Download recommendations for tissue preparation and formalin or

Experimental Pathology Research Laboratory
Division of Advanced Research Technologies
C. Loomis, M. Alu-Protocol: 12-16-2016
DetailedDiscussionofTissuePrepandFormalin/FormaldehydeFixationRecommendations:
1. Timetofixationiscritical.Thequickerthebetter,asstressresponsesareinitiatedalmostimmediatelyafterthe
tissueisdeprivedofitsbloodsupply.Ideally,thetimebetweendeathandinitiationoffixationshouldbelessthan20
minutes,andthisshouldberecorded.Keepingtissuesnear(4o)priortofixationslowsdownbiologicalprocesses
inducedbyoxygenandnutrientdeprivation.Priortofixation,tissuesshouldbemaintainedinaneutral,isotonic
solutionwithphysiologicCaandMgconcentrations(e.g.PBSplusCa&Mg)toavoidunnecessarydisruptionof
adhesionsystemsandactivationofsignalingcascades.
2. Typeoffixative:paraffinembeddedtissuesaremostoftenfixedineither10%(v/v)neutralbufferedformalin(NBF)
orfresh4%(w/v)formaldehydesolution(“PFA”)madefromparaformaldehydepower.Itisimportanttorealizethat
theconcentrationsofformaldehydeinboth10%(v/v)NBFand4%(w/v)PFAarealmostidentical.Confusionarises
becausetheconcentrationofformaldehydeinNBFisgivenasapercentvolumeperfinalvolume(v/v)whereasthe
concentrationofformaldehydeinPFAisgivenasapercentweightperfinalvolume(w/v).Theworkingsolutionof
commerciallyavailableNBFisa1:10dilutionofa37%(w/v)formalinstock.Thisresultsina10%volume/volume,
whichisalmostequivalentto3.7%weightpervolumeformaldehydeconcentration–similartothe4%
(weight/volume)PFAmadefrompowderparaformaldehyde.Thereare,however,differencesbetweenthesetwo
relatedfixativesolutions,whichcanimpactontheultimatechoice.(SeediscussionunderFixativeOptions).
3. Modesofformaldehydefixation(immersionandcardiacperfusion).Immersionfixationistheeasiestandmost
commonmodeoffixation.Humansamplesareexclusivelyimmersionfixed.Foranimaltissues,cardiacperfusionis
advantageousinsomecasesbecausefixationinitiatesmorerapidlythanimmersionperfusion,butonlyifperformed
byaskilledindividualwhoknowshowtoevaluatewhetherornottheprocedureiseffective.Perfusionismore
effectivebecausethefixativeisdelivereddirectlytotissuecapillarybeds,andsinceallcellsarewithinafewcell
diametersofacapillary,theyareawashinfixativewithinsecondstominutes.Toensurecompleteness,mostperfused
tissuesarealsosubjectedtoimmersionfixationforsomeperiod.PERFUSIONISSTRONGLYRECOMMENDEDFOR
ADEQUATEFIXATIONOFRODENTBRAINS,unlessthebrainisslicedintosmallpieces(bread-loafed)immediatelyafter
dissection.Perfusionisunnecessaryandimpracticalinmanyotherinstances.
4. Volumeoffixativeshouldbe10-20timesthevolumeofthetissuesforimmersionfixation.Inadequatevolumeisa
commonmistakeandleadstopoorandvariablefixation.
5. Sizeoftissues:samplesmustbelessthan0.5cm(5mm)inonedimensionforadequateimmersionfixation.Some
recommendnomorethan2-3mm,especiallyfordensetissues.Formadehydepenetratestissuesatarateof1
mm/hr,atbest.Iftissuesamplesarethickerthan4mm,thenfixationinthecenterwillnotinitiateuntilfewhours
afterdissection.Bythistime,thecentralcellswillbequitehypoxicandpossiblynecrotic.Therecommendedoverall
dimensions:1.5x1.5x0.4cm.
6. Timeoffixation:6-18hrsforbiopsyspecimensand24-72hrsforstandardsamples.Bothunderandoverfixation
alterthequalityofmolecularanalytes.Arecentreportrecommendsthattissuenotbefixedformorethan36hrsto
avoidoverfixationandexcessivecross-linking.Wehavealsofoundthatextendedfixationcancauseexcessive
brittlenessofparaffin-embeddedmousetissues,whichareinherently“dryer”thanhumantissues.Afterfixation,
tissuescanbedehydratedfirstin50%EtOHandthenin70%EtOHandsubsequentlystoredforseveraldays(atleast)
beforeprocessingintoparaffin.
