DETECTIONOFß-GALACTOSIDASE

TheE.colilacZgeneencodingß-galactosidase(ß-gal)istheclassicalhistochemicalreportergene(Beckwith,1980).Itcanbedetectedusingavarietyofsubstrates,allofwhichhavegalactoselinkedthroughaß-D-glycosidiclinkagetoamoietywhosepropertieschangeuponliberationfromgalactose(WallenfelsandWeil,1972).Severalsubstratesyieldcoloredorfluorescentsolubleproductswhichareusefulwhenquantifyingß-galactivity(McCamanandRobins,1959,Miller1972)orvisualizingtransducedcellsliveinvivo(Krasnowetal.1991,NirenbergandCepko,1993,Linetal.1994).Thefluorescentproductscanevenbeusedtokillcellsinvivo(NirenbergandCepko,1993).However,forlocalizationofcellscontainingtransducedlacZ,chromogenicsubstratesthatyieldaprecipitatedproductaredesirable(HoltandO"Sullivan,1958,Pearse1954,Pearsonetal1963).Themostcommonsuchsubstrateisanindolederivative,5-bromo-4-chloro-3-indolyl-ß-D-galactoside(X-gal,HoltandSadler,1958).

Whenß-galcleavestheglycosidiclinkageinX-gal,asoluble,colorlessindoxylmonomerisproduced.Subsequently,2oftheliberatedindoxylmoietiesformadimerwhichisnonenzymaticallyoxidized(Figure1).Theresultanthalogenatedindigoisaverystableandinsolublebluecompound(HoltandSadler,1958).Thedimerizationandoxidationreactionsrequiretransferofanelectron,whichisfacilitatedbyelectronacceptorsoftheproperredoxpotential(CotsonandHolt,1958).TheferricandferrousionsincludedinmostX-galreactionbuffersprovidethisfunction(Lojda,1970).Tetrazoliumsalts,whichcanserveasthefinalelectronacceptors,alsocanbeadded,andprecipitatewhenreducedtoformcoloredformazancompounds(Altman,1972).Phenazinemethosulfate(PMS)canfurtherincreasethisreactionratebyquantitativelyreducingtetrazoliumsalts(Altman,1972).Alternativestainingprotocolsthatyielddifferentcoloredproductshavebeendevelopedbasedupontheseconsiderationsandwillbediscussedattheend.

X-GALSTAININGOFINTACTTISSUE(WHOLEMOUNTS)

1. Dissectembryosorsmallpiecesoftissue(e.g.thesizeofaretinafromanadultratworkswellwiththisprocedure)intoPBScontaining2mMMgCl2(PBS+Mg2+)onice.PBS(10X)
   80gNaCl
   2gKCl
   11.5gNa2HPO4
   2gKH2PO4
   in1literH20
2. Fixin0.5%glutaraldehydeinPBS+Mg2+or2.0%-4.0%paraformaldehydeinPBS+Mg2+onicefor30"toseveralhours.Theamountoftimeshouldbedeterminedempirically.Generally,itiswisetominimizetimeinfixativeasthelacZencodedenzymecanbeoverfixed.Fixationwith0.5%glutaraldehydegivessuperiorstainingrelativetoparaformaldehydefixation,butcanalsopreserveendogenousenzymeactivitytothepointwherethesignalfromendogenousactivityisconfounding.0.5%glutaraldehyde
   Makefroma25.0%stockimmediatelybeforeuse.Youcanbuya25%solution(Sigma)andfreeze-thawitmanytimes.4.0%paraformaldyde
   4gparaformaldehyde
   2mMMgCl2(0.2mlofa1Mstock)
   1.25mMEGTA(0.25mlofa.5MEGTAstock,pH8.0)
   in100mlPBS,pH7.2-7.4Heatabout80mlH₂Oto60⁰Candaddparaformaldehyde;add1-2dropsof10MNaOHtogetparaformaldehydeinsolution.Cooltoroomtemperature,add10ml10XPBS,adjustpHwithHCl,addMgCl2andEGTA,andmakeupto100mlwithH2O.Thesolutioncanbestoredat4⁰C1-2weeks.

