1

Monome r i z a t i o n o f A β p ep t i d e Materials: β‐Amyloid(1‐42)(AmericanPeptide,62‐0‐80)

[Note:ThepeptideshouldbetheTFApreparation;theHClpreparationssometimesprecipitateduringoligomerization.ItisalwaysbesttospecifyTFAwhenorderingthepeptide.Therearebatch‐to‐batchdifferencesinpeptidequality.Asmallamountofaspeci icbatchistestedforoligomerization,analyzedbySDS‐PAGEsilverstainandWesternblot,hot‐spotbindingtohippocampalneurons,and,wheneverpossible,toxicitypriortopurchasingalargerquantityforlongtermuse.]

1,1,1,3,3,3‐Hexa luoro‐2‐propanol(HFIP)(Sigma,52517)

MAXYMumRecoveryMicrotubes(Axygen,MCT‐175‐L‐C)

1. SolidAβ1‐42isdissolvedinice‐coldHFIP.

[Note:HFIPisveryvolatileandevaporatesquickly.UsingIce‐coldHFIPreducestheevaporationrate.]

2. Thepeptideisincubatedatroomtemperatureforatleast1hrtoestablishmonomerizationandrandomizationofstructure.

[Note:seeChromyetal,Biochemistry,42,12749‐12760,2003Figure2showingthemonomericstateofAβ1‐42afterHFIPtreatment.]

3. Placethemonomerizedpeptide/HFIPsolutionbackoniceforatleast1hr,toreduceHFIPevaporationduringaliquotting.

4. TheHFIPisaliquotted(typically~0.226or0.451mgpeptide/tube)intomicrotubesandallowedtoevaporatewithcapsopenovernightinahood.

5. Thetubesspunfor10mininaSavantSpeedVac.Theresultingpeptide ilmsarestoredat‐80°C.

[Note:ThepeptideshoulddissolvetoaclearsolutionwhenmonomerizinginHFIP.WehavefoundbatchesofHFIPthatdonotdissolvepeptideproperly.TheHFIP/peptideshouldbecompletelydryfollowingtheovernightevaporationinthehood.Thespeedvacstepisjustanextraprecaution.]

P r e p a r i n g Aβ o l i g ome r s ( AβO s ) Materials: β‐Amyloid(1‐42) ilm(seeabove)

Dimethylsulfoxide(DMSO),Hybri‐Max(Sigma;D2650)

Ham’sF12,withoutphenolred,withL‐glutamine(CaissonLabs,HFL05‐500)

MAXYMumRecoveryMicrotubes(Axygen,MCT‐175‐L‐C)

1. Bringatubeofpeptide ilmtoroomtemperature.UsingafreshlyopenedvialofDMSO,dissolvethepeptide ilmto~5mM(10μlDMSO/0.226mgpeptide)usingamicropipettorwithlowretentiontiptowash/scrapesidesoftube(toaboutthe0.5mllevelofthetube)tocompletelydissolve ilm.Taketimeatthissteptobesure ilmhasdissolvedcompletely;youshouldnotbeabletosee inedropletsonthesideofthetube.

[Note:Dissolvingthepeptide ilminDMSOisaveryimportantstep.Iftherearesmallbeadsofliquidonthesideofthetubethepeptideisprobablynotcompletelydissolved.]

2. AddcoldHam’sF12toa inalpeptideconcentrationof~100mM(490μlbuffer/0.226mgpeptide).Mixonvortex~10sec.Incubateat4°Covernight.

3. PrepareaDMSO/F12control(vehicle)asaboveinasteriletube.

4. Centrifugetubesat14,000gx10minat4°;thereshouldbeminimalpellet.Carefullytransfersupernatant(Aβoligomers)tosteriletube.Storeat4°C.

[Note:DonotvortexAβOsoncetheyareprepared(vortexingproducesproto ibrils/ ibrils).Gentlyrocktubeend‐to‐endtomixifnecessary.Shouldbeusedassoonaspossiblebutwithin3‐4days.Areadilyvisiblepelletfollowingthe inalcentrifugationofthepeptidepreparationusuallyindicatesaproblemwithoneofthereagents.]

