常用荧光探针小结

常用荧光探针小结一、异硫氰酸荧光素（Fluoresceinisothiocyanate,FITC）FITC有两种异构体，性质稳定，低温下干燥保存，其性状多年不变，室温下也能保存两年以上。异构体I、II均能与蛋白质良好结合，但异构体I的荧光效率更高，与蛋白质的结合也更稳定。FITC的最大吸收光谱为490-495纳米，最大发射光谱为520-530nm，呈明亮的黄绿色荧光。FITC含有异硫氰基，在碱性条件下能与IgG的自由氨基（主要是赖氨酸的-氨基）形成荧光抗体结合物。,1,Exitationmax:495nm;Emissionmax:519nm;SolventpH:8.00,2,3,Immunocytochemistry/Immunofluorescence-alphaTubulinantibodyDM1A(FITC)(ab64503)ICC/IFimageofab64503stainedhumanHeLacells.Thecellsweremethanolfixed(10min),permabilisedin0.1%PBS-Tween(20min)andincubatedwiththeantibody(ab64503,1g/ml,FITCconjugated(green)for1hatroomtemperature.1%BSA/10%normalgoatserum/0.3Mglycinewasusedtoblocknon-specificprotein-proteininteractions.AlexaFluor594WGAwasusedtolabelplasmamembranes(red).DAPIwasusedtostainthecellnuclei(blue).,4,二、荧光素酯,Carboxyfluoresceindiacetatesuccinimidylester(CFDA-SE)isacellpermeabledyegenerallyusedinanimalcellproliferationresearch.CFDA-SEenterscellsbydiffusionandiscleavedbyintracellularesteraseenzymestoformanamine-reactiveproduct,CFSE.Thisproductproducesadetectablefluorescenceandcovalentlybindstointracellularlysineresiduesandotheraminesources.,酯酶,5,视频：TheUseofCarboxyfluoresceinDiacetateSuccinimidylEster(CFSE)toMonitorLymphocyteProliferation,6,四甲基异硫氰酸罗达明，它是一种紫红色粉末，较稳定。其最大吸收光谱为550nm，最大发射光谱620nm，呈橙红色荧光，与FITC的黄绿色荧光对比清晰，与蛋白质结合方式同TITC。它可用于双标记示踪研究。,三、异硫氰酸罗丹明（TMRITC）,7,Detectionof-tubulininA549cellsdemonstratesuseofrhodamine-labeledsecondaryantibody.Cellswereprobedwithamouseanti-tubulinprimaryantibody(0.4g/mL)andRhodamine-goatanti-mousesecondaryantibody(2g/mL).NucleiwerelabeledwithHoechstDye.Imageswereacquiredbyfluorescencemicroscopy.A.Fluorescenceimageshowsadelicatenetworkof-tubulin(pseudo-coloredgreen)locatedexclusivelyinthecytoplasm.B.NuclearcounterstainwithHoechstDye(pseudo-coloredblue)C.Mergedimage.,8,AC41323-0010,RB200，也称丽丝胺罗丹明B无定形褐红色粉末，不溶于水，易溶于酒精和丙酮，性质稳定，可长期保存，最大吸收光谱为570nm，呈明亮的橙色荧光，因与FITC的黄绿色有明显区别，故被广泛用于对比染色或用于两种不同颜色的荧光抗体的双重染色。标记方法方法：取1gRB200及五氯化磷（PCL5）2g放乳钵中研磨5min（在毒气操作橱中），加10ml无水丙酮，放置5min，随时搅拌，过滤，用所得溶液进行结合。将每亳升血清用1ml生理盐水及1ml碳酸盐缓冲液（0.5mol/L，pH9.5）稀释，逐滴加入0.1mlRB200溶液，随加随搅拌，在0-4继续搅拌12-18h。,四、罗丹明200,9,ConfocalimageofdoubleimmunostainingforAktinhippocampalCA1pyramidalneurons.Sectionisshownfromanormalrat.TheredcolorderivedfromlissaminerhodamineconjugatedsecondaryantibodyrepresentsMAP2,thegreencolorderivedfromfluoresceinindicatesthelabelingofAkt.Yellowrepresentsoverlayofredandgreen.,10,五、溴化乙锭详见第四节“应用于核酸检测的荧光探针技术”,11,六、DAPI（4,6-diamidino-2-phenylindole）,DAPIwasfirstsynthesisedinaspartofasearchfordrugstotreattrypanosomiasis.Althoughitwasunsuccessfulasadrug,furtherinvestigationindicateditboundstronglytoDNAandbecamemorefluorescentwhenbound.Whenboundtodouble-strandedDNADAPIhasanabsorptionmaximumatawavelengthof358nm(ultraviolet)anditsemissionmaximumisat461nm(blue).ThereforeforfluorescencemicroscopyDAPIisexcitedwithultravioletlightandisdetectedthroughablue/cyanfilter.TheemissionpeakisfairlybroadDAPIwillalsobindtoRNA,thoughitisnotasstronglyfluorescent.Itsemissionshiftstoaround500nmwhenboundtoRNA.,12,DAPI(magenta)boundtotheminorgrooveofDNA(greenandblue).,DAPIcanbeusedforfixedcellstaining,theconcentrationofDAPIneededforlivecellstainingisgenerallyveryhighandrarelyusedforlivecells.ThoughitwasnotshowntohavemutagenicitytoE.coli,itislabelledasaknownmutageninmanufacturerinformation.AsitisaDNAbindingcompounditislikelytohavesomelowlevelmutagenicpropertiesandcareshouldbetakeninitshandlinganddisposal.,13,Atypeofsimplefluorescence:DAPIisexcitedinnearUVwith365nmandemittedintheviolet-bluespectralrange.Themarkingrevealscellnucleiandparticularlythechromosomes,DAPIisadyewhichbindstoDNAandshowsanincreasedbluefluorescencewhenboundtoDNA.Thebluefluorescentspotsinthepictureshowthenucleiinthemycel.,14,Endothelialcellsunderthemicroscope.NucleiarestainedbluewithDAPI,microtublesaremarkedgreenbyanantibodyandactinfilamentsarelabelledredwithphalloidin.,15,七、Hoechst,HoechststainsarepartofafamilyofbluefluorescentdyesusedtostainDNA.TheseBis-benzimideswereoriginallydevelopedbyHoechstAG.TherearethreerelatedHoechststains:Hoechst33258,Hoechst33342,andHoechst34580.ThedyesHoechst33258andHoechst33342aretheonesmostcommonlyusedandtheyhavesimilarexcitation/emissionspectra.,16,17,TransmissionimageofHeLacells,withoverlayofHoechst33258staining(blue).,Hoechst33258(magenta)boundtotheminorgrooveofDNA(greenandblue),18,八、碘化丙啶(PropidiumIodide,PI）,Propidiumiodideisanintercalatingagentandafluorescentmoleculethatcanbeusedtostaincells.WhenPIisboundtonucleicacids,thefluorescenceexcitationmaximumis535nmandtheemissionmaximumis617nm.Excitationenergycanbesuppliedwithaxenonormercury-arclamporwiththe488lineofanargon-ionlaser.PropidiumiodideisusedasaDNAstainforbothflowcytometry,toevaluatecellviabilityorDNAcontentincellcycleanalysis,andmicroscopytovisualisethenucleusandotherDNAcontainingorganelles.Itcanbeusedtodifferentiatenecrotic,apoptoticandnormalcells.,PI,EB,19,ConfocalimmunofluorescentanalysisofHeLacellsusing-Tubulin(DM1A)MousemAb(green).Red=PropidiumIodide.,P.marneffeiyeastcellswithpropidiumiodidelabellednuclei,20,九、DPH（Diphenylhexatriene）,Diphenylhexatrieneisusedinthestudyofcellmembranes.Itisalmostnon-fluorescentinwater,butitexhibitsstrongfluorescencewhenitisintercalatedintolipidmembranes.Itincorporatesitselfintothelipidbilayerandactslikealipid.,21,十、Cy系列-Cy5.5、Cy7,1、Cy5.5,Cy5.5isafar-red(andnear-infrared)emittingdyewhichisidealforfluorescencemeasurementswherebackgroundfluorescenceisaconcern.Itisalsosuitableforinvivoimagingexperiments.,22,23,2、Cy7,NIRfluorophoresc