| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | None/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | RT |
| Category | MicroBIOLOGy FluorescenceImaging |
| Related | BiochemicalAssays |
1. Platecellsonslidechambersorsterileglasscoverslips,andtreatthecellsasdesired
2. Performformaldehydefixation.Incubatecellswith3.0–4.0%formaldehydeinPBSatroomtemperaturefor10–30minutes.
3. Washthefixedcells2–3timesinPBS.
Optional:Add0.1%TritonX-100inPBSintofixedcellsfor3to5minutestoincreasepermeABIlity.Rinsethecells2–3timesinPBS.
4. Blockwithblockingagentsuchaswith5%BSAinPBSfor30min.
5. Diluteprimaryantibodyindilutionbufferasrecommendedinthespecificproduct’sdatasheet.Overlayenoughdilutedantibodytocovercellsoncoversliporeachchamberofthechamberslides.
6. Wash2-3timeswithPBS.
7. WarmupFluoroQuest™fluorescencesignalenhancingsolutionatroomtemperaturebeforeuse.
8. DilutefluorescentsecondaryantibodyinFluoroQuest™fluorescencesignalenhancingsolutionandthenstaincellsfor1houratroomtemperature.Generalrangeforsecondaryantibodiesisbetween0.1-10µg/mLforIgGconjugatesformostapplications. Keepslipscoveredorinahumidifiedchambertoavoidevaporation.
9. WashthecellsthreetimeswithPBS.
Note:Additionalstainingwithfluorescentnuclearstainsorphalloidinscanbedoneatthisstep.
10. Inverteachcoverslipontoacleanedslidewithmountingmedia,preferablyonewithananti-fadepreservative.Sealedgeswithclearpolishifdesired.
11. Imagingthecellsundermicroscopewithcorrectfilters.Storeslidesinthedarkat4°C.
| References&Citations |  PrinterFriendlyVersion |
1. CordesT,VogelsangJ,TinnefeldP.(2009)OnthemechanismofTroloxasantIBLinkingandantibleachingreagent.JAmChemSoc,131,5018.
2. Gallardo-EscarateC,Alvarez-BorregoJ,VonBrandE,DupreE,DelRio-PortillaMA.(2007)RelationshipbetweenDAPI-fluorescencefADIngandnuclearDNAcontent:AnalternativemethodtoDNAquantification?BiolRes,40,29.
3. WidengrenJ,ChmyrovA,EggelingC,LofdahlPA,SeidelCA.(2007)Strategiestoimprovephotostabilitiesinultrasensitivefluorescencespectroscopy.JPhysChemA,111,429.
4. DikiciogluE,MeteogluI,OkyayP,CulhaciN,KacarF.(2003)Thereliabilityoflong-termstorageofdirectimmunofluorescentstainingslidesatroomtemperature.JCutanPathol,30,430.
5. El-RifaiWE,KnuutilaS.(2001)Comparativegenomichybridizationtechnique.MethodsMolMed,50,25.
6. BerriosM,ConlonKA,ColfleshDE.(1999)Antifadingagentsforconfocalfluorescencemicroscopy.MethodsEnzymol,307,55.
7. BrockR,HamelersIH,JovinTM.(1999)ComparisonoffixationprotocolsforadherentculturedcellsappliedtoaGFPfusionproteinoftheepidermalgrowthfactorreceptor.Cytometry,35,353.
8. KolankoCJ,PyleMD,LoatsH,PartonJ,BlakelyWF,NathJ.(1999)Fast-insituhybridizationandimmunoenzymaticcolorpigmentdetectionofmousebonemarrowmicronucleus.BiotechHistochem,74,111.
9. BenchaibM,DelormeR,PluvinageM,BryonPA,SouchierC.(1996)Evaluationoffivegreenfluorescence-emittingstreptavidin-conjugatedFluorochromesforuseinimmunofluorescencemicroscopy.HistochemCellBiol,106,253.
10. SwartzDJ,SantiPA.(1996)ImmunofluorescentartifactsduetothepHofantifadingmountingmedia.Biotechniques,20,398.