[Note:Forlong‐termstorage,Aβoligomerpreparationmaybealiquottedintosingleusequantitiesandfrozenat–80°C.Aliquotscanthenbethawedoutoniceandcentrifugedat14,000xgfor10minat4°Cpriortouse.WehavefoundthatAβOpreparationsarestableforatleast6monthswhenstoredat–80°C.]

P r o t e i n d e t e rm i n a t i o n Materials: CoomassiePlusProteinAssay(Pierce)withBSAstandard.1. Dilute10ml2mg/mlBSA(Pierce)with70mlofvehicle

controltogive0.25mg/mlBSA.

2. PipetddH2O,CoomassiePlusreagent,vehiclecontroland

0.25mg/mlBSAintodisposablecuvettes(seetable).3. Covercuvetteswithpara ilmandmix.4. Readat595nm.

AβOconcentration=[mgprotein(averageforAβOsamples)¸10(μltested)]¸4514(MW).Theconcentrationisusuallybetween65and85mM.

P r e p a r i n g l ow ‐ do s e AβO s Materials: β‐Amyloid(1‐42) ilm(seeabove)

Protein  ddH2O Coomassie 

Plus Vehicle control  0.25 mg/ml BSA 

0 mg BSA  0.98 ml  1 ml  20 ml  0 ml 

1 mg BSA  0.98 ml  1 ml  16 ml  4 ml 

2 mg BSA  0.98 ml  1 ml  12 ml  8 ml 

3 mg BSA  0.98 ml  1 ml  8 ml  12 ml 

4 mg BSA  0.98 ml  1 ml  4 ml  16 ml 

5 mg BSA  0.98 ml  1 ml  0 ml  20 ml 

AβOs  0.98 ml  1 ml  10 ml  10 ml AβOs 

Dimethylsulfoxide(DMSO),Hybri‐Max(Sigma;D2650)

Neurobasalmedia,phenolredfree(Gibco12348‐017)

Incubatorsetat37°C

1. CarefullysnapopenananhydrousDMSOampule(Sigma;D2650)andtransfer1mltoeachofseveralEppendorftubes,clearlylabeled“DMSO”.Capthetubesandstoreatroomtemperatureuntiluse.

[Note:AnewtubeshouldbeusedtoprovidefreshanhydrousDMSOateachofthefollowingsteps.Donotreuseatubeonceithasbeenopened.]Aftereachpreparation,thetubesusedandtheanhydrousDMSOcontainedthereinshouldbeproperlydisposedof.

2. RemoveatubeofAβ1‐42peptide ilm(0.045mg)fromthefreezerandwarmtoroomtemperaturefor15min,

3. Add100μloffreshanhydrousDMSOtothetubeofpeptideilm.Vortexvigorouslyfor30to60seconds,spindowninthemicrofugebrie ly,thenscrapethesidesoftheEppendorftubewithasmallmetalspatulatoremove ilmadheringtotheside.Repeatvortexing,spinningdown,andscrapingthesidestwomoretimes,butomitthescrapingonthethirdcycle.Afterthe inalspindown,letstandfor15minutesatroomtemperature.Examine ilmareaforundissolvedresidue,andrepeatvortex/spindown/scrapingifnecessarytodissolveall ilm.Add400μloffreshanhydrousDMSO,bringingthe inalvolumeto500μl.Vortex30to60seconds,theninvertseveraltimestomix.Brie lyspindowninthemicrofuge.Holdatroomtemperaturefor15min.Examine ilmareaforundissolvedresidueonthesidesoftheEppendorftubebyinvertingthetube;repeatvortexing/spindownifnecessarytodissolveall ilm.ThisstepprovidesastockofDMSOsolutioncontaining20μMofAβ1‐42in100%DMSO.

5. LabelanemptyEppendorftube“10μM”andpipettein250μlofanhydrousDMSO.Add250μlofthe20μMstockDMSOsolutionfromthepreviousstep.Inverttomixandspindowninthemicrofugebrie lytoremoveliquidfromthecap.This10μMstocksolutionin100%DMSOisstablefortheentiredayandonlyneedstobemadeonce.Thetubeshouldbecappedwhennotinuse.Thissolutiondoesnotneedtobevortexed.Thisstepprovidesastockof10μMAβ1‐42in100%DMSO.

