Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis.

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by  Goldschlager, Tony

JournalofVisualizedExperiments www.jove.com
VideoArticle
Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome
Analysis
Tony Goldschlager1,Amany Abdelkader1,Jeffrey Kerr2,Ian Boundy2,Graham Jenkin1
1MonashImmunologyandStemCellLaboratories,MonashUniversity
2AnatomyandDevelopmentalBiology,MonashUniversity
Correspondenceto:[email protected]
URL:http://www.jove.com/details.php?id=1707
DOI:10.3791/1707
Citation:Goldschlager T.,Abdelkader A.,Kerr J.,Boundy I.,Jenkin G.(2010).UndecalcifiedBonePreparationforHistology,HistomorphometryandFluorochromeAnalysis.JoVE.35.
http://www.jove.com/details.php?id=1707,doi:10.3791/1707
Abstract
Undecalcifiedbonehistologydemonstratesthemicro-architectureofbone.Itshowsboththemineralisedandcellularcomponentsofbone,which
providesvitalinformationonboneturnoverorboneformationandresorption.Thishastremendousimportanceinavarietyofclinicalandresearch
applications.Ityieldsbeautifulimages1andallowsfortechniquessuchasfluorochromeassessmentandhistomorphometry2.Fluorochrome
analysisisatechniquewherefluorescentdyesthatbindtocalciumareinjectedataparticulartimepoint,whichallowsforquantificationofthe
amountofmineralisationatthatgiventime.Histomorphometryisaprocessofbonequantificationatthemicroscopiclevel.
Performingundecalcifiedbonehistologyistechnicallychallenging,particularlywithlargesizespecimens.Itrequiresvariationsintechniquefrom
thoseusedinstandardparaffinembeddedhistology.Thisvideoillustratestheprocessofproducinggoodqualitysectionsanddemonstratesthe
technicaldifficultiesandmethodswithwhichtoovercomethem.Specimenpreparation,fixationandprocessingareachievedwithamannersimilar
toothersofttissues,howeverduetothedensityandlowerpermeabilityofboneconsiderablylongerfixationandprocessingtimesarerequired,
oftentakingseveralweeks.Embeddingisachievedusingasupportingmediumwithsimilarorequalhardnessanddensitytothebonesuchas
methacrylate-basedresins,butunlikeparaffininfiltrationandembedding,thisisanirreversiblestep.Sectioningcanbeachievedbygrindingwhich
producesathickersection,whichisoptimalforstudiessuchasfluorochromeanalysis.Thisisbestachievedusingadiamondbladeona
macrotome.Alternatively,thinnersectionscanbeproducedforlightmicroscopyandthisisachievedusingasledgemicrotomewithaverysharp
blade.Thesledgemicrotomeprovidestheadditionalstrengthandstabilityrequiredforlarge,hardblocks.Resinembeddedsectionscanbestained
withavarietyofstains,whicharedemonstrated.
Protocol
1. Thetissueisplacedinasealedcontainercontaining10%phosphatebufferedformalinsolution3.Ensureminimalexposuretolight,if
fluorochromeanalysisistobeperformed,asthefluorochromesarelightsensitive.
2. Establishwhichorientationofthetissuewillberequired.
3. Trimthespecimentosize,inthecorrectorientation,usingabandsaw.Ensuresafetymeasuresareadheredto.Thespecimenisthenplaced
intoanopaquecontainer,thevolumeofwhichshouldbeapproximatelytentimesthesizeofthespecimentoachieveadequatefixation.The
specimenshouldremainintheformalinsolutionatotalofbetweenoneandtwoweeksdependingonitssize.Itissuggestedusingcontrol
tissuefortestingandoptimisationpurposes.
