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Preprint:Pleasenotethatthisarticlehasnotcompletedpeerreview.
Brainandbloodextractionforimmunostaining,protein,andRNAmeasurementsafterlong-termtwophotonimaginginmice.
CURRENTSTATUS:POSTED
NancyE.Ruiz-UribeCornellUniversity
OliverBrackoCornellUniversity
DOI:10.21203/rs.3.pex-838/v1
SUBJECTAREASBiologicaltechniques Neuroscience
KEYWORDSNeuroscience,cellbiology,immunofluorescence,proteinextraction,RNAextraction,mice.
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Abstract
Thisprotocoldescribesamethodtoextractbraintissueandwholebloodsamplesto
performimmunostaining,proteinextractionforELISA,westernblot,orRNAextractionfor
qPCRafterlong-terminvivoimaging.Thisprotocolisinparticularusefultoprocessand
maintainvaluabletissuesamples,allowingforabroadspectrumofanalysisand
techniqueswithoutcompromisingthequalityofthesamples.
Introduction
Withtheincreasingdevelopmentoftechnologiestostudyneuroscienceand
neurophysiologyinvivo,anincreasingdemandforsurgicalpreparationstogainoptical
accesstothebraininrodentshasemerged.Cranialwindowsurgeriesonrodentshave
becomeastandardprocedureforinvivoimagingofthebrain,forexample,tomeasure
neuronalactivity,bloodflow,andcellulardynamics.However,suchinvivostudiesoften
requireexvivoanalysisoftissuetofurthercorroboratehypothesisandbiological
mechanisms.Theamountoftissueavailablefortheseexperimentsmaybelimited,andit
isoftenimportanttogetthedatafromthesameanimalsusedforimaging.Here,we
illustrateaprotocolthatcanbeusedtotakeadvantageofbraintissueandbloodofmice
thathavebeensubjectedtoacranialwindowandthathavegonethroughlong-termin
vivoimaging.Ourprotocolcanbereplicatedindifferentmousemodelsandresultsinhigh
proteinandhighRNAyield,aswellashighantigenbindingforimmunostaining.This
allowsfurtherexplorationofbiologicalmechanismsinvolvedinthebiologicalprocesses
thatwerestudiedwithinvivoimaging.
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Reagents
-Experimentalanimals(C57BL/6Jormousemodelofpreference)
CAUTION:Allanimalexperimentsshouldbehandledunderaninstitutionalmouseprotocol.
OurexperimentswereconductedunderproceduresapprovedbytheCornellUniversity’s
InstitutionalAnimalCareandUseCommittee(IACUC),protocolnumber2015-0029.
-Pentobarbital(10mg/kg)
-4%Paraformaldehyde(PFA,Sigma-Aldrich,cat.no.P6148)
CAUTION:PFAiscarcinogenicandmustbealwayshandledwithglovesandproper
protection
-1XPhosphatebufferedsaline(PBS)
-30%sucrosein1xPBS
-10%TritonX-100(MilliporeSigma)
-Goatserum(MilliporeSigma)
-Primaryantibodies:Anti-chickenGFAP(Rockland),Anti-rabbitIba1(Wakochemicals)
-Secondaryantibodies:AlexaFluor488anti-chicken(ThermoFisher),AlexaFluor594anti-
rabbit(ThermoFisher)
-Methoxy-X04(TOCRISBioscience)
-Razorblades
-Liquidnitrogen
-80%ethanol
-Proteininhibitors(AESBF)(MilliporeSigma)
-cOmplete(MilliporeSigma)
-Cryoprotectionsolution
-RNAzap(ThermoFisher)
-TrizolLS(ThermoFisher)
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-Trizol(ThermoFisher)
-Blockingmarker(Part#6505,NewcomerSupply)
-Prolonggoldmountingmedia(ThermoFisher;P10144)
Equipment
-Eppendorfs
-Coverglass
-Onesidedcoatedglassslides(ElectronMicroscopySciences;71863-01)
-Smallsizedbrushes
-Douncehomogenizer(Sigma-Aldrich;D9063)
-Petridishes(ThermoFisher;FB0875712)
-Distilledwater
-Curvedforceps(FineScienceTools;11271-30)
-Finehemostats(FineScienceTools;13007-12)
-Finescissors(FineScienceTools;14160-10)
-3mLsyringes
-22-25Gneedles(BDBiosciences)
-Polystyrenetubes
-EDTAtubes(BDVacutainer®EDTAtubes)
-Stainlesssteelmousebrainmatrix(Harvardapparatus;72-5032)
-Perfusionpump(Thermofisher)
-Rectangularbucketandgratetoholdthemouse
-Cryotome
-Centrifuge
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-Sonicator
-Confocalmicroscopy(ZeissLSM710Confocal)
-Pipettes
Procedure
STEP1:Euthanasia
Whenmicehavereachedtheirendpoint,euthanizethemwithalethalinjectionof
pentobarbital(i.p.10mg/kg).
