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DETECTION OF ßGALACTOSIDASE AND ALKALINE PHOSPHATASE ACTIVITIES IN TISSUE
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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,2oftheliberatedindoxylmoietiesformadimerwhichisnonenzymaticallyoxidizedFigure 1(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.

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

  4. 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,65OCfor30min.Paraffin,2x15min.Embedinparaffin.

        X-GALSTAININGOFTISSUESECTIONS

        Theprocedureforstainingtissuesectionsisverysimilartotheprotocolforintacttissue.Sectionstainingshouldbeusedwhenawholemountcannotbeusedduetothesizeofthetissue.

        1. Fixtissueinfixativeslistedaboveusingperfusionifpossibleandfollowwithimmersioninfixativeat4OCforseveralhours.RinsebrieflyinPBS,thensinkin30%sucroseinPBS+Mg2+at4OC.

          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%paraformaldehydefor10-15minutesat4OC.

        5. RinseslidesinPBS+Mg2+twice,for10minuteseach,at4OC.

        6. StainslidesinX-galReactionBufferfor1-24hoursat37OC.

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

        8. Coverslipingelvatol.
        9. 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. StoreGelvatolsolutionat4oC.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%paraformaldehydeinPBSfor2-4hoursat4OC.Inareaswherebackgroundalkalinephosphataseactivityisaproblem,increasingthetimein4%paraformaldehyde,evenuptoseveraldays,candecreaseendogenousbackgroundwithoutsignificantlydecreasingPLAPactivity.
            2. ChickretinasandcerebellahavebeenkeptinPBSat4OCforatleastonemonthafterfixationandrinsinginPBSwithnoappreciablelossofsignalinX-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.TransferslidestopreheatedPBSat65OCandheatfor30minutes.

            7.RinseslidesinroomtemperaturePBSfor5minutes.

            8.RinseslidesinX-Phos/NBTDetectionBufferfor10minutes.

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

            10.RinseslidesinPBS+20mMEDTA,3x10minutes.

            11.MountinGelvatol(+20mMEDTAifdesired).

            Storingslidesat-80oChelpedpreventbackgroundstainingfromincreasing.

            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,evenat37OC.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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