6. Add3000μlofNeurobasalmediatoaseparate5mlEppendorftube,capandincubateat37°Cforatleast15minutes.

7. Add9μlof10μMAβstockDMSOsolutiontothetube,vortexvigorouslyfor30to60seconds,spindownbrie lyinthemicrofugeandincubateat37°Cfor15minpriortouse.ThisgivesanAβoligomersolutioncontaining30nMAβpeptideinNeurobasalmediacontaining0.3%DMSO.

2

1. Assemblegelwithnitrocelluloseintransfercassette

[Note:TransferofproteinsfromgelstoPVDFwillresultsinheavyimmunostainingmonomerstaining(Figure2c).]

2. Runtransferincoldtransferbufferat100Vfor1hrat4°

[Note:Transferofproteinsfromgelstonitrocelluloseusingtransferbufferthatdoesnotcontain0.02%SDSwillresultinheavyimmunostainingofmonomerevenwithanti‐oligomerantibodies(Figure2a).]

3. Removenitrocelluloseandplaceinblockingbuffer

[Note:Heat‐treatmentofnitrocellulosefollowingtransferbutpriortoblocking(i.e.immediatelyaftertransferringgeltonitrocellulose,membraneisimmersedinboilingPBS,heatsourceisturnedoff,andblotallowedtositinhotPBSfor5min.)willresultinheavyimmunostainingofmonomerbandsandreducedstainingofoligomers≥4mers(Figure2b).]

4. IncubateatRTfor1hrorovernightat4°C.5. 1°antibody:diluteinmilk/TBS‐TandincubateatRTfor90

mina. Monoclonalanti‐oligomerantibodies(Lambertetal.,J.

Neurochem.100,23‐34,2007):1µg/mlNU1orNU4;1.5µg/mlNU2

b. 6E10(Covance):1:20006. Wash:3x10minwithTBS‐T7. 2°antibody:ECLHRP‐linkedanti‐mouseorrabbitwhole

moleculeIgG(GE;NA934orNA931);dilute1:40,000inmilk/TBS‐T;incubateatRTfor1hr.

8. Wash:3x10minwithTBS‐T9. Rinse3xwithdH2O10. Substrate:SuperSignalWestFemtoMaximumSensitivity

Substrate(Pierce);dilute2partsddH2O+1parteachoftwo‐partsubstratekit

11. Poursubstrateoverdrainedblotandincubateaminute12. TransferblottoKodakImageStationandvisualize.

[Note:AboveWesternblotprotocolshouldshowminimalWesternblotimmunostainingwithanti‐oligomerantibodies,particularlyNU2,whilerecognizingtrimers,tetramers,andlargeroligomers(Figure1right).Heavierproteinloadingorhigherconcentrationsofantibodywillresultinheaviermonomerimmunostaining.]

Figure1.AnalysisofsolubleAβoligomers.(left)SilverstainedSDS‐PAGEgelofAβoligomersshowsmonomer,trimer,andtetramerbands.(Right)Westernblotimmunostainingwith6E10oranoligomerspeci icmonoclonalantibody(NU2).NU2detectsthepresenceoflargeSDS‐stableoligomersinadditiontotetramerandtrimer,butnomonomer.NU2detectsnobandsintheAβ1‐40monomer.

AdaptedfromLambertetal,JNeurochem.,100,25‐35,2007.

S D S ‐ PAGE a n a l y s i s o f A βO s Materials: Gel:Novex10‐20%Tris‐Tricinegels,1mm,15‐well

(Invitrogen)

Electrophoresisbuffer:Tris‐Tricine‐SDS(Bio‐Rad,dilutedto1X)

Samplebuffer:Tricinesamplebuffer(2X,Bio‐RadorInvitrogen)

1. Samplepreparation:

a) Novexsharpprestainedproteinstandards(Invitrogen#LC5800),warmtoRT

ForWesternblotload5μl/well

Forsilverstaindilute50‐foldin1XTricinesamplebufferandload5μl/well

b) AβOsaredilutedto6mMwithF12bufferandthenmixed1:1with2XTricinesamplebuffertogive3mMAβOs.