STEP2:Extractionofblood
Theextractionofbloodisperformedviacardiacpuncture.Scruffthemousefromtheback
withthebodyhangingstraightvertically.Inserta22-25Gneedlewitha3mLsyringejust
undertheribsatthebodycenterline.Theneedleshouldhittheheartapproximatelyat
theleveloftheelbow(Figure1).Gently,withdrawthesyringeplungeruntilbloodstartsto
comeout.Ifblooddoesn’tcomeout,movethesyringeslightlyupwardsordownwards
untilbloodisreadilywithdrawn.Avolumeof1-2mLcanbeextractedwiththistechnique.
Donotapplyadditionalbackpressureuntilthebloodhasfilledthesyringe1,2.
CRITICALSTEP:propermouserestrainisimportanttoensuresuccessfulbloodcollection.
Thisstepneedstobedoneasfastaspossibletoensurethatvesselswon’tcollapsebefore
thetranscardialperfusion.
CAUTION:applyingtoomuchbackpressurecancollapsetheheartmusclethusinterfering
withthebloodextraction.
RapidlytransferthebloodtoavacutainerbloodcollectiontubewithEDTA.Ifthebloodwill
beusedforRNAextraction,applythreepartsofTrizolLSperonepartofblood,and
processimmediatelyorsnapfreezeforlaterprocessing.Ifthebloodwillsitformorethan
aminuteinthesyringebeforetransferringtoacontainerwithEDTA,thenitmaybe
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advisabletoaddsomeEDTAtothesyringetocoattheinside.
STOPPOINT:samplescanbestoredat-80ºCuntilfurtherprocessingorcanbeprocessed
fresh.ProceedtoextractRNAwithapreferredmethod.
STEP3:TranscardialPerfusion
Placethemouseonitsbackonagratesittingontopoftherectangularbucketandtape
theforelimbsandhindlimbsfirmlytothegrate.Performacutfrombelowtheribstothe
upperchestinordertoexposethechestcavity.Attachhemostatstotheribsandretract
inordertofullyexposetheheart.Inserta21Gneedleattachedtoaperfusionpump
containing1XPBSintotheleftventricle,andthenperformasmallcutintherightatrium.
Starttopump1XPBStothemouse,untilthefluidrunsclearoutofthecutintheright
atrium.Theliverchangingfromadeepredtoapalershadeisanindicatorofagood
perfusion.
CRITICALSTEP:furtherperfusionwith4%PFAshouldbeavoidedashalfofthebrainwill
beusedforproteinextractionandimmunostainingresultshaveshownsignificant
improvementwhenskippingthisstep.
STEP4:Extractionofbrainforimmunostainingandprotein/RNA
Afterperfusionwith1XPBS,thebrainthroughthecranialwindowshouldlookpale.
