常用熒光探針小結演示文稿

常用熒光探針小結演示文稿本文檔共24頁；當前第1頁；編輯于星期六\4點20分Exitationλmax:495nm;Emissionλmax:519nm;SolventpH:8.002本文檔共24頁；當前第2頁；編輯于星期六\4點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本文檔共24頁；當前第3頁；編輯于星期六\4點20分二、熒光素酯Carboxyfluoresceindiacetatesuccinimidylester(CFDA-SE)isacellpermeabledyegenerallyusedinanimalcellproliferationresearch.CFDA-SEenterscellsbydiffusionandiscleavedbyintracellular

esteraseenzymestoformanamine-reactiveproduct,CFSE.Thisproductproducesadetectablefluorescenceandcovalentlybindstointracellularlysineresiduesandotheraminesources.酯酶4本文檔共24頁；當前第4頁；編輯于星期六\4點20分視頻：TheUseofCarboxyfluoresceinDiacetateSuccinimidylEster(CFSE)toMonitorLymphocyteProliferation5本文檔共24頁；當前第5頁；編輯于星期六\4點20分

四甲基異硫氰酸羅達明，它是一種紫紅色粉末，較穩(wěn)定。其最大吸收光譜為550nm，最大發(fā)射光譜620nm，呈橙紅色熒光，與FITC的黃綠色熒光對比清晰，與蛋白質(zhì)結合方式同TITC。它可用于雙標記示蹤研究。三、異硫氰酸羅丹明（TMRITC）6本文檔共24頁；當前第6頁；編輯于星期六\4點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本文檔共24頁；當前第7頁；編輯于星期六\4點20分

AC41323-0010

RB200，也稱麗絲胺羅丹明B無定形褐紅色粉末，不溶于水，易溶于酒精和丙酮，性質(zhì)穩(wěn)定，可長期保存，最大吸收光譜為570nm，呈明亮的橙色熒光，因與FITC的黃綠色有明顯區(qū)別，故被廣泛用于對比染色或用于兩種不同顏色的熒光抗體的雙重染色。標記方法方法：取1gRB200及五氯化磷（PCL5）2g放乳缽中研磨5min（在毒氣操作櫥中），加10ml無水丙酮，放置5min，隨時攪拌，過濾，用所得溶液進行結合。將每亳升血清用1ml生理鹽水及1ml碳酸鹽緩沖液（0.5mol/L，pH9.5）稀釋，逐滴加入0.1mlRB200溶液，隨加隨攪拌，在0-4℃繼續(xù)攪拌12-18h。四、羅丹明2008本文檔共24頁；當前第8頁；編輯于星期六\4點20分ConfocalimageofdoubleimmunostainingforAktinhippocampalCA1pyramidalneurons.Sectionisshownfromanormalrat.TheredcolorderivedfromlissaminerhodamineconjugatedsecondaryantibodyrepresentsMAP2,thegreencolorderivedfromfluoresceinindicatesthelabelingofAkt.Yellowrepresentsoverlayofredandgreen.9本文檔共24頁；當前第9頁；編輯于星期六\4點20分五、溴化乙錠詳見第四節(jié)“應用于核酸檢測的熒光探針技術”10本文檔共24頁；當前第10頁；編輯于星期六\4點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本文檔共24頁；當前第11頁；編輯于星期六\4點20分DAPI(magenta)boundtotheminorgrooveofDNA(greenandblue).DAPIcanbeusedforfixedcellstaining,theconcentrationofDAPIneededforlivecellstainingisgenerallyveryhighandrarelyusedforlivecells.

ThoughitwasnotshowntohavemutagenicitytoE.coli,

itislabelledasaknownmutageninmanufacturerinformation.

AsitisaDNAbindingcompounditislikelytohavesomelowlevelmutagenicpropertiesandcareshouldbetakeninitshandlinganddisposal.12本文檔共24頁；當前第12頁；編輯于星期六\4點20分Atypeofsimplefluorescence:DAPIisexcitedinnearUVwith365nmandemittedintheviolet-bluespectralrange.ThemarkingrevealscellnucleiandparticularlythechromosomesDAPIisadyewhichbindstoDNAandshowsanincreasedbluefluorescencewhenboundtoDNA.Thebluefluorescentspotsinthepictureshowthenucleiinthemycel.13本文檔共24頁；當前第13頁；編輯于星期六\4點20分Endothelialcellsunderthemicroscope.NucleiarestainedbluewithDAPI,microtublesaremarkedgreenbyanantibodyandactinfilamentsarelabelledredwithphalloidin.14本文檔共24頁；當前第14頁；編輯于星期六\4點20分七、Hoechst

Hoechststainsarepartofafamilyofbluefluorescent

dyesusedtostain

DNA.TheseBis-benzimideswereoriginallydevelopedbyHoechstAG.TherearethreerelatedHoechststains:Hoechst33258,Hoechst33342,andHoechst34580.ThedyesHoechst33258andHoechst33342aretheonesmostcommonlyusedandtheyhavesimilarexcitation/emissionspectra.15本文檔共24頁；當前第15頁；編輯于星期六\4點20分16本文檔共24頁；當前第16頁；編輯于星期六\4點20分TransmissionimageofHeLacells,withoverlayofHoechst33258staining(blue).Hoechst33258(magenta)boundtotheminorgrooveofDNA(greenandblue)17本文檔共24頁；當前第17頁；編輯于星期六\4點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本文檔共24頁；當前第18頁；編輯于星期六\4點20分ConfocalimmunofluorescentanalysisofHeLacellsusingα-Tubulin(DM1A)MousemAb(green).Red=PropidiumIodide.P.marneffeiyeastcellswithpropidiumiodidelabellednuclei19本文檔共24頁；當前第19頁；編輯于星期六\4點20分九、DPH（

Diphenylhexatriene

）Diphenylhexatrieneisusedinthestudyofcellmembranes.Itisalmostnon-fluorescentinwater,butitexhibitsstrongflu