ForWesternblotload5μl/well(15pmoles)

Forsilverstainload15μl/well(45pmoles)

[Note:Wedonotboiloursamples.Boilingthesampleswilldisrupttheconformationoftheoligomersandresultsindetectingonlymonomer.]

2. Loadgelusinggelloadingtips3. Rungelatroomtemperature:125V(shouldtake~90‐100

min)4. Stoprunwhendyefrontis~0.5cmfrombottomofgel

S i l v e r s t a i n : SilverXpressSilverStainkit(Invitrogen),Tris‐tricineprotocolprovidedinmanufacturer’sinstructions.

[Note:SilverstainofSDS‐PAGEgelsoffreshlypreparedAβOsshouldshowaheavymonomerbandandlightertrimerandtetramerbands(Figure1left).]

We s t e r n B l o t : Materials:

Transferbuffer:25mMTris‐192mMglycine‐20%(v/v)methanol,0.02%SDS,pH8.3(pHdoesnotneedtobeadjusted),storeat4°

TBS‐T:20mMTris‐HCl,pH7.5,0.8%(w/v)NaCl,0.1%(v/v)Tween20

Blockingbuffer:5%(w/v)non‐fatdrymilkininTBS‐T

Velascoetal,ACSChem.Neurosci.,3,972‐981,2012

Figure2:DifferencesinWesternblotprocessingaffectimmunolabelingofAβmonomerbandbysomeantibodies.Aβ1‐42oligomers,preparedat100μMpeptide,weresubjectedtoSDS‐PAGE.Panela:Gelswereelectroblottedtonitrocellulosewith25mMTris,192mMglycine,and20%methanolintheabsence(lefttwolanes)orpresence(righttwolanes)of0.02%SDS,followedbyimmunostainingwith6E10orNU1.Panelb:FollowinggeltransfertonitrocellulosewithSDS‐containingtransferbuffer,lanes2‐4wereheat‐treatedinPBSat100°Cfor5min,andimmunostainedwith6E10,NU1orNU2.Lane1wasnotheat‐treatedpriortoimmunostainingwithM70/2.Panelc:Thegeloffresh(left)and2‐weekoldoligomers(right)wastransferredtoPVDFwithSDS‐containingtransferbufferandimmunostainedwithNU1.

For2Dgelanalysis,seeGongetal,PNAS,100,10417‐10422,2003.

ForNativegelanalysis,seeChromyetal,Biochemistry,42,12749‐12760,2003.

D o t b l o t a s s a y f o r A βO d e t e c t i o n : 1. SyntheticAβOsinF12aredilutedwithF12.2. EndogenousAβOsarecollectedaccordingtothemethods

detailedinGongetal,PNAS,100,10417‐10422,2003;Changetal,J.Mol.Neurosci.,20,305‐313.

3. 1μlaliquotsarespotted,intriplicate,ondryHybondECLnitrocellulose(Amersham)andallowedtodry10minfollowingthe inalspotting.

4. Theblotisblockedwith5%non‐fatdrymilkinTBS‐T[20mMTris‐HCl,pH7.5,0.8%(w/v)NaCl,0.1%(v/v)Tween‐20]for1hratroomtemperature.

5. Primaryantibodiesaredilutedinmilk/TBS‐Tandblotsincubatedforovernightat4°C.

6. Blotsarewashed3x10minwithTBS‐T.7. HRP‐linkedsecondaryantibodies(Amersham)arediluted

1:40,000inTBS‐Tandblotsincubatedfor1hratroomtemperature.

3

8. Blotsarewashed3x10minwithTBS‐T.9. Blotsarerinsedbrie ly3XwithdH2O.10. SuperSignalWestFemtoMaximumSensitivitySubstrate

(Pierce)isdilutedtohalf‐strengthwithddH2Oandblotsincubated1minatroomtemperature.

