Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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 Experimental Pathology Research Laboratory Division of Advanced Research Technologies 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 Experimental Pathology Research Laboratory 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.