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On the History and Mechanism of Alizarin and Alizarin Red s Stains

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http://jhc.sagepub.com/ Journal of Histochemistry & Cytochemistry http://jhc.sagepub.com/content/17/2/110 The online version of this article can be found at:   DOI: 10.1177/17.2.110 1969 17: 110 J Histochem Cytochem HOLDE PUCHTLER, SUSAN N. MELOAN and MARY S. TERRY ON THE HISTORY AND MECHANISM OF ALIZARIN AND ALIZARIN RED S STAINS FOR CALCIUM     Published by: http://www.sagepublications.com On behalf of:     Official Journal of The Histochemical Society can be found at: Journal of Hi
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    http://jhc.sagepub.com/  Journal of Histochemistry & Cytochemistry  http://jhc.sagepub.com/content/17/2/110The online version of this article can be found at: DOI: 10.1177/17.2.110 1969 17: 110 J Histochem Cytochem  HOLDE PUCHTLER, SUSAN N. MELOAN and MARY S. TERRY ON THE HISTORY AND MECHANISM OF ALIZARIN AND ALIZARIN RED S STAINS FOR CALCIUM  Published by:  http://www.sagepublications.com On behalf of:  Official Journal of The Histochemical Society  can be found at: Journal of Histochemistry & Cytochemistry  Additional services and information for http://jhc.sagepub.com/cgi/alerts Email Alerts: http://jhc.sagepub.com/subscriptions Subscriptions:  http://www.sagepub.com/journalsReprints.nav Reprints:  http://www.sagepub.com/journalsPermissions.nav Permissions: What is This? - Feb 1, 1969Version of Record >>  at CHRISTIAN UNIV on December 18, 2013 jhc.sagepub.comDownloaded from at CHRISTIAN UNIV on December 18, 2013 jhc.sagepub.comDownloaded from   110 THE JOURNALOF HI8TOCHEMISTRY AND CYTOCHEMI8TRY Copyright © 1969byThe HistochemicalSociety, Inc.Vol.17,No.2 Printedin U S A ONTHEHISTORYANDMECHANISMOFALIZARINANDALIZARIN REDSSTAINSFORCALCIUM”2 HOLDEPUCHTLER,SUSANN.MELOAN AND MARYS.TERRY DepartmentofPathology MedicalCollegeof Georgia,EugeneTalmadgeMemorialHospital,Augusta,Georgia ReceivedforpublicationOctober18,1968 Alizarin (madder)hasbeenusedintextiledyeingsinceearlyantiquitycInhistologycal-cium-alizarin or calcium-alizarinredScompounds are oftenreferredtoas“lake”or“com-plex.”Chemicalandinfraredspectroscopic data showed thatthesecompoundsaresalts, notchelates. In dyechemistrythe termlake denotes a poorlysolubleorinsolublesaltof a water-solubledye. Salt formationbetweencalciumdepositsintissues and alizarin oraliza- rnredSisindicatedbythesensitivity ofthesecompounds towarddiluteaceticacid. Acid dyesforlakes,whichdonotcontainchelatinggroups,alsostained calcium depositsselec- tively.  lizarin stainedcalciumdepositsintenselyonly around pH12. Alizarin redScolored calcium depositsselectivelyaroundpH9; neutral andaciddyesolutionsproducedsevere diffusion artifacts. Chemicaldataindicatethat alizarin red  can react with calcium via its sulfonicacidand/oritsOHgroups. InhistologyalizarinredSandvonKossa’stechniquearecommonlyusedfordemonstrationofcalciumsalts.VonKossa’sprocedureisbelievedtovisualizephosphateandcarbonateanions,whereasalizarinredSreactswithcalciumandothercations.Bothmethodshavebeenusedfordiagnosisofcalciumdeposits.However,duringinvestigationsofearlyarterioscleroticlesionsweobservedstrikingdiscrepanciesbe-tweentheamountsof“calcium”demonstratedbyalizarinredSandbyvonKossa’stechnique.Forexample,insomearteriesvonKossa’sprocedurecoloredsegmentsoftheinternalelasticmembraneblack,yetadjacentsectionsutterlyrefusedtobindalizarinred5,and viceversa. Obviously,foranunderstandingoftheprocessesoccurringinthesearterioscleroticlesions,itwasessentialtoobtaininformationconcerningthehistochemicalsignificanceofthese procedures. Apreliminaryperusaloftheliteratureshoweddifferencesbetweenchemicalandhisto-logicconcepts; e.g. chemistsclassifythecalcium-alizarinoralizarinredScompoundsasasalt,whereasinhistology and histoehemistryithas‘DedicatedwithadmirationtoDr.R.D.Lillie, whoby his farranging knowledgeofthehistory andchemistry ofdyes,elevatedstainingfroma crafttoabranchofhistochemistry. 