常用荧光探针小结复习进程

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。1Exitationλmax:495nm;Emissionλmax:519nm;SolventpH:8.002Immunocytochemistry/Immunofluorescence-alphaTubulinantibody[DM1A](FITC)(ab64503)

ICC/IFimageofab64503stainedhumanHeLacells.Thecellsweremethanolfixed(10min),permabilisedin0.1%PBS-Tween(20min)andincubatedwiththeantibody(ab64503,1µg/ml,FITCconjugated(green))for1hatroomtemperature.1%BSA/10%normalgoatserum/0.3Mglycinewasusedtoblocknon-specificprotein-proteininteractions.AlexaFluor®594WGAwasusedtolabelplasmamembranes(red).DAPIwasusedtostainthecellnuclei(blue).3二、荧光素酯Carboxyfluoresceindiacetatesuccinimidylester(CFDA-SE)isacellpermeabledyegenerallyusedinanimalcellproliferationresearch.CFDA-SEenterscellsbydiffusionandiscleavedbyintracellular

esteraseenzymestoformanamine-reactiveproduct,CFSE.Thisproductproducesadetectablefluorescenceandcovalentlybindstointracellularlysineresiduesandotheraminesources.酯酶4视频：TheUseofCarboxyfluoresceinDiacetateSuccinimidylEster(CFSE)toMonitorLymphocyteProliferation5

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）67

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2008ConfocalimageofdoubleimmunostainingforAktinhippocampalCA1pyramidalneurons.Sectionisshownfromanormalrat.TheredcolorderivedfromlissaminerhodamineconjugatedsecondaryantibodyrepresentsMAP2,thegreencolorderivedfromfluoresceinindicatesthelabelingofAkt.Yellowrepresentsoverlayofredandgreen.9五、溴化乙锭详见第四节“应用于核酸检测的荧光探针技术”10六、DAPI

（4‘,6-diamidino-2-phenylindole）DAPIwasfirstsynthesisedinaspartofasearchfordrugstotreattrypanosomiasis.Althoughitwasunsuccessfulasadrug,furtherinvestigationindicateditboundstronglytoDNAandbecamemorefluorescentwhenbound.Whenboundtodouble-strandedDNADAPIhasanabsorptionmaximumatawavelengthof358

nm(ultraviolet)anditsemissionmaximumisat461

nm(blue).ThereforeforfluorescencemicroscopyDAPIisexcitedwithultravioletlightandisdetectedthroughablue/cyanfilter.TheemissionpeakisfairlybroadDAPIwillalsobindtoRNA,thoughitisnotasstronglyfluorescent.Itsemissionshiftstoaround500

nmwhenboundtoRNA.11DAPI(magenta)boundtotheminorgrooveofDNA(greenandblue).DAPIcanbeusedforfixedcellstaining,theconcentrationofDAPIneededforlivecellstainingisgenerallyveryhighandrarelyusedforlivecells.

ThoughitwasnotshowntohavemutagenicitytoE.coli,

itislabelledasaknownmutageninmanufacturerinformation.

AsitisaDNAbindingcompounditislikelytohavesomelowlevelmutagenicpropertiesandcareshouldbetakeninitshandlinganddisposal.12Atypeofsimplefluorescence:DAPIisexcitedinnearUVwith365nmandemittedintheviolet-bluespectralrange.ThemarkingrevealscellnucleiandparticularlythechromosomesDAPIisadyewhichbindstoDNAandshowsanincreasedbluefluorescencewhenboundtoDNA.Thebluefluorescentspotsinthepictureshowthenucleiinthemycel.13Endothelialcellsunderthemicroscope.NucleiarestainedbluewithDAPI,microtublesaremarkedgreenbyanantibodyandactinfilamentsarelabelledredwithphalloidin.14七、Hoechst

Hoechststainsarepartofafamilyofbluefluorescent

dyesusedtostain

DNA.TheseBis-benzimideswereoriginallydevelopedbyHoechstAG.TherearethreerelatedHoechststains:Hoechst33258,Hoechst33342,andHoechst34580.ThedyesHoechst33258andHoechst33342aretheonesmostcommonlyusedandtheyhavesimilarexcitation/emissionspectra.1516TransmissionimageofHeLacells,withoverlayofHoechst33258staining(blue).Hoechst33258(magenta)boundtotheminorgrooveofDNA(greenandblue)17八、碘化丙啶(PropidiumIodide,PI）Propidiumiodideisanintercalatingagentandafluorescent

moleculethatcanbeusedtostain

cells.WhenPIisboundtonucleicacids,thefluorescenceexcitationmaximumis535

nmandtheemissionmaximumis617

nm.Excitationenergycanbesuppliedwithaxenonormercury-arclamporwiththe488lineofanargon-ionlaser.PropidiumiodideisusedasaDNAstainforbothflowcytometry,toevaluatecellviabilityorDNAcontentincellcycleanalysis,

andmicroscopytovisualisethenucleusandotherDNAcontainingorganelles.Itcanbeusedtodifferentiatenecrotic,apoptoticandnormalcells.PIEB18ConfocalimmunofluorescentanalysisofHeLacellsusingα-Tubulin(DM1A)MousemAb(green).Red=PropidiumIodide.P.marneffeiyeastcellswithpropidiumiodidelabellednuclei19九、DPH（

Diphenylhexatriene

）Diphenylhexatrieneisusedinthestudyofcellmembranes.Itisalmostnon-fluorescentinwater,but