4. Checkthetissuefitsintotheembeddingmouldandplacethetissueintoasealedopaquecontainercontaining70%ethanol.
5. Processthetissueinascendingconcentrationsofethanol,shieldingfromlightandunderconstantagitation.
a. 70%ethanol1week
b. 80%ethanol1week
c. 90%ethanol1week
d. 100%ethanol1week
6. Thetissueisthenclearedinbutanolfor1week,againshieldingfromlightandunderconstantagitation.
7. Thedensityoftheresinandboneshouldbecloselymatched.Theresindensitycanbealtered(seestep8)andwerecommend
embeddingatestspecimenfirsttoensurecorrectdensity.WeuseTechnovit7100(HeraeusKulzer)forlightmicroscopyandfluorochrome
analysis.IfimmunohistochemistryisrequiredanalternativeresinmustbeusedsuchasTechnovit9100(HeraeusKulzer).
8. TheTechnovit7100preparationsolutionmaybemixedwithsmallaliquotsofthesofteningsolutionprovidedwiththekit,ifrequired,buttono
morethan5%thevolumeofpreparationsolution;inourexperiencethesofteningsolutionisrarelyrequired.Equalpartsofabsoluteethanol
andbaseliquidTechnovit7100aremixedandappliedtothetissueasapre-infiltrationsolutionfor24hoursminimisinglightexposureand
withagitation.
9. Theinfiltrationsolutionisthenmadeupusing1ghardenerI(=1bag)whichisdissolvedin100mlbaseliquid.Thisreplacesthe
pre-infiltrationsolutionandisallowedtoinfiltratefor1weekunderconstantagitation.
10. Placethetissueintothemould,withthecuttingsurfacefacedown.Ensurethereisamargin(ideallyatleast5millimetres)aroundthetissue
fortheresin;thisensuresblockstrength.Mix15mlsofTechnovit7100preparationsolutionwith1mlofhardenerIIandpouraroundspecimen
inthemouldandcoveritbyatleast5millimietres.Curingwillcommencewithin2hoursbutwillbecompleteovernight.
11. MountingisthenperformedbyfashioningabackingblockusingTechnovit3040(HeraeusKulzer).MixTechnovit3040inavolumeratioof2
partspowderto1partliquidtoobtainaviscousliquid.PourTechnovit3040intotherecessatthebackoftheblocktoalevelofatleast2mm
abovethebaseoftheblock.Afterabout10minutesthemouldcanbepeeledawayandtheblockisready.
12. Thedensityandthereforethecuttingpropertiesofmethacrylateresinaresusceptibletotheambienthumidityintheroom.Theblocksshould
thereforebestoredinadessicator.
13. Groundsectioningproduceslargersectionsbetween20-50micrometresinthickness.Itisusefulforfluorochromeanalysis(seesection
below)asthickersectionsproducebrighterfluorescence.Securethediamondbladetomacrotome.Addlubricant,suchaspetroleumspiritto
thereservoironthemacrotome.Securetheblockintheclampandorientatetoblade.Allowgrindingtooccurslowly(approximately20-30
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minutespersection)ensuringadequatelubrication.Thefirstsectionexposesthecuttingfaceandorientatesthesection-itshouldbe
discarded.
14. ThenextsectionisthenplacedonaSuperfrostPlusslide.Thesectionhasatendencytocurlsowerecommendplacingaportionofa
sandwichbagoveritandthenholdingitflatwithanotherslideclampedinplace.Itisthenplacedintoanincubatorofbetween60-80degrees
Celsiusfor1hour.Thissoftensthemethacrylateandhelpsthesectionadheretotheslideinaflatmanner.
15. Thegroundsectionmaybecleanedandpolishedasrequiredusingfinegritsandpaperinagentlemanner.
16. Thesledgemicrotomecutsthinsections,whichprovidesthebestsectionsforlightmicrosocopy.Useasledgemicrotomeasithasadded
rigidity,whichisrequiredforcuttingthesehardblocks.Alternativelyapoweredrotarymicrotomecanbeused.Useasharpstainlesssteel
blade.Itispreferabletohavetwobladesavailableasonecanbesharpenedastheotherisused.Thebladeshouldmakea45degreeangle
withtheblock.Surfacesofteningcanbeachievedbyapplyingawetpapertotheblockifrequired.Itmaytakeseveralsectionstoachievethe
desiredcuttingconditionstoobtainaqualitysection.Thesectionisthenfloatedonawaterbathandplacedonaslideasin16above.