2 Thisinvestigationwassupportedbyagrant-in-aidfromtheGeorgiaHeartAssociationandUnitedStates PublicHealthServiceResearch GrantHE 12147fromtheNationalHeartInsti-tute.beenreferredtoasalakeorcomplex.Further-more,sincethepublicationofthereviewsbyHarms(22)andMcGee-Russell(32),newchemicalandinfraredspectroscopicdataoncalcium-alizarin-alizarinredSandrelatedcom-poundshavebecomeavailable.ItwasthereforedeemedofinteresttoreinvestigatethemechanismofalizarinandalizarinredSstainsforcalciumandtosearchforamethodwhichwouldavoiddiffusionartifacts.Sincethisprojectwaspartofastudyofearlyarterioscleroticlesions,investiga-tionswerelimitedtocalcificationsinsofttissues;boneandteethwereexcluded.Thesestudiesandareviewofpertinentliteraturearepresentedinthisreport.ObservationsonthemechanismofvonKossa’sprocedurewillbepublishedsepa-rately. MATERIALSANDMETHODS Experimentswerecarriedoutonhumanautopsymaterial.Blocksoftissues werefixedinabsolute alcohol,Carnoy’sfluid(absolutealcohol-chloro- form-glacialaceticacid 6:3:1),10 unbuffered formalinorZenker-formol(Spuler,Maximow) andembedded inParaplastbyAutotechnicon. Sectionswerecutat5 p. Onebatchofalizarinsiccum(Chroma,C.I.58000),fourbatchesofthebiologicstainalizarinredS(Chroma,C.I.58005),onesampleofthecorrespondingtextiledyeDiamondredW(VeronaDyestuffs)andonesampleofalizarinblueS  Chroma,C.I.67415) wereused.Solutions,0.1 and0.5 ofthesulfonateddyesindistilledwater  at CHRISTIAN UNIV on December 18, 2013 jhc.sagepub.comDownloaded from   ALIZARINANDALIZARIN REDSSTAINSFORCALCIUM 111andin 0.5%aqueoussolutionsofBrook’sbuffers, pH4.8 7.2and9.0 wereprepared.Todetermine possibleeffectsofthebuffer saltsonthestainingreaction,Sorensen’sphosphatebufferandbarbitalbuffer,pH7.2and9,weresubstitutedforBrook’sbuffer.Owingtothepoorsolubilityofalizarin,onlysaturatedsolutionsofthisdyeinpH7.2and9buffersand in0.3and 1%aqueousNaOHweretested.Sectionswerestainedfor5mmor1hr,rinsedinbuffersolutionforabout5sec,dehy-dratedinthreechangesofabsolutealcohol, clearedinxyleneand mountedinPermount.OtherserieswerestainedwithalizarinredSasrecom-mendedbyMcGee-Russell(32).Toobtaininformationconcerningtheselec-tivityofalizarinandalizarinredSforcalcium,thereactivityofvarioussaltswasstudiedinmodelexperiments.Toavoidlengthyrepetition,thecompoundsusedareitemizedonlyinTableI.Approximately500mgofthesubstance(A.C.S.reagentgrade)tobestudiedwereplacednearoneendofaslide.Twoto3dropsofthedyesolu-tionwereadded;anotherdropwasplacedattheotherendoftheslidetoserveasacolorstandard.Theinteractionbetweentestsubstanceanddyesolutionwasobservedbetweenparallelandcrossedpolaroidsfor2-5mmoruntilnofurtherchangesoccurred.ThisprocedureisamodificationofthetechnicrecommendedbyMcGee-Russell(32)forstainingofcalciumintissuesections.Inanotherseriesthesaltswereplacedonslides, mixed with gelatin air-dried fixed informalde-hydevapororCarnoy’ssolutionandstainedasdescribedabovefortissuesections.ToinvestigatetheroleofthesulfonicacidgroupofalizarinredSinthestainingofcalciumdepositsintissues,thestainingpatternsofsal-fonateddyes withoutchelatinggroupsrecom- mendedbyPratt(38)orthe ColourIndex  10 forpreparationofbariumorcalciumlakeswerestudied.Thefollowingdyeswereemployed:BordeauxredB(C.I.16180),brilliantblack(C.I.27260),croceineorangeY(C.I.15970),fastlightrubineBL(C.I.17065),fastredS(C.I.15620)andPonceau2R(C.I.16150).Todeterminewhetherornothighaffinityforearthalkalisisapeculiarityofcertainaciddyes,aseriesofsulfonateddyes(withoutchelatinggroups)wasselectedforcom-parison,namelyAcilancosineE(C.I.14710),AcilanredS2B(C.I.23910),amaranth(C.I.16185),azophloxinGAandfastcrimsonGR(C.I.18050),azorubinS(0.1.14720),Benzofastscarlet4BS  