7. Temperatureoffixation:isimportant,butcontroversial.Metabolicartifacts–inducedbytheabruptcessationof
oxygenandnutrientdeliveryaswellaswasteremovalwhenthetissueisremovedfromitsbloodsupply–canbe
minimizedbyslowingdowncellularmetabolicprocessesand/oracceleratingtherateoffixation.Toslowdown
metabolicprocessespriortocompletefixation,manyresearchersfixat4o.Clinicallabs,ontheotherhand,typicallyfix
atroomtemperatureinpartbecauseofwork-flowconsiderationsandbecauseoftheincreasedrateoffixation.
Somegroupsrecommendusingaspecialmicrowaveprocesstosimultaneouslyfixthetissueandinactivate
endogenousphosphatases,proteasesandnucleases.Fortissuesdestinedforparaffinprocessing,wecurrently
Experimental Pathology Research Laboratory
Division of Advanced Research Technologies
C. Loomis, M. Alu-Protocol: 12-16-2016
recommendfixationfor24-48hrsatroomtemperatureor48-72hrs–butthisassumesthethicknessofthetissueis
4mmorlessandthevolumeoffixativeisat10Xthevolumeofthetissue.
8. Werecommendgentlenutationorslowshakingtoreplenishfreshfixativearoundthetissue.Exhaustionof
surroundingfixativeisparticularlyproblematicwhentissuesareatthebottomofconicaltubes.Avoidbigbubbles,as
shearforcesattheair-surfaceinterfacewillshredthetissueedges.
Typesoffixatives:
Therearetwobasicclassesoffixative:cross-linkingfixativesandcoagulantfixatives.Theybothhaveadvantagesand
disadvantages–andeachproducesitsownsetofartifacts.
Cross-linkingfixatives:Formaldehyde-basedfixativesarethemostcommontypeofcross-linkingfixativeused,and
theyworkbycovalentlycouplingmoleculestoeachotherandcreatingastablemeshworkwithintissues.Twosimilar
thoughnotidenticalformaldehydefixativesareneutralbufferedformalinandfreshlypreparedparaformaldehyde.(See
Freshlypreparedparaformaldehydevsneutralbufferedformalinfixatives).Glutaraldehydeisanothercross-linking
fixative.ThelatterisusedforEM,butrarelyforstandardparaffin-embeddedtissues,asitmakesthetissuesverybrittle
andismorelikelytoproduceautofluorescenceifdoingIF.Thelatterproblemcanbeavoidedifthefreealdehydesare
reducedusingsodiumborohydrideorsimilarreagent.Themajordisadvantageofcross-linkingfixativesisthattheycan
maskepitopesrecognizedbycertainantibodies–eitherbychemicallymodifyingacriticalaminoacidorbyblocking
antibodyaccesstotheantigenbecauseofthedensecross-linkedmeshwork“cage”thatisgenerated.Manyofthelatter
epitopescanbere-exposedafterantigenretrieval,althoughthismaycauseunwantedsecondarytissuedamage.The
latterisespeciallyproblematicforcryosections.
Coagulantfixatives:organicsolventsaretypicalcomponentsofcoagulantfixatives.Theyfunctionbyprecipitatingand
oftendenaturingproteinsinsitu.Examplesincludeethanol,methanol,acetoneoracombination.Organicsolvent
fixativesworkwellwhenimmunostainingcytoskeletalproteinsandothercomponentsofinsolublelarge
macromolecularcomplexes.However,lipidsandmanylipid-modifiedproteinsareextracted,andmanyaqueous-soluble
cytoplasmicproteinsarelostduringsubsequentwashsteps.Organicsolventscausemuchgreatertissueshrinkagethan
cross-linkingfixativesandusuallydestroytheintegrityofcellorganelles.Ontheotherhand,theypenetratetissues
rapidly,initiatingthefixationprocessfasterthanformaldehydefixatives.Also,becausetheydestroycellmembranes,
theypermeablizecellsatthesametimetheyfixthecells.Iftheantigensarenotextracted,epitopesmaybebetter
preservedthanwithcross-linkingfixatives.
Somecomplexfixativescontainmultiplecomponents,includinganorganicsolvent,anacidandacross-linkingfixative
(e.g.Bouin’sfixative).
Formaldehydebasedfixatives:
Neutralbuffered(NB)formalinvsfreshlypreparedparaformaldehyde(PFA):
Forparaffinembeddedtissues,neutralbufferedformalin(NBF)isthemostcommonlyusedfixative.(Note,itmustbe
bufferedtoavoidprecipitatesandotherartifacts).NBFisconvenienttostore(liquidatroomtemperature)andcanbe
orderedasaworkingsolutionandthereforerequiresnopriorpreparation.Inadditiontoitsconvenience,somepreferit
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C. Loomis, M. Alu-Protocol: 12-16-2016
becausethemethanoladditivepenetratesthetissuefasterthanformaldehyde,thisinitiatingfixationmorequicklythan
withpureformaldehydesolutionsfreshlypreparedfromparaformaldehydepower.