   RinseinmanychangesofPBS.Residualfixativecaninhibittheenzyme.Rinsingovernightisfine,butwaitingforseveraldaysatthisstepmaydecreaseß-galactivity,andparaformaldehydefixedtissuecanbecome"unfixed".

   DilutetheX-galstockintoX-galReactionBufferandincubatewiththetissue2-4hoursat37⁰C.

   X-galReactionBuffer

   • 35mMpotassiumferrocyanide(canvaryfrom5-35mM)
   • 35mMpotassiumferricyanide(canvaryfrom5-35mM)
   • 2mMMgCl2
   • 0.02%NonidetP-40(NP-40)(dilutedfrom10%stocksolution)
   • 0.01%Nadeoxycholate(dilutedfrom10%stocksolution)inPBS

   X-galreactionbuffercanbestoredforatleastoneyearatroomtemperatureinfoil-coveredcontainer.

   • X-galstock(40X)40mg/mlX-galindimethylformamide
   • Storeat-20⁰Cinfoilcoveredglasscontainer.

   • RinsemanytimesinPBSuntilthesolutionnolongerturnsyellow.Thisusuallytakesabout5changes.O/Nrinseisfine.

   • Viewunderbrightfieldopticsforoptimaldetection.

   Notes

   1. Althoughwehaveseenreducedactivityofß-galaftertreatmentwithorganicsolvents,lyophilizationfollowedbypermeABIlizationinacetonehasbeenusedsuccessfullyinplaceofaldehydefixationforX-galstaininginC.elegans(Fire,1992).
   2. Theamountofferricyanideandferrocyanidecanbevaried.Amorerapidprecipitationisachievedwiththehigherconcentration,butthehigherconcentrationcanleadtoagreenishprecipitate(PrussianGreen)intissueuponprolongedstaining.
   3. Theferricyanideandferrocyanidecanformablueprecipitate(Prussianblue)uponreactionwithfreeferricion.DonotusemetalforcepstomanipulatethetissuewhileitisintheX-galdetectionbuffer.
   4. iv.Alltissueshaveendogenous,lysozomalß-gal.ItspHoptimumisverylow,andthusitisnotveryactiveinthepH7.4bufferusedhere.However,sometissuesalsohaveacytosolicformofß-galwhichcanshowenoughactivitytobeconfounding.Thevariablesthataffectbackgroundstainingincludethefixativetype,lengthoftimeinfixative,pHofthebuffer,andamountoftimeinthestainingsolution(Rosenbergetal1992).Ifaftervaryingtheseparameters,backgroundisstillaproblem,onecandetectthelacZenzymebyimmunohistochemistry.Monoclonalandpolyclonalantibodiesareavailable(e.g.from5prime,3prime,Boehringer-MannheimandCappel).
   5. TrisbufferwastriedinplaceofPBSinthereactionbuffer,withnosuccess.
   6. ThedetergentsintheX-galreactionbufferneednotbeincluded,butusuallydonotreducestaining,andforsometissuestheyincreasestaining.
   7. Theindigoproductissolubleinorganicsolventsandthusoneshouldminimizeexposuretosuchsolventsafterformationoftheproduct.Nonetheless,fixationwithglutaraldehyde(butnotformaldehyde)allowedpreservationofenoughindigotomakeitthroughthevarioussolventsrequiredforembeddingforelectronmicroscopy(EM)(Snyderetal.1992).
   8. Doublestainingofcellsforß-galwithX-galandacellularantigenusingantibodiesisdifficult,thoughpossIBLe(Snyderetal1992,VaysseandGoldman,1990).TheindigoproductofX-galabsorbsinthewavelengthsemittedbythestandardfluorescent-conjugatedantibodiesandissodarkthatitcanobscuretheproductsfromeitherhorserADIshperoxidaseoralkalinephosphatase-conjugatedantibodies.However,bycarefullycontrollingthereactiontimeinX-galsothatonlyasmallamountofindigoisproduced(VaysseandGoldman,1990),byhavingß-gallocalizedtothenucleuswhenthecellularantigenisnon-nuclear(Bonnerotetal1987),orbyusingantibodiestodetectbothß-galandthecellularantigen,onecanovercometheseproblems.
   9. Ifnitrobluetetrazolium(NBT)saltisaddedtotheX-galreactioninplaceofiron,apurpleprecipitatewillresult.ThiscanbeafasterandmoresensitivereactionthanX-galalone.AstocksolutionofNBTcanbepreparedbyadding50mgNBTpermlof70%dimetnylformamideandstoredat-20oC.Finalworkingconcentrationsare0.25-1.0mg/ml.Phenazinemethosulfate(PMS)canbeaddedinconjunctionwithNBTtoincreasethereactionratefurther.Preparea100Xstockof2mg/mlinH2Oanduseimmediately.PMSisveryunstable.
   10. Toaidinidentificationofstainedcells,thetissuecanbeprocessedforcryostatsectionsasdiscussedbelow.Inaddition,stainingcryostatsectionsmaygivehighersignalthanwholemountssoifyouarenotsurethatthewholemountproceduregavethemaximalstaining,stainsectionsasbelow.Alternatively,forsimplyvisualizingthestainedcellsproducedduringstainingofwholemounts,paraffinsectionscanbemade(paraffinembeddingdestroysß-galactivitysothereisnopointinstainingparaffinsections).Embedinparaffinusingminimumnecessarytimesforthetissueofinterestasthesolventscanpartiallydissolveindigo.Forglutaraldehyde-fixedmouseretina,whichisapproximately250micronsthick,thefollowingprocedurewasused.Dehydratethroughgradedethanols(50%,70%,95%,100%,100%)for20mineach.Clearinxylene,2x15min.Infiltratewith1:1mixofxyleneandparaffin,65^OCfor30min.Paraffin,2x15min.Embedinparaffin.