C.I. 29160),Benzofastscarlet4GS(C.I.29185),BiebrichscarletWS(0.1.26905), brilliant scarlet6R(C.I.16255),croceinescarlet6R(C.I.16255),croceinescarletMOOP(0.1.27290),di-phenylgreenGPD(C.I.30295),durolblack2B(0.1.26370),fastwoolredGL(C.I.17045),levanolfastscarletFGN(0.1.18020),levanolyellow6G  C.I. 23900), Niagaraskyblue6B(0.1.24410),orangeGandwoolorange2G(0.1.16230),Siriussuprared4BLA(0.1.29065),Siriussupratur-quoiseLG(0.1.74180),SolophenylorangeTGL(0.1.40215and40220)andtartrazine0(0.1.19140).Sectionswerestainedin0.5%solutionsofthesedyesfor5mmor1hr.Thedyenamesgivenabovearethetradenamesusedbythemanufacturers.Previouspaperchro-matographicstudiesshowedsignificantdiscrep-anciesbetweenthecompositionofsomedyesamplescarryingthesame Colour Index numberobtainedfromdifferentmanufacturers(44).Itwasthereforedeemedexpedientto use thetradenamesofthedyesemployedratherthansyno-nyms.Otherbrandsofthesedyesmayormaynotyieldsimilarstainingpatterns.Calciumandrelatedsaltswereremovedfromsectionsbytreatmentwith5%aqueousaceticacidfor5or10mm.Sectionswerethenwashedindistilledwaterandstainedasdescribedabove.Sincealizarin-metalchelatesareinsolubleinaceticacid,whereassaltsofalizarinarereadilysoluble(22),stainedsectionsweretreatedwith5%aqueousaceticacidfor5 mm toobtainin-formationconcerningthenatureofthebondbetweendyesandcalciumdepositsintissues.PaperchromatogramsofthefourbatchesofalizarinredSandofDiamondredWwerepre-paredaccordingtothemethoddescribedbyRosenthal,PuchtlerandSweat(44).AReichertZetopanmicroscopeequippedwithatwinlampunit(tungstenandmercuryvaporlamp),brightfieldanddarkfieldcondensers, was usedforcomparisonofstainingandfluorescencemicroscopicpatterns.Thecombinationofultra-violetbluepassfilterBG12/3mmandultravioletbluebarrierfilterSp.3(GG9/1mm+OG1/1.5mm)wasemployed.Dyeswhichwerenotfluores-centundertheseconditionswerestudiedalsowithultravioletpassfilterUG1/1.5mmandbarrierfilterSp.2(GG13/1+3mm+Wrattenfoil2B). Theterm “calciumdeposits”isusedinade-scriptivesensewithoutstrictchemicaldenota-tions;theseareasmayormaynotcontainothercationsinadditiontocalcium. RESULTS Effectoffixatives:Comparisonofsectionsfromthesameorganfixedinthesolutionslistedaboveandstainedunderidenticalconditionsshowedstrikingdifferences.Colorationofcalciumdepositswasmostintenseinalcohol-andCarnoy-fixedsections;nodifferencewasobservedbetweenthesetwofixatives.Informalin-andZenker-  at CHRISTIAN UNIV on December 18, 2013 jhc.sagepub.comDownloaded from   112PUCHTLER,MELOANAND TERRY fixedmaterialcalciumdepositswereweaklytomoderatelycolored.Stainingofcalciumdepositsdecreasedwiththedurationofstorageinformalinandwasabolishedafter 2-3 weeks.ThestainingpropertiesofmaterialfixedinZenker-formolvariedsignificantlyandseemedtodependonthedurationoffixationandsubsequentwashing.Therefore, unless otherwisestated,alldescriptionsandcommentshereafterreferonlytoalcohol-andCarnoy-fixedtissues.Binding of alizarin:AlizarinwaspracticallyinsolubleinbuffersofpH7.2anddidnotstaincalciumdeposits.SaturatedsolutionsofalizarininbuffersofpH9orin0.3%aqueousNaOHcoloredmostcalciumdepositsmoderately;verysmalldepositswereonlyfaintlystained.Veryintensebluish red topurplecolorationofallcalciumdepositswithin 5mmwasobtainedwith saturatedsolutionsofalizarinin1%aqueousNaOH;othertissuestructuresremainedun-stained.Smallintracellulardepositswereclearlydelimitedandtherewerenodiffusionartifacts.Sectionsstainedwiththisprocedurewereusedasstandardsinassessingthespecificityandintensityofcolorationobtainedwithotherstainingtech-niquesdescribedbelow.Unfortunately,inthisstronglyalkalinesolutionsectionstendtobecomedetachedfromslidesandthemethodisthereforeinconvenientforgeneral