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Basic Method for Direct Immunofluorescence Labeling

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BasicMethodforDirectImmunofluorescenceLabeling

Background

Thisisthemethodfordirectimmunofluorescencelabeling;thatis,theantibodieshavethefluorescentdyeattached.Directlabelingissimplerandquickerthanindirectlabeling.Westronglyrecommenddirectlabelingifyou"replanningtosimultaneouslylabelcellswith2ormoreantibodies.Stilltherearesomereasonstogowiththeindirectmethod:

YoucannotobtainyourspecificantibodyinaFluorochrome-conjugatedform;
Fluorochromeconjugationinterfereswiththespecificityoftheantibody;
Thenumberofreceptorspercellislow(indirectmethodsgenerallycanrenderbrighterlabeling);
Youneedveryaccuratecomparisonsoflabelingwithdifferentantibodies.
Ifanyoftheaboveapply,seeourprotocolforindirectlabeling.
Materials
1. Fluorochrome-ConjugatedAntibodies:
  - Testantibody:MousemonoclonalconjugatedtoFITC,PE,PerCPorPE-CY5Tandem(tradenames:Cy-ChromeorTri-Color).Ifyou"replanningtolabelcellswith2ormoreantibodiessimultaneously,youneedanegativecontrolforeachfluorochromeconjugate.
    Forsimultaneouslabeling,choosethefluorochromescarefully.Youwantthefarthestred-emittingantibodytolabeltheantigenwiththegreatestdensity(mostreceptorspercell).
  - Negativecontrolsera:Usually,purifiedmouseIgMorIgGofthesamesubclassasthetestantibodiesandconjugatedtothesamefluorochrome.Oneusuallypurchasesthemfromthesamemanufacturertogetasimilarfluorochrome-to-proteinratioasthetestantibodies.
    Inflowcytometry,fluorescenceisrelative.Weneedanegativecontroltodeterminewhere"positivity"begins.
  - PBSwith0.1%sodiumazideadded.Thesodiumazideassistsinpreventingcappingandsheddingorinternalizationoftheantibody-antigencomplexaftertheantibodiesbindtothereceptors.
  - CellsinsUSPension,countedandviABIlity-checked.Keeptheminculturemediumsupplementedwithantibioticsand2-5%fetalbovineserum,onice.Ifviabilityislessthan90%,consideraddinganotherfluorochrometoidentifydeadcellsduringanalysis.
    Equipment
    1. Centrifuge.YoushouldknowhowtheRPMtranslatesintoG-force.
    2. Precisionadjustablemicropipet.Youwillprobablyneedtwo:oneintherangeof10-100microliters,andanotherrangingfrom100-1000microliters.
    3. Vortexmixer.Youcouldmixbytappingorshakingthetubes,butamixerwillgivemuchmorereproducIBLeresultsinmostcases.
    4. 12x75mmpolystyrenetubes.Theclearplastickind.Ifyoucan,buytheFalconbrandbecausetheyfittheinstrumentbest.Ifyoucan"t,don"tworry-wewillsupplythemwhenyoubringyoursamplestothelab.
    5. Icebucketwithcover.Generally,cellsaremorestableandtolerateinsultbetterwhenthey"recold.Thecoverkeepslightout,whichcouldbleachthefluorochromes.
    6. Flowcytometer.Ifyouanalyzeyoursamplesinourlab,theinstrumentyouusewillmostlikelybeaFACScanorFACSort,madebyBecton-Dickinson.(Seeourinstrumentlistformoredetails.)
      Procedure
      1. Adjustthecellconcentrationto1millionperml.withculturemediaorPBS.
      2. Place1ml.ofthecellsuspensionintoeachofthe12x75tubes.
        Youwillneedatubeforeachantibodyplusthenegativecontrol.Ifyou"redoingsimultaneouslabeling,useonetubeforeachcombinationofantibodiesorcontrols,butwewillprobablyneedsingle-antibodylabeledcellsforeachcombinationaswell.Confused?Let"stalk!
      3. Centrifugeat250xgfor5minutes.Useapipettoremovetheliquid.Becarefulnottodisturbthepellet.Aslightamountofliquidcanremain.
        Thisforceandtimeworkswellforlymphoidcells.Youmayhavetoadjustasrequiredifyourcellsaredifferent.
      4. Addtheappropriateamountofmonoclonalantibodyorcontrolsera.Theamountisusuallygivenbythemanufacturer.Ifnot,itshouldhavebeendeterminedpreviouslybytitration,usingtargetcellswithalargenumberofreceptors.
      5. Vortex.Keepthetubesoniceforaround30minutes.Covertheicebucket.
      6. Firstwash-Add1ml.ofthePBS+azide.Vortex.
      7. Centrifugeandremoveliquidasabove.
      8. Secondwash-Add1ml.ofthePBS+azide.Vortex.
      9. Centrifugeandremoveliquidasabove.
      10. Add1ml.ofthePBS+azide.Vortex.