlargenumbersofsamples,butthesehavetobepolystyrene,NOTpolypropylene.Pullvacuumfor10minutesatroomtemp,thenfixatroomtemperaturefor20-30minutes.Inthemeantimema

3、keupstainingbufferwithoutX-Gluc(seebelowforrecipe)onicewithcoldsolutions.Removeacetonefromthesamples,andaddstainingbufferwithoutX-Gluconice.Pullavacuumfor10minutesandthenreleasePreparestainingbufferwithX-glucinanewtubetoafinalconcentrationof2mM-froma100mMstocksolutionofX-GlucinDMF-thismus

4、tbekeptinthedarkat-20°C.RemovestainingbufferwithoutX-gluc(washbuffer)fromsamplesandaddstainingbufferwithX-Gluconice.Infiltratethesamplesundervacuum,onice,for15to20minutes.Releasethevacuumslowlyandverifythatallthesamplessink.Iftheydon't,infiltrateagainuntiltheyallsinktothebottomwhenthevacu

5、umisreleased.Mayrequiretwoorthreevacuum/releasecyclesIncubateat37°C(Iusuallydoitovernight,butitdependsontransgenestrength.Itisnotadvisablefrommyexperiencetogotolong(overtwodays)asthetissueseemstobegindeterioratingduringlongincubations.Removesamplesfromincubatorandremovestainingbuffer.Goth

6、roughanEthanolseriesinwhichsamplesareincubatedsuccessivelyin20%,35%and50%ethanolatroomtemperaturefor30minuteseach.IncubateinFAA(recipebelow)for30minutesatroomtemperaturetofixthetissue.RemoveFAAandadd70%ethanol.Atthispointthetissuecanbestoredat4°Cforlongperiods,orexaminedunderthemicroscope

7、.Twoadditionalchoicesatthispoint:Ifthetissueistobesectioned,leavein70%ethanolfor30minutesandcontinuebelow.ifyouwanttolookatwholemountedclearedtissuesthenincubate30minat80%incubate30minat90%ETOH6E:file-serverfs_tmpdownload80068e7646d61e7ed906d3a1a020e9b8.doc15Mar