use inhospitalpathology.Attemptstosubstitute1%NaOHin70%ethanolasadyesolventwereunsuccessful;undertheseconditionscalciumdepositswereonlyweaklytomoderatelycolored,evenwhenstainingwasprolongedupto1hr. Aqueousunbufferedsolutionsofalizarinred S:Thestainingpropertiesof0.1%solutionsofalizarin red Svariedwidelyfrombatchtobatch.Onebatch,whichhadbeenpurchasedseveralyearsbefore,yieldeddeeporange-redcolorationof calcium depositswithin5mm;othertissuestructureswerestainedlightpink.Whenstainingtimewasextendedto1hr,theintensityofcolora-tionofcalciumdeposits was strikinglydecreasedandothertissuestructuresweremoderatelystained. This lossofcontrastmadeitdifficulttoidentifysmallfociofcalcification.PracticallyidenticalstainingpatternswereobtainedwiththecorrespondingtextiledyeDiamondredW.AsampleofalizarinredSboughtinthecourseofthisstudystainedcalciumdepositsyellowishorange; all othertissuestructureswerecoloredbrightyellow(stainingtime5mm).TwofurthersamplesfromdifferentbatchesofalizarinredSyieldedsimilarstainingpatterns.Notraceofthereddyecouldbefoundinsectionsstainedfor1hrinthethreedyesamplescontainingyellowimpurities;tissueswerecoloreduniformlyyellow.Withalldyesamplestested,diffusionartifactsaroundcalciumdepositswerealreadysevereinsectionsstainedfor5 mm. Individualgranules, e.g., inmusclefibersorrenalepithelialcells,were notrecognizable;thecellswerecoloreduniformlyorange-red. AlizarinredSinbuffersolutions,pH4.8: Thestainingpatternsobtainedwiththesesolu-tionswereverysimilartothoseproducedbyunbufferedaqueoussolutions.PaperchromatogramsoftheoldbatchofalizarinredSandofDiamondredWwereverysimilar.Thedyemovedinawelldefinedbandwhichwasframedbytracesofbluishpinkmaterial.TheotherthreedyesamplescontainedlessalizarinredSandshowedsignificantamountsofyellowimpuritieswhichmovedmoreslowlythanthedye;thedyebandwasnothomogeneousbutincludedabrownishsubstance.ThepHvaluesof0.1%aqueoussolutionsofthesedyeswere:oldbatchofalizarinredS,3.5;DiamondredW,3.7;otherbatchesofalizarinred5,2.7,2.9and2.9;thustheimpuritieswereapparentlymoreacidthanalizarinredS.McGee-Russell’sprocedure:Underthecondi-tionsofMcGee-Russell’sprocedure(32)alldyesamplesstainedcalciumdepositsdeeporange.Intensityandhueofbackgroundcolorationvariedwiththeamountofimpuritiesinthedyesamples,asdescribedabovefor0.1%aqueoussolutionsofthesedyes.Whenstainingtimewaslimitedto1mm,diffusionoftheorangesubstancewasmoder-ate.However, small intra-orextracellulargran-ules,whichstoodoutclearlyinadjacentsectionsstainedwithalkalinesolutionsofalizarin,alizarinredSoralizarinblueS(seebelow),couldnotbeidentified;suchareaswerecoloreduniformlyorange.AlizarinredS inbuffersolutions,pH 7.2:Calciumdepositswerecoloredorange-red.Sec-tionsstainedfor 5mmshowedmoderate diffusion;granulesofcalcifiedmaterialincellsandmusclefiberswerenotdistinguishable.Allothertissuestructureswerestainedfaintlypink;bindingoftheyellowimpuritiesinthreedyebatcheswasabolished.However,someyellowcolorationpersistedinandaroundcalciumdeposits,particu-larlybetweencloselyspacedlesions.Insectionsstainedfor1hrtheintensityofcolorationofcalciumdepositsandbackgroundstainingweremoderatelyincreased,anddiffusionaroundcalcifiedareaswasmoremarked. AlizarinredS inbuffersolutions,pH9:In sectionsstainedfor5mmcalciumdepositswerestainedredwithoutayellowtinge;allotherstructureswereunstainedorwerecoloredfaintlypink.Evensmallgranulesofcalcifiedmaterialstoodoutclearlyagainstthepracticallycolorlessbackground.Notraceofyellowcouldbefoundin  at CHRISTIAN UNIV on December 18, 2013 jhc.sagepub.comDownloaded from 
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