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Preprint:Pleasenotethatthisarticlehasnotcompletedpeerreview.

Brainandbloodextractionforimmunostaining,protein,andRNAmeasurementsafterlong-termtwophotonimaginginmice.

CURRENTSTATUS:POSTED

NancyE.Ruiz-UribeCornellUniversity

OliverBrackoCornellUniversity

ob84@cornell.eduCorrespondingAuthor

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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