Carefullyremovethecranialwindowwithforceps.Decapitatewithscissors,thencutthe
musclesoftheneckandtheskinthatcoversthecranium.Insertthescissorsthroughthe
foramenmagnum,andgentlystartcuttingthecranialbone,uptotherostralside.
CAUTION:Takecaretonotdamagethebrainduringthisprocess.
Withforceps,removethelateralboneflapsandexposethebraincompletely.Slidecurved
narrowforcepsunderthebrainandcarefullytiltitupward.Whenthebrainissufficiently
loose,slideitoutofthecraniumwiththehelpoftheforcepsandaddtoacleanpetridish
sittinginanicebathandwashthebrainwithicecold1XPBStoremoveallhairsorother
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debris.Withacleanrazorblade,sectionthebraininhalfalongthesagittalaxis.Withthe
helpofcurvedforceps,carefullyseparatethecortexfromthehippocampus(formore
detailssee5)ordissectotherbrainregionsaredesired.
Immerseonehalf(forimmunostaining)inatubecontaining4%PFAandfixovernightat
4°C.After24hours,transferthebraintoasolutioncontaining30%sucrose.Theother
halforremainingbrainsectionscanbesnapfreezeinliquidnitrogenorprocessed
immediatelyforRNAorproteinextraction.
STOPPOINT:Ifsamplescannotbeimmediatelyprocessed,werecommendhomogenizing
thetissueimmediatelyusingaDouncehomogenizer(orothermethods,suchasbeadmill)
intheappropriatebuffer,makingsuretoaddRNAaseorproteaseinhibitorsdependingon
need,andthensnapfreezeandstoreat-80ºC.
STEP5:Samplepreparation
ForRNAanalysis,prepareatubecontainingTrizoloralysisbufferwithanRNAase
inhibitorofpreference3.
Forproteinextraction,prepareextractionbufferina50mLconicaltubecontaining1mM
AESBF(orotherappropriateproteinaseinhibitors),1tabletofcOmpleteand2mLofRIPA
bufferanddilute1:25with1XPBS.
PlacehalfbraininsideacleanDouncehomogenizerandhomogenizethebrainwith500uL
ofextractionbuffer.Usesmallstrokestoavoidbubbles.Atotalof20-30strokesshouldbe
enoughtohomogenizethetissue.Withapipette,transferthehomogenizedtissuetoan
Eppendorfandkeeponice.WashthehomogenizerwithMiliQwaterbetweensamples.
Sonicatetubesonwaterfor10minutes.Transfersamplestoa4ºCcentrifuge,and
centrifugeat14000rpm(tabletopcentrifuge)for30min.Transferthesupernatanttoa
newtubeandifdesired,thepelletcanbestoredforfurtherprocessing.Ifextracting
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proteinorRNAfromcerebralmicrovesselsisofinterest,followthisdetailedprotocolon
extractingcerebralmicrovesselsfromcortex4.
Aftersamplepreparation:
·Proceedwithpreferredprotocolforproteinmeasurement(Figure2).WeusePierce
BCAProteinAssayKit(ThermoFisherCATNo.23225)
·IfperformingRNAextraction,processsamplesasquicklyaspossibleandcleanall
surfaceswithRNAzap(ThermoFisher,CATNo.AM9780).Proceedwithpreferredprotocol
forRNAextraction.(Figure3)
CRITICALSTEP:Theextractionandsectioningofthebrainhastobeperformedasquickly
aspossibleinordertoensuretheleastamountofRNAorproteindegradation.
STEP5:Brainprocessingandslicingforimmunostaining
Thehalfbrainstoredinsucrosewillbeusedforimmunostaining.Extractthebrainfrom
thesucrosesolutionandaddtoapetridish.Placethebraininacoronalorsagittal
positioninsidethemetalliccryotomeholderwithOCTcompoundasanadhesive.Let
freezeinsidethecryotome.
CRITICALSTEP:thebrainshouldbeorientedstraightandparalleltothebladeinorderto
ensurecorrectslicing.
