常用荧光探针小结演示文稿

常用荧光探针小结演示文稿本文档共24页；当前第1页；编辑于星期六\4点20分Exitationλmax:495nm;Emissionλmax:519nm;SolventpH:8.002本文档共24页；当前第2页；编辑于星期六\4点20分Immunocytochemistry/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本文档共24页；当前第3页；编辑于星期六\4点20分二、荧光素酯Carboxyfluoresceindiacetatesuccinimidylester(CFDA-SE)isacellpermeabledyegenerallyusedinanimalcellproliferationresearch.CFDA-SEenterscellsbydiffusionandiscleavedbyintracellular

esteraseenzymestoformanamine-reactiveproduct,CFSE.Thisproductproducesadetectablefluorescenceandcovalentlybindstointracellularlysineresiduesandotheraminesources.酯酶4本文档共24页；当前第4页；编辑于星期六\4点20分视频：TheUseofCarboxyfluoresceinDiacetateSuccinimidylEster(CFSE)toMonitorLymphocyteProliferation5本文档共24页；当前第5页；编辑于星期六\4点20分

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）6本文档共24页；当前第6页；编辑于星期六\4点20分Detectionofα-tubulininA549cellsdemonstratesuseofrhodamine-labeledsecondaryantibody.

Cellswereprobedwithamouseanti-α-tubulinprimaryantibody(0.4µg/mL)andRhodamine-goatanti-mousesecondaryantibody(2µg/mL).NucleiwerelabeledwithHoechstDye.Imageswereacquiredbyfluorescencemicroscopy.A.Fluorescenceimageshowsadelicatenetworkofα-tubulin(pseudo-coloredgreen)locatedexclusivelyinthecytoplasm.B.NuclearcounterstainwithHoechstDye(pseudo-coloredblue)C.Mergedimage.7本文档共24页；当前第7页；编辑于星期六\4点20分

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2008本文档共24页；当前第8页；编辑于星期六\4点20分ConfocalimageofdoubleimmunostainingforAktinhippocampalCA1pyramidalneurons.Sectionisshownfromanormalrat.TheredcolorderivedfromlissaminerhodamineconjugatedsecondaryantibodyrepresentsMAP2,thegreencolorderivedfromfluoresceinindicatesthelabelingofAkt.Yellowrepresentsoverlayofredandgreen.9本文档共24页；当前第9页；编辑于星期六\4点20分五、溴化乙锭详见第四节“应用于核酸检测的荧光探针技术”10本文档共24页；当前第10页；编辑于星期六\4点20分六、DAPI

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

nm(ultraviolet)anditsemissionmaximumisat461

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

nmwhenboundtoRNA.11本文档共24页；当前第11页；编辑于星期六\4点20分DAPI(magenta)boundtotheminorgrooveofDNA(greenandblue).DAPIcanbeusedforfixedcellstaining,theconcentrationofDAPIneededforlivecellstainingisgenerallyveryhighandrarelyusedforlivecells.

ThoughitwasnotshowntohavemutagenicitytoE.coli,

itislabelledasaknownmutageninmanufacturerinformation.

AsitisaDNAbindingcompounditislikelytohavesomelowlevelmutagenicpropertiesandcareshouldbetakeninitshandlinganddisposal.12本文档共24页；当前第12页；编辑于星期六\4点20分Atypeofsimplefluorescence:DAPIisexcitedinnearUVwith365nmandemittedintheviolet-bluespectralrange.ThemarkingrevealscellnucleiandparticularlythechromosomesDAPIisadyewhichbindstoDNAandshowsanincreasedbluefluorescencewhenboundtoDNA.Thebluefluorescentspotsinthepictureshowthenucleiinthemycel.13本文档共24页；当前第13页；编辑于星期六\4点20分Endothelialcellsunderthemicroscope.NucleiarestainedbluewithDAPI,microtublesaremarkedgreenbyanantibodyandactinfilamentsarelabelledredwithphalloidin.14本文档共24页；当前第14页；编辑于星期六\4点20分七、Hoechst

Hoechststainsarepartofafamilyofbluefluorescent

dyesusedtostain

DNA.TheseBis-benzimideswereoriginallydevelopedbyHoechstAG.TherearethreerelatedHoechststains:Hoechst33258,Hoechst33342,andHoechst34580.ThedyesHoechst33258andHoechst33342aretheonesmostcommonlyusedandtheyhavesimilarexcitation/emissionspectra.15本文档共24页；当前第15页；编辑于星期六\4点20分16本文档共24页；当前第16页；编辑于星期六\4点20分TransmissionimageofHeLacells,withoverlayofHoechst33258staining(blue).Hoechst33258(magenta)boundtotheminorgrooveofDNA(greenandblue)17本文档共24页；当前第17页；编辑于星期六\4点20分八、碘化丙啶(PropidiumIodide,PI）Propidiumiodideisanintercalatingagentandafluorescent

moleculethatcanbeusedtostain

cells.WhenPIisboundtonucleicacids,thefluorescenceexcitationmaximumis535

nmandtheemissionmaximumis617

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

andmicroscopytovisualisethenucleusandotherDNAcontainingorganelles.Itcanbeusedtodifferentiatenecrotic,apoptoticandnormalcells.PIEB18本文档共24页；当前第18页；编辑于星期六\4点20分ConfocalimmunofluorescentanalysisofHeLacellsusingα-Tubulin(DM1A)MousemAb(green).Red=PropidiumIodide.P.marneffeiyeastcellswithpropidiumiodidelabellednuclei19本文档共24页；当前第19页；编辑于星期六\4点20分九、DPH（

Diphenylhexatriene

）Diphenylhexatrieneisusedinthestudyofcellmembranes.Itisalmostnon-fluorescentinwater,butitexhibitsstrongflu