11. Thewell‐drainedblotsareimagedwithaKodakImageStation440CFwithKodak1Dimagesoftware.

Lambertetal,J.Neurochem.,100,25‐35,2007

Figure3.Dot‐blotanalysisshowsselectiveantibodiesdiscriminatebetweenAlzheimer'sdiseaseandcontrolbraintissue.SyntheticADDLs(1pmol)andhumanbrainextractsinF12(fromhumanAlzheimer'sdiseaseandcontrolbrain;0.5μg)werespottedontonitrocelluloseinduplicate.Afterperoxidasetreatment(3%H2O2,20min)andblocking,eachverticallanewasthenprobedwiththeindicatedmonoclonalsorrabbitpolyclonal(M71/2)for1hatroomtemperature.Afterwashing,thenitrocellulosewasincubatedwiththeappropriateHRP‐conjugatedsecondaryantibody;boundsecondarywasthenvisualizedwithchemiluminescence.NotethatNU‐1,NU‐4,andM71/2bindstronglytoAlzheimer'sdiseasebrainsamplesbutnottocontrolbrainsamples.

Figure4.Region‐speci icaccumulationofADDLsinAPPtransgenicmousebrain.Twotransgenicmiceandonenon‐transgeniccontrolmouseweretestedblindforADDLformationinbrainhomogenatesusingthedot‐blotassay.SyntheticADDLsasastandard(A),andsolubleprotein(1ug)fromhomogenatesofdifferentbrainregions(B)wereappliedtonitrocelluloseandanalyzedwiththeoligomer‐speci icantibodyM93/3.

Changetal,J.Mol.Neurosci.,20,305‐313.

A βO immuno c y t o c h em i s t r y (Ho t S p o t A s s a y f o r S y n ap t i c B i n d i n g o f Aβ 1 ‐42 O l i g ome r s t o H i ppo c ampa l N eu r on s )

Primaryhippocampalcultures(Lacoretal.,J.Neurosci.24,10191−10200),18‐21dayold(35mmculturedishes,5‐12mmcoverslips/dish)

A βO I n c uba t i o n : 1. Equalizemediaindishes(for35mm,weequalizeto1

ml/dish).2. AddAβOsorvehiclecontroltodesired inalconcentration

(usually100‐500nM).3. Incubateat37°Cfordesiredtime(usually30‐60minfor

simplebindingassay).

[Note:GlialcellsendocytoseAβOsinthemedia.Ahighpercentageofglialcellsinthecultureswillsigni icantlyreducethelevelsofAβOsavailabletobindtothehippocampalneurons.]

[Note:AβOsbindtohippocampalcellsasearlyas1weekafterculturing(seeGongetall.,PNAS,2003),butdonotlocalizetosynapsesuntilthecultureshavematured(>14daysinculture)(seeLacoretal,JNeurosci,2004).]

C e l l F i x i n g : 1. Aspirateincubationmediafromdish2. Washdishes1‐3Xbrie lywith1mlwarmNeurobasal

media(Invitrogen);leavein inalwash3. To inalwash,addequalvolume(1ml)of3.7%

formaldehydeinNeurobasalmediaandincubatefor5‐10min;remove.

4. Add1ml3.7%formaldehydeinNeurobasalmediaandincubatefor5‐10min;remove.

5. Wash2Xrapidlyin2mlPBS;remove.6. Washadditional3x5minin2mlPBS.

[Note:Insuf icientquenchingofthefreealdehydesfrom ixationcanresultinnon‐speci iclabelingofthecellbody.Wewashextensivelytoremove/reducefreealdehydes.Ifnon‐speci iccellbodylabelingpersists,thefreealdehydescanbequenchedbypre‐incubatingthecoverslipswith0.5mg/mlsodiumborohydridefor5minutesbeforeblockingwiththeNGSsolution(beforeimmunostaining).]

7. Cellscanbestored,sealedwell,at4°Cin inalPBSwash

Immuno s t a i n i n g : 1. Prepareblockingbufferusing10%normalgoatserum

(NGS)inPBS.