Ifsectionsareusedasserialsectionspreparesixtubesforahalfbrain,oriftheywillbe
storedasregionsofinterestlabelthetubesaccordinglyandfillthemupwith1.7mLof
cryoprotectionsolution.Preparecryoprotectionsolutionbymixing150gofsucrose,200
mLof1XPBS,150mLofethyleneglycol,andcompletetoafinalvolumeof500mLwith
1XPBS.Storeat4ºC.
Positionthemetallicholderintothecryotome.StarttrimmingtheOCTadhesiveuntilthe
brainappears.Startslicingthebrainwithathicknessof30µm,orasdesired.Selecteach
slice,submergeinPBSandalternatethestorageofeachsliceintodifferenttubes.This
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willensurethatarepresentationofthewholebrainisstoredoneachtube.
STOPPOINT:Slicescanbestoredat4ºCforaslongasneededbeforemounting.
STEP6:Immunostainingforastrocytes,microgliaandamyloidplaques
Takeoneofthetubesstoredat4ºCandplacethebrainslicesonapetridishwith1XPBS.
Mounttheslicesonaglassslidewithabrush.Atotalof3-6slicescanbemountedona
glassslide,dependingonthesizeoftheslices.Ensurethattheslicesarecompletely
straightandnotfolded.Makesuretoleaveenoughspacesintheedgesfortheliquid
blockermarker.Allowtothoroughlydry.
Aftersliceshavedried,drawarectanglewithliquidblockingmarkertopreventleakageof
solutions.Washslices6timeswithPBSfor5minutes.Prepareablockingsolutionby
adding250µLofgoatserum,50µLof10%TritonXandcompletetoa5mLvolumeusing
1XPBS.Blockslidesfor30minwiththeblockingsolution.Aspiratetheblockingsolution
andapplyprimaryantibody(1:500dilution,ortheappropriatedilutionfortheantibody,in
blockingsolution).Letincubateovernightat4ºC(orasappropriatefortheantibody)with
awetpapertoweltoavoiddesiccation.
Thefollowingdayaspiratetheprimaryantibody.Washslidesthreetimeswithfor5min
with1XPBS.
CRITICALSTEP:oncetheslicesarethoroughlyadheredafterthefirstdryingstep,donot
allowslidestocompletelydryagainbecausethiscancompromisetheintegrityofthe
tissueandthequalityofthestaining.Makesuretocoverslidescompletelywiththe
solution.
Applysecondaryantibody(1:500dilution,orasappropriate,inblockingsolution)and
incubatefor2hoursatroomtemperature(orasappropriate)inalightshieldedareawith
awetpapertoweltoavoiddesiccation.
Aspiratethesecondaryantibodyandwashthreetimeswith1XPBS.Thiswouldbean
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appropriatetimetoapplyDAPIorotherwater-solublecounterstains.Forexample,ifthis
wasanAlzheimer’sdiseasemousemodelandlabelingofamyloidplaqueswasdesired,
applyMethoxy-X04diluted1:250in1XPBSandincubateatroomtemperaturefor15
minutes.Wash2timeswith80%ethanol.Then,allowslidestothoroughlydrybefore
mounting.Useamountingmediumofpreference(werecommendProlongGold).Our
imageswereacquiredwithaZeissLSM710Confocal,usingfluorescently-labeled
secondaryantibodies(Figure4).
Troubleshooting
·Thesyringewon’tfillupwithblood:Don’tapplyfurtherbackpressuretothesyringe.
Movetheneedleupanddownuntilbloodcomesout.Itispossibletotrytheneedle
insertionwiththemouseinapositionofdorsalrecumbency,withthemouselayingtoon
onesideandtheneedleinsertedparalleltothetable,perpendiculartothechestwall,and
betweentheribs.Applybackpressureuntilbloodcomesout.