   X-GALSTAININGOFTISSUESECTIONS

   Theprocedureforstainingtissuesectionsisverysimilartotheprotocolforintacttissue.Sectionstainingshouldbeusedwhenawholemountcannotbeusedduetothesizeofthetissue.

   1. Fixtissueinfixativeslistedaboveusingperfusionifpossibleandfollowwithimmersioninfixativeat4^OCforseveralhours.RinsebrieflyinPBS,thensinkin30%sucroseinPBS+Mg2+at4^OC.

      Fixationtimeswillvarywiththesizeandnatureofthetissue.ForembryonicchickbrainsofE10orolderandforpostnatalratormousebrainsweincubateupto8hoursinfixative.PerfusionmaynotbenecessaryforalltissuesandshorterfixationtimesmaybepreferableasX-galstainingmaybedecreasedbylengthyfixation.

   2. EmbedtissueinOCT(Miles)orgelatin/sucrosemountingmediumandfreezeonmetalchuckscooledinliquidN2.

      Gelatin/sucroseembeddinggivesbetterfrozensectionsforembryonictissuethandoesOCT.

      Gelatin/sucroseembeddingsolution
      • 7.5%gelatin(porcineskin,Sigma)
      • 15%sucrose
      • 0.05%sodiumazide
      • in1XPBS
      Dissolvegraduallyat60oC,withstirring.Themediumsolidifiesatroomtemperaturetoatransparentgel.Storeatroomtemperature.Liquefyinmicrowavewithfrequentswirlingbeforeembeddingsamples.

   3. Cutcryostatsectionsandmountonsilane-coated(Rentropetal.,1986)orgelatin-coatedslides;air-dryO/N.Sectionsupto90mmthick(thethickestwehavetried)havebeensuccessfullystained.
      • GelatinSolutionforsubbingslides
      • 2ggelatin
      • 0.1gchromiumpotassiumsulfate(chromealum)
      • in200mlH20

      HeatH20to60oC.Dissolvechromealum,thengraduallydissolvegelatin.Filterbeforeuse.Canincreaseordecreasethepercentageofgelatin.Loadslidesinracks,dipquickly,andair-dryovernight.