[Note:Ifyouaregoingtobedouble‐labelingwithanantibodyagainstanintracellularprotein,0.1%TritonX‐100canbecombinedwiththe10%NGS:PBSsolutionforbothblockingandprimaryantibodyincubation.Wehavefoundthat0.1%TritondoesnotappeartoimpactAβOdetection.]

1. Inalightblockingchamber,af ixasheetofpara ilmbigenoughtoholdallcoverslipsandplaceadamppapertoweltoonesidetomaintainhumidity.NOTE:Oncecoverslipsareplacedonthepara ilm,theymustnotsitdryformorethanacoupleofsecondsatatime.

2. Placecoverslips,cellsideup,onpara ilmandadd125‐150μlofblockingsolutiontoeachcoverslip;incubateatRTfor30‐45min.

3. Diluteprimaryantibodyinblockingsolution(usually1μg/mlforNU1,NU2orNU4)andincubate100‐150μl/coverslipovernightat4°C.

4. Washcoverslipswith3x125µlPBSfor5‐10mineach.5. Dilutesecondaryantibodyin1%NGSinPBS[for

AlexaFluoranti‐mouseIgG488(Invitrogen),1:2000]andincubate125μl/coverslipfor2hratRT

6. Washcoverslips3XinPBSfor5‐10min7. Add1‐5dropsofProLongGoldwithDAPI(Invitrogen),one

foreachcoverslip,toaslideandplacecoverslipscellsidedown.

8. LetdryatRTovernightinthedark.9. Storeslideinthedark.10. CoverslipsareimagedonaKodakepi luorescent

microscopeat60Xmagni ication(seeFigure4).

Figure5.SynapticbindingbyAβ1‐42oligomerstohippocampalneuronsdetectedwithNU4antibody.Neuronstreatedfor3hwith100nMADDLsfollowedbydoublelabelingforinsulinreceptor(red)andADDLs(green).

AdaptedfromDeFeliceetal,PNAS,106,1971‐1976,2009.

Figure6.ADDLsshowpunctatebindingtoneuronalcell‐surfaceproteins.CulturedhippocampalneuronswereincubatedwithsyntheticADDLs.Bindingwasvisualizedbyimmuno luorescencemicroscopyusingM93antibody.Smallpuncta,typically<1μm,andlargelydistributedalongneurites,areevident.(Bar,10μm.).

AdaptedfromGongetal,PNAS,100,10417‐10422,2003.

4

[Note:Weusefree loatingorfrozentissuesections.Wehavefoundthatharshorexcessiveprocedurescanalterthenativeconformationoftheantigensandresultinanegativeresultwhenprobingwithourconformation‐sensitiveAβOantibodies.Suchproceduresincludeembeddinginparaf inoracrylic.Ifsuchproceduresarenecessary,tissueshouldbelabeledwithantibodiespriortoembedding.]

3. Slicesaretransferredtonewwellscontaining1mlofextractionbuffer/wellandincubatedfor10mins.Thisisrepeated2moretimes,transferringslicestonewwellseachtime(3x10minswithextractionbuffer).

4. Slicesaretransferredtonewwellscontaining1ml/wellblockingbufferandincubatedfor30minsatroomtemperaturewithverygentleagitationororbitalshaking.

5. Slicesaretransferredtonewwellcontainingthe1mloftheprimaryantibody(ies)dilutedinblockingbuffer.Slicesareincubatedovernightat4°Cwithverygentleagitation.

6. Slicesarerinsed3timeswithTBSasinstep#2,transferringslicestocleanwellseachtime.

7. Slicesaretransferredtocleanwellcontaining1mloftheappropriatesecondaryantibody(ies)inadilutedblockingbuffersolution(1:10blockingbuffer:TBS)andincubatedfor3hoursatroomtemperature.Ifsecondaryantibody(ies)arenotneeded,skiptostep#9.

8. Slicesarerinsed3timeswithTBSasinstep#2,transferringslicestocleanwellseachtime.

9. SlicesareplacedoncleanslidesandtheexcessliquidwickedawaywithKimwipes.

10. 1‐2dropsofProlongareplaceddirectlyonthetissue,withouttouchingdroppertothetissue,andthetissueiscoveredwitha0‐thicknesscoversliplargerenoughtocompletelycovertheslice(s).

