Journal of Histochemistry & Cytochemistry
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
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110
THE
JOURNALOF
HI8TOCHEMISTRY
AND
CYTOCHEMI8TRY
Copyright
©
1969byThe
HistochemicalSociety,
Inc.Vol.17,No.2
Printedin
U S A
ONTHEHISTORYANDMECHANISMOFALIZARINANDALIZARIN
REDSSTAINSFORCALCIUM2
HOLDEPUCHTLER,SUSANN.MELOAN
AND
MARYS.TERRY
DepartmentofPathology MedicalCollegeof
Georgia,EugeneTalmadgeMemorialHospital,Augusta,Georgia
ReceivedforpublicationOctober18,1968
Alizarin
(madder)hasbeenusedintextiledyeingsinceearlyantiquitycInhistologycal-cium-alizarin
or
calcium-alizarinredScompounds
are
oftenreferredtoaslakeorcom-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.
InhistologyalizarinredSandvonKossastechniquearecommonlyusedfordemonstrationofcalciumsalts.VonKossasprocedureisbelievedtovisualizephosphateandcarbonateanions,whereasalizarinredSreactswithcalciumandothercations.Bothmethodshavebeenusedfordiagnosisofcalciumdeposits.However,duringinvestigationsofearlyarterioscleroticlesionsweobservedstrikingdiscrepanciesbe-tweentheamountsofcalciumdemonstratedbyalizarinredSandbyvonKossastechnique.Forexample,insomearteriesvonKossasprocedurecoloredsegmentsoftheinternalelasticmembraneblack,yetadjacentsectionsutterlyrefusedtobindalizarinred5,and
viceversa.
Obviously,foranunderstandingoftheprocessesoccurringinthesearterioscleroticlesions,itwasessentialtoobtaininformationconcerningthehistochemicalsignificanceofthese
procedures.
Apreliminaryperusaloftheliteratureshoweddifferencesbetweenchemicalandhisto-logicconcepts;
e.g.
chemistsclassifythecalcium-alizarinoralizarinredScompoundsasasalt,whereasinhistology
and
histoehemistryithasDedicatedwithadmirationtoDr.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.ObservationsonthemechanismofvonKossasprocedurewillbepublishedsepa-rately.
MATERIALSANDMETHODS
Experimentswerecarriedoutonhumanautopsymaterial.Blocksoftissues
werefixedinabsolute
alcohol,Carnoysfluid(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
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ALIZARINANDALIZARIN
REDSSTAINSFORCALCIUM
111andin
0.5%aqueoussolutionsofBrooksbuffers,
pH4.8 7.2and9.0
wereprepared.Todetermine
possibleeffectsofthebuffer
saltsonthestainingreaction,Sorensensphosphatebufferandbarbitalbuffer,pH7.2and9,weresubstitutedforBrooksbuffer.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-hydevapororCarnoyssolutionandstainedasdescribedabovefortissuesections.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
calciumdepositsisusedinade-scriptivesensewithoutstrictchemicaldenota-tions;theseareasmayormaynotcontainothercationsinadditiontocalcium.
RESULTS
Effectoffixatives:Comparisonofsectionsfromthesameorganfixedinthesolutionslistedaboveandstainedunderidenticalconditionsshowedstrikingdifferences.Colorationofcalciumdepositswasmostintenseinalcohol-andCarnoy-fixedsections;nodifferencewasobservedbetweenthesetwofixatives.Informalin-andZenker-
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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-Russellsprocedure:Underthecondi-tionsofMcGee-Russellsprocedure(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
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