   4. Fixsectionstoslidesin4%paraformaldehydefor10-15minutesat4^OC.

   5. RinseslidesinPBS+Mg2+twice,for10minuteseach,at4^OC.

   6. StainslidesinX-galReactionBufferfor1-24hoursat37^OC.

   7. RinseslidesinPBS3times,for10minuteseach,oruntilsolutionisnolongeryellow.SlidescanbeleftinPBSO/N.

   8. Coverslipingelvatol.

   GELVATOLPREPARATION

   WehaverevisedtheprotocolpublishedbyRodriguezandDeinhardt(1960).

   1. Take200mlof0.01MKH2PO4(aboutpH5.0)andaddenough0.01MNa2HPO4tobringthepHupto7.2.
   2. Take250mlofthe0.01MKH2PO4/Na2HPO4andadd2.05gNaCLtogivea0.14MNaCLconcentration.
   3. Dissolve62.5gGelvatol(AirProducts)inthe250mlof0.01MKH2PO4/Na2HPO4/.14MNaCL.Stironmagneticstirrerinwarmroomforafewhours.
   4. Addglycerolinanamountequaltoone-halfthetotalvolumeoftheGelvatolbufferedsalinesolutionandstirovernightatroomtemperature.
   5. CentrifugetheGelvatolsolutionat12,000rpmfor15min.in30mlCorextubesinBeckmanJ2-21centrifugeatroomtemperaturetoremoveundissolvedparticles.
   6. Pipettethesupernatantintosmallscrewcapbottles.CheckpHofGelvatolsolution.ItshouldbebetweenpH6and7.
   7. StoreGelvatolsolutionat4^oC.Screwcapsontightlytopreventevaporation.DonotleaveGelvatoluncappedforlongerthannecessarywhenworkingwithit.

   Notes

   1. TheamountoftimefortheX-galreactionwillvaryaccordingtotheconcentrationoflacZandtheamountofendogenousß-gal.
   2. Ifculturedcellsaretobestained,fixin0.5%glutaraldehydeinPBSorin4.0%paraformaldehydeinPBSfor5"atroomtemperatureandproceedfromstep5above.Fixationfor>5minutescanleadtodecreasedenzymeactivity.

   STAININGFORALKALINEPHOSPHATASEACTIVITY

   Phosphatasegenesareusefulreportergenesasseveralhistochemicalmethodsthatyieldprecipitated,highlycoloredand/orelectrondenseproductshavebeendevised.Achromogenicsubstrate,5-bromo-4-chloro-3-indolylphosphate(X-Phos),whichisverysimilartoX-gal,canbeemployedfordetectionandleadstoproductionofablueprecipitate(Figure1).Inaddition,thetetrazoliumsaltNBT,(Altman,1972)canbeusedinconjunctionwithX-Phosasthefinalelectronacceptorfortheindoxyldimerizationreaction.Whenreduced,NBTformsapurpleprecipitate.

   Oftheclonedphosphatases,thehumanplacentalalkalinephosphatasegene,PLAP(Kametal1985),isperhapsthemostusefulasitisveryheatstableandisresistanttosomechemicalinhibitorsthatareactiveonotherendogenousalkalinephosphatases.PLAPhasbeenusedasahistochemicalreporterfairlyrecently(Bergeretal1987,Henthornetal1988,Fields-Berryetal1992).IthasnotbeenusedaswidelyaslacZandthusitsneutralityneedstobeestablished.LacZandPLAPhavedifferentstrengthsandweaknessesregardingtheirdistributionandhistologicaldetection.PLAPisnormallyassociatedwithmembranesandthusPLAPactivityanddefinestheoutersurfaceoftransducedcells,includingneuronalprocesses.WehavefoundPLAPstaininginretinalganglioncellaxonsseveralcentimetersfromcellbodies(FeketeandCepko,unpublished).However,therearetimeswhenneuronalcellbodiesarenotwelldefined,whichcanmakeitdifficulttocountcells(HallidayandCepko,1992andFeketeetal,1994).ß-galactivityvisualizedbyX-galtypicallydoesnotfillcells,particularlylongprocesses,butoftengivesgoodstainingofcellbodiesandthusitiseasytocountcells.Frequently,X-galstainingisfairlyperinuclearandsometimesitispunctateaswell.WhenX-galprecipitateisviewedundertheEM,itappearsthatitlocalizestothenuclearenvelopeandthegolgiandendoplasmicreticulum(Snyderetal1992).ForPLAP,thereexistsaprocedureinwhichleadisprecipitatedveryclosetothelocationoftheenzyme(HugonandBorgers,1966).TheleadprecipitateissuperiortothatofX-galandthusPLAPistheenzymeofchoicewhenelectronmicroscopyisrequired.