11. Slidesareallowedtodryatroomtemperatureovernight(inthedark).Slidescanbestoredinthedarkuntilreadytoimage.

Figure7.NU4andNU4MNSdiscriminateADhumanfrontalcortexsectionsfromagedcontrols.Humanfrontalcortexsectionswereprobedwith luorescentlytaggedNU4.NU4detecteddiffuseplaque‐likestructuresandsmallerneuronaldeposits,consistentwithtypicalADpathology.Thesestructureswerenotseenincontrolbrain,demonstratingthatNU4iscapableofdiscriminatingbetweenADandnon‐dementedcontrols.Scalebar=100µm

OR12. FORNON‐FLUORESCENTSLICES:IfanHRP‐conjugated

secondarywasused,avisualizationreactionisperformed(i.e.DABreactionoralkalinephosphatasereaction,kitsavailablefromVectorLabs)tolocalizethesecondaryantibody(ies).Slicesarethencounterstained,dehydratedthrough2washesof100%ethanol,washedtwicewithXylenes,andcoverslippedusingEukittmountingmedium.

13. Slidesareallowedtodryatroomtemperatureovernight(inthedark).Slidescanbestoredatroomtemperatureuntilreadytoimage.

AdaptedfromLacoretal,J.Neurosci.,24,10191‐10200,2004andFerreiraandKleinNeurobiol.Learn.Mem.,96,529‐543,2011

Figure8.Dendriticoligomerpathologyoccursearlyindiseaseprogression.(Left)Lowmagni icationofhumanbrainsectionstainedwithanti‐oligomerantibody.Patternindicatesassociationwithonlyscatteredindividualneuronsearlyindisease,priortoamyloidplaques.(Right)Perineuronallocalizationofoligomersdetectedusingoligomer‐speci icantibody,enlargedfrom ieldatright(box).Stainisassociatedwithdendriticarbor,notsomaticcytoplasm.

Immunoh i s t o c h em i s t r y ( t i s s u e s l i c e s ) T i s s u e p r ep a r a t i o n ( human o r a n ima l ) : 1. Tissueisdissectedoutandsubmergedin3.7%(animal)or

10%(human)formalinsolutioninPBS,pH7.3for1‐2days,untiltissuesinkstobottomofcontainer.

2. Tissueistransferredtoa10‐40%sucrosegradient.3. Free loatingsections(40‐50umthick)areobtainedand

transferredtosterilePBSforuse.

M a t e r i a l s a nd Bu f f e r s : 12‐wellor24‐wellplatesVery inepaintbrushesEndogenousperoxidasebuffer: 10%methanol 3%H2O2 InTBSTBS 20mMTris,pH7.6 0.2MNaClExtractionbuffer: 0.3%TritonX‐100 InTBSBlockingbuffer 10%NGS(normalgoatserum) 0.3%TritonX‐100 InTBS [Note:PBSmaybesubstitutedforTBSinthisprocedure.]

Immuno s t a i n i n g ( f r e e ‐ f l o a t i n g s e c t i o n s ) :

[Note:Mountedfrozensectionscanbeimmunolabeledusingthisprocedure,butincubationtimesmayneedtobeincreasedordecreaseddependingonthethicknessoftheslices.]

1. Slicesareincubatedin1mlEndogenousperoxidasebufferfor5minsina12‐or24‐wellplate(upto2slices/well).

[Note:Thisstepcanbeomittedifvisualizingantibodybyluorescence.]

2. Usinga inepaintbrush,slicesaretransferredtonewwellscontaining1mlofTBS/wellandincubatedfor5minutes.Thisstepisrepeated2moretimes,transferringslicestonewwellseachtime(3x5minswithTBS).

5

Figure10.Surface‐boundAβOexistinthe5xFADmouse.Immunohistochemicaldetectionofendogenously‐expressedAβOinthe5xFADhippocampususingNU4AβO‐speci icantibody(left).DirectdetectionofAlexa luor‐555‐conjugatedNU4appliedintranasallytotargetoligomerspresentintheextracellularspace(right).