“Paraformaldehyde”(PFA)ispreferredforcertainprotocolsbecausemostcommercialNBformalinsolutionscontain
methanolasanadditiveinordertopreventformaldehydepolymerizationovertime.SincePFAiseitherfreshlyprepared
fromparaformaldehydepowderorfreshlydilutedfromafrozenstockorstockstoredunderoxygen-freegas,this
polymerizationprocessisnotanissue.(Note,althoughthesolutionistypicallycalled“Paraformaldehyde”,itisreallya
formaldehydesolution.)Moreover,NBFdegradeswithage,withtheproductionofformicacid,andthisissueisalso
avoidedwithfreshlypreparedPFAsolutions.PFAistheformaldehydefixativeofchoicefor:1)cardiacperfusions;2)
shortfixationforcryo-embeddedtissueswhereantigenretrievalneedstobeavoided;3)insituhybridizationsand4)
TEM,asanalternativetoglutaraldehyde.SomeresearchersalsopreferPFAforimmersionfixationoftissuesdestined
forparaffinembedding,iftheyareperformingsubsequentimmunostaining.BecausePFAlacksadditivesandis
preparedfreshlyeachtime,officinadosfeeltheycanmorerigorouslystandardizetheextentofcross-linking,facilitating
consistencyofsubsequentantigenretrievalprotocols.Note,ithasrecentlybecomepossibletobuyMeOH-free
formaldehydesingle-useampulestocks,whichiseasierandsafertodilutethanmakingupsolutionsfrom
paraformaldehydepowder.
RecipesforpreparingPFA-basedfixatives:
20%(wt/vol)Paraformaldehyde(PFA)StockinPBS(plusCa++andMg++)
• Mixandheattonogreaterthan60oC.Itwilltakesometimeforthepolymerizedparaformaldehydetobreak
downtoformaldehydeandgointosolution.
• CheckpHtomakesureapprox7.4-7.6.UsepHpaperNOTpHmeter.DoNOTstickpaperintothesolution–
putdroponpaper.AdjustpHasnecessary.
• Filterifparticlesremain.
• Store10mlin50mlconicaltubesat-20oC.Note:ifpossiblestoreinexplosion-prooffreezer.
• Whenneeded,freshlythawandresuspendinPBSplusCa++&Mg++(50mltotal).Finalworkingsolution:4%
w/v.Heatto37oCifnecessarytogetallPFAintosolution.
• Useforfixationafterchillingonice.
• FreshlypreparedPFAisideallyusedthesameday.Ifnecessarystoreat4oandusewithin24-48hrs.Canalso
storelongeranduseasafinalpost-fixstep,afterimmunohistochemistryorRNAinsitustepsarecomplete.
• IfPFAistobeusedtofixtissuesforanydownstreamRNA-dependentanalyses(e.g.insituhybridizations),
thenallaboveproceduresshouldutilizesolutions,chemicalsandcontainersthatareRNasefree.
Alternativefixativewithglutaraldehyde(goodforsubsequentβ-galhistochemicalstaining):
Alt Fixative:
Fixative:
1% formaldehyde
0.2% glutaraldehyde
2mM MgCl2
5mM EGTA
Recipe (20 ml)
0.540 ml 37% stock
0.160 ml 25% stock
0.040 ml 1M stock
0.200 ml 0.5M stock
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Division of Advanced Research Technologies
0.02% NP-40 or equivalent
C. Loomis, M. Alu-Protocol: 12-16-2016
0.200 ml 2% stock
Volume to 20 ml with PBS
Reagents:
From Sigma
Addfresh:
a. Glutaraldehyde,0.2%v/vfinalconcentration(1:800dilutionofa25%stock—concentrationinmost
commercialglutaraldehydesolutions).
b. Formalin,1.5%v/vfinalconcentration(1:25dilutionofa37%stock–concentrationofcommercial
formaldehydesolutions).
2. Fixtissuefor1-2hrs(preferablyonice).
3. Wash3xinPBStoremovefixative.
Notesonfixationandfixatives:
Note,ifwe(Loomislab)wanttokeepourtissuesforalongtimeafterfixation,wewillkeepinPBSat4owithadropor
twoof4%PFAtopreventbacterialovergrowth.Thismethodisnotpublished,butwehavenotobservedproblemswith
epitopessensitivetocross-linkingfixativesnorbacterial/fungalovergrowth.
Althoughexcellentwhenstainingforβ-galactivity,glutaraldehyde-containingfixativesoftenresultinautofluorescence
duetothemanyresidualfreealdehydessoitshouldnotbeusedwhenplanningtodoIFunlessyoufirstreducewith
sodiumborohydrideorequivalent.Glutaraldehydealsomakestissuealittlemorebrittleanddistortsmorphology.