   ThereareendogenousalkalinephosphataseactivitiesthatmayleadtodifficultiesindetectionoftransducedPLAP(McCombetal.,1979).Inmostcases,backgroundactivitiescanbeminimizedwithheatandchemicalinhibitorsthatleavePLAPactive(ZoellnerandHunter,1989).Inaddition,monoclonalandpolyclonalantibodiesspecifictoPLAPcanbepurchased(Dako,Zymed,Medix,andAccurateChemicalandScientificCo.)whenthebackgroundisnotsurmountableusingthevariousinhibitors.

   X-PHOS/NBTSTAININGOFWHOLEMOUNTS

   SeeaboveprotocolforX-galstainingofwholemountsandperformsteps1and2.3.Heattissueforatleast30"at65⁰C.

   Forstainingofembryonicchickdiencephalon(oneoftheareasofthebrainwiththehighestbackground),thisstepwasincreasedto1.5hours.Itmaybethatevenlongerheattreatmentcouldbenefitspecificstaininginareaswithhighbackground.

   4.IncubatetissueinX-Phos/NBTDetectionBufferfor15"atroomtemperature.

   XPhos/NBTDetectionBuffer(Buffer3asdescribedforGeniuskitbyBoehringer-Mannheim)

   • 100mMTris-HCl,pH9.5
   • 100mMNaCl
   • 50mMMgCl2

   Storeatroomtemperature.Tendstoprecipitateoverseveralweeks.Thisdoesnotseemtonoticeablyaffectthestaining.

   5.IncubateinX-Phos/NBTReactionSolutionfor1toseveralhoursatroomtemperature.Coverthereactionwithfoiltoreducebackground.
   X-Phos/NBTReactionSolution
   • 50ulof100XX-Phosstock
   • 100ul50XNBTstock
   • 50ulof100Xlevamisolestock,optional
   • in5mlX-PhosDetectionBuffer
   • MixX-Phos,NBT,andlevamisolewiththeDetectionBufferimmediatelybeforeusing.

   Stocks

   • X-Phos(100X):10mg/ml5-bromo-4-chloro-3-indolyl-phosphate(alsoreferredtoasBCIP)inH20.Storeinthedarkasaliquotsat-20⁰C.Canbefrozenandthawedseveraltimes.
   • NBT(50X):50mg/mlnitrobluetetrazoliumin70%dimethylformamide,30%H2O.Storeat-20⁰Cinaglasscontainercoveredwithfoil.Doesnotfreezeatthistemperature.
   • Levamisole(100X):50mMinH20.Storeat-20⁰C.
   • PMS(100X):2mg/mlphenazinemethosulfateinH20.Useimmediately.

   6.Rinsein20mMEDTAinPBSfor2-4hours.

   Tissuecanbestoredinthedarkat4⁰CinPBS+EDTAor30%sucroseinPBS+EDTA+0.05%sodiumazideformanymonths,althoughthebackgroundclearlyincreasesovertime.TissuecanthenbesectionedasaboveforX-galstainedwholemounts.