R e f e r en c e s Chang,L.,Bakhos,L.,Wang,Z.,Venton,D.L.,andKlein,W.L.(2003).Femtomoleimmunodetectionofsyntheticandendogenousamyloid‐βoligomersanditsapplicationtoAlzheimer'sdiseasedrugcandidatescreening.Journalofmolecularneuroscience:MN20,305‐313.

Chromy,B.A.,Nowak,R.J.,Lambert,M.P.,Viola,K.L.,Chang,L.,Velasco,P.T.,Jones,B.W.,Fernandez,S.J.,Lacor,P.N.,Horowitz,P.,etal.(2003).Self‐assemblyofAβ(1‐42)intoglobularneurotoxins.Biochemistry42,12749‐12760.

DeFelice,F.G.,Vieira,M.N.,Bom im,T.R.,Decker,H.,Velasco,P.T.,Lambert,M.P.,Viola,K.L.,Zhao,W.Q.,Ferreira,S.T.,andKlein,W.L.(2009).ProtectionofsynapsesagainstAlzheimer's‐linkedtoxins:insulinsignalingpreventsthepathogenicbindingofAβoligomers.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica106,1971‐1976.

Ferreira,S.T.,andKlein,W.L.(2011).TheAβoligomerhypothesisforsynapsefailureandmemorylossinAlzheimer'sdisease.Neurobiologyoflearningandmemory96,529‐543.

Gong,Y.,Chang,L.,Viola,K.L.,Lacor,P.N.,Lambert,M.P.,Finch,C.E.,Krafft,G.A.,andKlein,W.L.(2003).Alzheimer'sdisease‐affectedbrain:presenceofoligomericAβligands(ADDLs)suggestsamolecularbasisforreversiblememoryloss.

ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica100,10417‐10422.

Klein,W.L.(2002).AβtoxicityinAlzheimer'sdisease:globularoligomers(ADDLs)asnewvaccineanddrugtargets.Neurochemistryinternational41,345‐352.

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I n v i v o l a b e l i n g b y i n t r a n a s a l ( I N ) a n t i b ody d e l i v e r y

[Note:ProcedureisbasedonprotocolpublishedbyXiaoetal,JAlz.Dis.,35,777‐788,2013.]

1. Animalsareanesthetizedbyi.p.injectionofketamine/xylazineorbyinhalationofiso luorane.

[Note:Animalsquicklyrecoverfromiso luoraneanesthetization.Onlyusethisiftheprocedurecanbecompletedquickly.]

2. Withtheuseofamicropipette,miceareINadministeredwithasinglebolusdose(5μl/naris,Total10μl)AlexFluor®568‐labeledNU4antibodyandallowedtosurvivefor6or12hpost‐injection.[Note:Theanimalisheldinanuprightpositiontomaximizeintranasal(IN)accessofIN‐administeredmaterialwhilesimultaneouslyminimizingextra‐nasalentryinthesurroundingareassuchasthethroat.]

3. Miceareeuthanizedandthebrainsareharvested.4. Thebrainsare ixedorsnapfrozen.5. Thebrainsareprocessedtoobtainfrozensagittalsections

of40‐50μmthickness.[Note:Snapfrozensectionsarelightly ixedwithformaldehydepost‐sectioning.]

6. BrainsectionsarethenmountedusingProlonganti‐fadereagentcontainingDAPI(blue)tolabelcellnucleiofthevariousbrainregions(Figures9and10).

AdaptedfromXaioetal,J.Alz.Dis.,35,777‐788,2013

Figure9.Distributionof luorescentlylabeledNU4at6and12hpostinjectionshowingdetectionofantibodywithinthepyramidalneuronslayerofthehippocampalCA1regionat6hpost‐injectionshowingperisomallabeling.Scalebar=50μm.

K L E I N L A B

NorthwesternUniversity2205TechDrive

Evanston,IL60208

Phone:847‐491‐2868Fax:847‐491‐5211

web:www.kleinlab.org

PreparedbyKirstenL.ViolawithcontributionsfromPaulineT.Velasco,PascaleN.Lacor,andKyleC.Wilcox