ting98

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Cell-Mate3DProtocol:

CryoctioningandStaining

RequiredEquipment

•WaterBathtto97°C

•1.5mLEppendorfTubes

•Bluntendforceps

•CoverWell™ImagingChambers(GraceBio-Labs#631051)

•InvertedConfocalMicroscope

•CoplinJar

•CryoMold

•Forceps

•RazorBlades

•ChargedSlides

•MountingChuck

•Cryostat

•Pencil

•Paintbrushes

RequiredReagents

•4%Formalin

•PBS

•DIWater

•SuperfrostPlusSlides,PositivelyCharged(Fisher#:6776214)

•NucBlueFixedCellStainReadyProbesDAPI(LifeTechnologies#R37606)

•OCTMountingMedium

•NucBlueLiveReadyProbesReagent(Invitrogen#R37605)orotherDAPIreagent

SuggestedBlockingReagents

•orewesuggesttryingveralblocking

reagentstooptimizewithyourspecificantibody.

•SuperblockblockingbufferinPBS(ThermoScientific#37515)

•100%NormalHumanSerum–Unconjugated(JacksonImmunoRearch

Laboratories#009-000-121)

•

SuggestedAntigenRetrievalReagents:Someantibodiesrequireanantigenretrieval

steptoimprovestainingspecificity

•TargetRetrievalSolution(10X)CitrateRetrievalBuffer(Dako#S1699)

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CryoctioningandStaining:

Prep

a)WashCell-Mate3D™samplewith1mLofPBSfor5min

b)Fill½ofthecryomoldwithmountingmedium

c)RemoveCell-Mate3DsamplefromPBS,drylightlywithpapertowelorKimwipe

d)PlaceCell-Mate3DsampleinmoldwithOCTinpreferredorientation

e)CovercocoonsamplewithOCT,avoidingairbubblesinthemountingmedium

f)Freezeovernightat-80˚C,orbyusinganalternativefreezingmethod

smaybeprocesdwithacryotomewitharollplate(e2A)orwithouta

rollplate(e2B)

singSampleWithRollPlate

a)Transferfrozensampleinmoldfrom-80˚Ctocryostatandletsitfor5min

b)Popsampleoutofmold,anddetermineorientationforcutting

c)Addasmallamountofmountingmediumtothechuckandquicklyplacesample

onwetmediumbeforeitsolidifies

d)Placechuckwithmoldattachedonalevelsurfacetoallowittocontinuetofreeze

andsolidify

e)Mountchuckonthecryotome,adjustingorientationandanglesasneeded

f)Securerazortobegintotrimthesample

g)Trimuntilthesampleisvisibleunderthemedium,thenswitchtoasharperblade

andtthectionmeasurementto6-10µm,dependingonpreferenceandsample

type

h)Whensampleixpod,trimexcessOCTmediumusingaboxcutterblade

i)Adjustrollplatepositionsothatthesampleeasilyctionsbeneathit

j)Collectctionsastheyarecutbygentlytouchingtheroomtemperatureslideto

thection

k)Ifstoringapartiallycutsample,coverexpodsamplewithathinlayerofOCT

andallowtosolidify

l)Storecollectedslidesat-20˚emainingsampleinOCTat-80˚C

singSampleWithoutRollPlate

a)Transferfrozensampleinmoldfrom-80˚Ctocryostatandletsitfor5min

b)Popsampleoutofmold,anddetermineorientationforcutting

c)Addasmallamountofmountingmediumtothechuckandquicklyplacesample

onwetmediumbeforeitsolidifies

d)Placechuckwithmoldattachedonalevelsurfacetoallowittocontinuetofreeze

andsolidify

e)Mountchuckonthecryotome,adjustingorientationandanglesasneeded

f)Securerazortobegintotrimthesample

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g)Trimuntilthesampleisvisibleunderthemedium,thenswitchtoasharperblade

andtthectionmeasurementto6-10µm,dependingonpreferenceandsample

type

h)Whensampleixpod,trimexcessOCTmediumusingboxcutterblade

i)Turnthewheelwithyourrighthandinacontinuousmotionatasteadypace

j)Movethepaintbrushdownthesamplemoldinpacewiththechuck

k)Uapaintbrushtoguidethecutsamplectionsoffthemoldbycatchingthe

curlingedgeofthectionwhileturningthewheel

l)Collectctionsastheyarecutbygentlytouchingtheroomtemperatureslideto

ction

m)Ifstoringapartiallycutsample,coverexpodsamplewithathinlayerofOCT

andallowtosolidify

n)Storecollectedslidesat-20˚C,andcontinuetostoreremainingsampleinOCTat

-80˚C

ng

a)Thawslidesatroomtemperaturefor15min

b)RehydrateslidesinPBSfor10minutesusingaCoplinjar

c)Optional:AntigenRetrievalStep:

trateretrievalbuffer(diluteto1XinDIwater)to65˚C

eslidesincitrateretrievalbufferat97˚Cfor2-5minsinaCoplin

stainingjarplacedinwaterbath

eslidesbackto65˚Cbyremovingjarwithcontentsfromthewater

bathandallowingtocooltoroomtemperature

washtheslidesinDIwaterfor1minute

washtheslidesinPBSfor5minutes,threetimes

d)Blockfortheslidesfor30minutesatroomtemperature

e)Staintheslidesfor1houratroomtemperature

f)GentlywashtheslidesinDIwaterfor1minute

g)GentlywashtheslidesinPBSfor5minutes,threetimesAddDAPItotheslides

andimageusingaConfocalMicroscope

HumanMenchymalStemcells

embeddedinCell-Mate3D™

cryoctionedandstainedwithCD44-

A488(VWR#103016-BL)andDAPI.

100%HSAudforblockingreagent.

Imagedwithinvertedconfocal

microscope.60X