   Notes

   1. 0.5%glutaraldehydedecreasedPLAPactivityinembryonicchickbraincellsstainedinwholemounts,butnotinchickenembryofibroblastsculturedinvitro.Fixationofchickbrainwholemountsthusistypicallydonein4%paraformaldehydeinPBSfor2-4hoursat4^OC.Inareaswherebackgroundalkalinephosphataseactivityisaproblem,increasingthetimein4%paraformaldehyde,evenuptoseveraldays,candecreaseendogenousbackgroundwithoutsignificantlydecreasingPLAPactivity.
   2. ChickretinasandcerebellahavebeenkeptinPBSat4^OCforatleastonemonthafterfixationandrinsinginPBSwithnoappreciablelossofsignalinX-Phosstaining.
   3. BackgroundstainingcanbeduetoendogenousalkalinephosphataseactivitiesorreductionofNBTfromothersources(e.g.NADPH).Itisalsoenhancedbylight.Themosteffectiveinhibitorisheat.Toreducebackgroundfurther,oneormoreofthefollowinginhibitorscanbetriedinadditiontoheat(theyareaddedtothereactionmix):0.5mMlevamisole(L[-]-2,3,5,6-tetrahydro-6-phenylimidazo{2,1-b}thiazole),2mMmercuricchloride,5mML-leucyl-glycyl-glycine,1mMEDTA,1mML-phenylalanine-glycyl-glycine,0.2MlysineHClor0.3mMsodiumarsenate(ZoellnerandHunter,1989).Wefoundthatlevamisolewasthesecondmostusefultreatment(afterheat)inreducingthebackgroundstaininginbrains,althoughitalsoreducedPLAPstainingslightlyinsomecases.
   4. Ifbackgroundstainingisnotaproblem,thereactioncanbecontinuedforupto2days.

   X-PHOS/NBTSTAININGOFSECTIONS

   CryostatsectionscanbestainedforPLAPactivity.FollowtheprotocollistedaboveforX-galthroughstep5.

   6.TransferslidestopreheatedPBSat65^OCandheatfor30minutes.

   7.RinseslidesinroomtemperaturePBSfor5minutes.

   8.RinseslidesinX-Phos/NBTDetectionBufferfor10minutes.

   9.StainslidesinX-Phos/NBTReactionMixfor1to12hoursatroomtemperature.Coverwithfoilduringandafterstaining.ThetimeinthereactionbufferwilldependonthelevelofPLAPexpressionandendogenousbackground.Ifbackgroundislow,stainingcancontinuefor2days.

   10.RinseslidesinPBS+20mMEDTA,3x10minutes.

   11.MountinGelvatol(+20mMEDTAifdesired).

   Storingslidesat-80^oChelpedpreventbackgroundstainingfromincreasing.

   Notes

   1. Ifculturedcellsaretobestained,fixin0.5%glutaraldehydeinPBSorin4.0%paraformaldehydeinPBSfor5"atroomtemperatureandproceedfromstep6above.
   2. ProcessingculturedcellsgrownonglassthroughtheprocedureforparaffinembeddingpriortostainingpreservedenoughPLAPactivitythatpositivecellswerevisible.Ifparaffinsectionsaredesirable,itisworthtryingpilotexperimentsusingthetissueofinterest,varyingthefixation,andminimizingthetimesinorganicsolvents.

   X-galANDX-PHOS/NBTSTAINING

   ß-galandPLAPcanbedetectedinthesametissueandeveninthesamecell.Inordertoprocesstissueforbothactivities,stainingwithX-galmustbedonefirstasthelacZ-encodedenzymeisdestroyedbytheheatingstepusedtoreduceendogenousalkalinephosphataseactivities.Tocombinetheprotocols,proceedthroughtheX-galreactionasdescribedaboveforeitherwholemountsorsections.YoumaywishtostopandexaminetheresultsbeforemovingtoPLAPstaining.TheindigoproductofX-galcanbeeasiertodetectpriortocarryingouttheX-Phosreactionasbackgroundalkalinephosphatasestainingcanobscureit,particularlyinwholemounts.RinsethetissueverywellwithPBSpriortoPLAPstainingasresidualß-galactivityinthepresenceofX-galandNBTcanenableß-gal+,PLAP-cellstoturnpurple.FollowtheaboveprotocolsforPLAPstaining.

   ACOMPARISONOFALTERNATIVESUBSTRATESFORß-galANDPLAP

   Asdescribedabove,thereareanumberofalternativesubstratesforbothenzymes.Thefollowingdiscussionconcernsthesesubstratesandsummarizesourexperienceswiththemrelativetothethosedescribedabove.