·Nosignalfromimmunostaining:Makesurethatslicesdonotdryoutduringthe
stainingprocess.Slicesshoulddryonlytwice,afterplacingontheslidesandbefore
mountingwithamountingmedium.Itmayalsobenecessarytoapplyantigenretrieval
protocolssuchasincubationwithformicacidorheat.
·RNAqualityisbadorthereisnoRNAatallfromtheextraction:Makesuretoworkina
cleanenvironment.SprayallsurfacesandpipetteswithRNAzap.UseRNAse-freetubes
andwaterandchangeglovesbetweenpreparations.DependingontheRNAextraction
protocolofpreference,itispossibletoaddß-mercaptoethanoltosamplestohelpavoid
fastRNAdegradation.
TimeTaken
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STEP1:2minutes(permice)
STEP2:3minutes(permice)
STEP3:5minutes(permice)
STEP4:brainextraction:2-3minutes.Dissection:2-3minutes.(permice)
STEP5:twodays
STEP6:4-5hours,dependingonthenumberofbrains.
References
1Dyer,S.M.&Cervasio,E.L.Anoverviewofrestraintandbloodcollection
techniquesinexoticpetpractice.VetClinNorthAmExotAnimPract11,423-443,v,
doi:10.1016/j.cvex.2008.03.008(2008).
2Diehl,K.H.etal.Agoodpracticeguidetotheadministrationofsubstancesand
removalofblood,includingroutesandvolumes.JApplToxicol21,15-23,
doi:10.1002/jat.727(2001).
3Shatzkes,K.,Teferedegne,B.&Murata,H.Asimple,inexpensivemethodfor
preparingcelllysatessuitablefordownstreamreversetranscriptionquantitativePCR.Sci
Rep4,4659,doi:10.1038/srep04659(2014).
4Lee,Y.K.,Uchida,H.,Smith,H.,Ito,A.&Sanchez,T.Theisolationandmolecular
characterizationofcerebralmicrovessels.NatProtoc14,3059-3081,doi:10.1038/s41596-
019-0212-0(2019).
5.Spijker,Sabine.“DissectionofRodentBrainRegions.”InNeuroproteomics,edited
byKaWanLi,57:13–26.Totowa,NJ:HumanaPress,2011.https://doi.org/10.1007/978-1-
61779-111-6_2.
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Acknowledgements
WeacknowledgetheCornellBRCImagingFacilityandgrantNIHS10RR025502forthe
ZeissLSM710Confocal.WethankChrisB.Schafferforthoroughlyreadingandeditingthe
manuscript.
Figures
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Figure1
Figure1:Cardiacpunctureofmice.Theredarrowpointstotheribcage.The
needlemustbeinsertedbetweentheribs,paralleltothefrontalaxisofthe
mouse.Theheartshouldbeattheleveloftheelbow.
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Figure2
Figure2:RNAisolatedfromwholemousebloodwiththisprotocolhadhighRNA
integrity.RNAintegritywasdeterminedusingaBioanalyzer.RINnumberinthis
examplewasdeterminedbythe28S/18SribosomalRNAratioandreached8.9
indicatingagoodRNAqualityforRT-PCRorotherapplications.
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Figure3
Figure3:Levelsofhumanamyloid-beta40and42fromold5XFAD(amouse
modelforAlzheimer’sDisease)andWTmiceanalyzedwithourproteinextraction
protocolviaELISA(HumanA�40andHumanA�42MouseElisaKit–Thermofisher
CATno.KHB3481andKHB3441,respectively)
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Figure4
Figure4.Stainingofbrainhippocampusfrom5XFADmiceformicroglia(red-
IBA1),astrocytes(green-GFAP)andplaques(blue-Methoxy-X04).
HighfatdietworsenspathologyandimpairmentinanAlzheimer’smousemodel,butnotbysynergisticallydecreasingcerebralbloodflowbyOliverBracko,LindsayK.Vinarcsik,JeanC.CruzHernández,+13
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