17. StainingreagentsarelistedinMaterialsbelow.ProtocolsarebasedonBancroft4etal.Duetothicknessvariationitisrecommendedto
optimisestainsontestslides.Rackstainingmaycausetissuetofloatofftheslidesoaddingthestainsdirectlytoaflatslidemayberequired.
Anidenticalprotocolorrackstainingisnecessaryforconsistentstainingoutcomesrequiredforhistomorphometry.
a. HaematoxylinandEosin
i. StainwithHaematoxyilin5-10minutes
ii. WashwellwithScottstapwater
iii. StainwithEosin5minutes
iv. Washintapwater
v. Clearinxylene
vi. Mount
vii. Results
1. Osteoid=pink
2. Calcifiedbone=purplishbrown
3. Nuclei=blue
b. VonKossa
i. Placeinsilvernitratesolutionandexposetostronglightuntilmineralisedboneturnsblack(approx10mins)
ii. Washindistilledwaterthreetimes
iii. Threatwithsodiumthiosulfatefor5minutes
iv. Washindistilledwater
v. CounterstainwithSafrininO
vi. Clearinxylene
vii. Mount
viii Results
. 1. Mineralisedbone=black
2. Osteoid=red/pink
c. MassonGoldnersTrichrome
i. Washwithalkalinealcohol(90mlsof80%ethanoland10mlsof25%ammoniafor20minutes)
ii. Rinseinwater
iii. Rinseindistilledwater
iv. StainwithWeigertsHaematoxylinfor10minutes
v. Rinseindistilledwater
vi. StainwithPonceau-Fuchsinfinalsolution5minutes
vii. Rinsewith1%aceticacid15seconds
viii Staininphosphomolybdicacid-orangeGsolution5minutes
.
ix. Rinsewith1%aceticacid15seconds
x. Stainwithlightgreen5minutes
xi. Rinsewith1%aceticacid3changes
xii. Rinseindistilledwater
xiii Mount
.
xiv Results
. 1. Mineralisedbone=green
2. Osteoid=red/orange
3. Nuclei=bluegrey
4. Cartilage=purple
18. Fluorochromepreparations:
19. Theseareadministeredbyslowintravenousinjection,atleasttwoweeksapart.
20. Doses
a. CalceinGreen10mg/kgIV
b. Oxytetracycline50mg/kgIV
c. AlizarinComplexone30mg/kgIV
21. PreparationofCalceinGreen
a. OnegramofCalceinGreenistitratedwith1MNaOHuntildissolved.
b. pHisadjustedwith1%NaOHuntilpH=7.1-7.2.
c. Filterandkeepshieldedfromthelight
22. PreparationofAlizarinComplexone
a. ThreegramsofAlizarincomplexoneistitratedwith1MNaOHuntildissolved.
b. pHisthenadjustedwith1%HClorNaOHuntilpH=7.1-7.2.
c. Filterandkeepshieldedfromthelight
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23. PreparationofOxytetracycline
a. Oxytetracyclineisavailableasanofftheshelfanti-microbialdrug.Nopreparationisrequired.
Acknowledgements
TheauthorswouldliketoacknowledgetheassistanceofMsSueConnellforherexpertiseinresinembeddingandMsStephaniaTombsforher
laboratoryexpertise.TheauthorswouldliketothankProfessorFrankKandzioraandDrMarie-AnnePolbothfortheiradvice.
References
1. Kerr,J.AtlasofFunctionalHistology,HarcourtPublishing,Melbourne,Australia.164-168(2000).
2. Parfitt,A.M.,etal.Bonehistomorphometry:standardizationofnomenclature,symbols,andunits.ReportoftheASBMRHistomorphometry
NomenclatureCommittee.JBoneMinerRes2,595-610(1987).
3. An,Y.H.&Martin,K.l.Handbookofhistologymethodsforboneandcartilage.HumanaPressInc,NewJersey,USA.222(2003).
4. Bancroft,J.D.&Gamble,M.TheoryandPracticeofHistologicalTechniques.ChurchillLivingston,Nottingham,UnitedKingdom.352-360
(2008).
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