   ß-galSUBSTRATES:

   X-galisagoodchoiceasaprecipitablesubstratewithironasanelectronacceptor.Itgivesabrightbluecolor,althoughthereactionisquiteslow,evenat37^OC.NBTaddedtoX-galgivesanintensepurplecolorwhichdevelopsmuchmorerapidly.PMSwillincreasethisreactionrateevenfurther.Tetrazoliumredmakestheproductlookgreenish-blue,butincreasesbackground,makingthestainingmoreequivocal.Othermodifiedindolyl-basedcompoundscanbeusedifalternativecoloredprecipitatesaredesired.Salmon-gal(BiosynthInternational)resultsinalightorange/pinkprecipitatewhichdevelopsslowly.Addingtetrazoliumsaltstendstoincreasethebackground.Magenta-gal(BiosynthInternational)givesalightpinkishpurpleprecipitate.Green-gal(BiosynthInternational)didnotgivediscernibleprecipitatesinourhands,exceptinthepresenceofNBT,whichgaveapurpleprecipitate.

   PLAPsubstrates

   X-PhosintheabsenceofNBTgivesabrightblueprecipitatewhichdevelopsslowlyovertime.Asaresult,theremaybesomediffusionawayfromthesiteofenzymeactivity.Irondoesnotworkasanelectronacceptorfortheindolyl-basedcompoundsathighpH,andresultsinlargeamountsoffloatingprecipitate.AddingNBTresultsinadeeppurpleprecipitatewhichdevelopsmorequickly.AddingPMSincreasestherateofthereaction(inthepresenceofNBT)withoutaddingbackgroundbyquantitativelyreducingthetetrazoliumsalt.Othermodifiedindolyl-basedcompoundscanbeusedifalternativecoloredprecipitatesaredesired.Magenta-phos(BiosynthInternational)givesapinkish-purpleprecipitatewhichdevelopsatintermediaterates.ThecolorcanbedistinguishedfromthepurplecolorderivedfromX-Phos+NBT.Addingtetrazoliumredorbluemakestheproductlookmorepurple(lesspink).Salmon-phos(BiosynthInternational)resultsinalightorange-pinkprecipitatewhichdevelopsslowly.AddingtetrazoliumredorbluegivesapurpleprecipitatesimilartoXP+NBT.TheVectorRedsubstratekit(VectorLaboratories)resultsinstainingthatishighlyvariablebetweenexperiments;theprecipitatecanbeintenselyredbutisusuallylightpink.Thesubstrateisunstable,andbeginstoformfloatingprecipitatesafter20-30minutes.Additionoftetrazoliumsaltsisnothelpful,andPMSactsasaninhibitorinthissystem.Thesubstrateaffordshighbackgroundlevelstointacttissuesandthusisnotusefulforstainingwholemounts.Thebluesubstratekit(VectorLaboratories)affordsaveryprettypeacockblueprecipitate,buthasthesameproblemsastheredsubstratekit,i.e.unstablesubstrateandhighbackgroundtointacttissues.Stainingisreproducibleandintense,however.AddingtetrazoliumsaltssuchasNBTincreasesthebackgroundandresultsincopiousamountsofnon-localizedprecipitate.

   LegendforFigure1.ProductionofindigofromX-galorX-Phos.

   A.Productionofthestableblueprecipitatefromsubstitutedindolesproceedsasshown.TwomoleculesofeitherX-PhosorX-galgenerate2moleculesofindoxylwhichthenformtheindicateddimer.The"X"moiety(5-bromo-4-chloro-indolyl)inbothX-galandX-Phosisthesameandthusoncecleavagebytherespectiveenzymehasoccurred,thedimerizationandoxidationreactionsleadtothesamehalogenatedindigocompound.

   B.Nitrobluetetrazolium(NBT)isanexampleofatetrazoliumsalt.Thesearearatherunstableclassofcompoundswhichprecipitatewhenreducedtoformhighlycoloredcompounds.Inthiscase,NBTisreducedbythehydrideproducedbydimerizationofthetwoindoxylsthatresultfromcleavageofeitherX-galorX-Phos(shownin(A)above).Whenreduced,NBTformsformazan,adarkpurpleprecipitate.

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