常用熒光探針小結(jié)演示文稿

常用熒光探針小結(jié)演示文稿當(dāng)前第1頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)Exitationλmax:495nm;Emissionλmax:519nm;SolventpH:8.002當(dāng)前第2頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)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當(dāng)前第3頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)二、熒光素酯Carboxyfluoresceindiacetatesuccinimidylester(CFDA-SE)isacellpermeabledyegenerallyusedinanimalcellproliferationresearch.CFDA-SEenterscellsbydiffusionandiscleavedbyintracellular

esteraseenzymestoformanamine-reactiveproduct,CFSE.Thisproductproducesadetectablefluorescenceandcovalentlybindstointracellularlysineresiduesandotheraminesources.酯酶4當(dāng)前第4頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)視頻：TheUseofCarboxyfluoresceinDiacetateSuccinimidylEster(CFSE)toMonitorLymphocyteProliferation5當(dāng)前第5頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)

四甲基異硫氰酸羅達(dá)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(duì)比清晰，與蛋白質(zhì)結(jié)合方式同TITC。它可用于雙標(biāo)記示蹤研究。三、異硫氰酸羅丹明（TMRITC）6當(dāng)前第6頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)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當(dāng)前第7頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)

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(zhǎng)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(duì)比染色或用于兩種不同顏色的熒光抗體的雙重染色。標(biāo)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(shí)攪拌，過濾，用所得溶液進(jìn)行結(jié)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當(dāng)前第8頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)ConfocalimageofdoubleimmunostainingforAktinhippocampalCA1pyramidalneurons.Sectionisshownfromanormalrat.TheredcolorderivedfromlissaminerhodamineconjugatedsecondaryantibodyrepresentsMAP2,thegreencolorderivedfromfluoresceinindicatesthelabelingofAkt.Yellowrepresentsoverlayofredandgreen.9當(dāng)前第9頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)五、溴化乙錠詳見第四節(jié)“應(yīng)用于核酸檢測(cè)的熒光探針技術(shù)”10當(dāng)前第10頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)六、DAPI

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

nm(ultraviolet)anditsemissionmaximumisat461

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

nmwhenboundtoRNA.11當(dāng)前第11頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)DAPI(magenta)boundtotheminorgrooveofDNA(greenandblue).DAPIcanbeusedforfixedcellstaining,theconcentrationofDAPIneededforlivecellstainingisgenerallyveryhighandrarelyusedforlivecells.

ThoughitwasnotshowntohavemutagenicitytoE.coli,

itislabelledasaknownmutageninmanufacturerinformation.

AsitisaDNAbindingcompounditislikelytohavesomelowlevelmutagenicpropertiesandcareshouldbetakeninitshandlinganddisposal.12當(dāng)前第12頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)Atypeofsimplefluorescence:DAPIisexcitedinnearUVwith365nmandemittedintheviolet-bluespectralrange.ThemarkingrevealscellnucleiandparticularlythechromosomesDAPIisadyewhichbindstoDNAandshowsanincreasedbluefluorescencewhenboundtoDNA.Thebluefluorescentspotsinthepictureshowthenucleiinthemycel.13當(dāng)前第13頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)Endothelialcellsunderthemicroscope.NucleiarestainedbluewithDAPI,microtublesaremarkedgreenbyanantibodyandactinfilamentsarelabelledredwithphalloidin.14當(dāng)前第14頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)七、Hoechst

Hoechststainsarepartofafamilyofbluefluorescent

dyesusedtostain

DNA.TheseBis-benzimideswereoriginallydevelopedbyHoechstAG.TherearethreerelatedHoechststains:Hoechst33258,Hoechst33342,andHoechst34580.ThedyesHoechst33258andHoechst33342aretheonesmostcommonlyusedandtheyhavesimilarexcitation/emissionspectra.15當(dāng)前第15頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)16當(dāng)前第16頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)TransmissionimageofHeLacells,withoverlayofHoechst33258staining(blue).Hoechst33258(magenta)boundtotheminorgrooveofDNA(greenandblue)17當(dāng)前第17頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)八、碘化丙啶(PropidiumIodide,PI）Propidiumiodideisanintercalatingagentandafluorescent

moleculethatcanbeusedtostain

cells.WhenPIisboundtonucleicacids,thefluorescenceexcitationmaximumis535

nmandtheemissionmaximumis617

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

andmicroscopytovisualisethenucleusandotherDNAcontainingorganelles.Itcanbeusedtodifferentiatenecrotic,apoptoticandnormalcells.PIEB18當(dāng)前第18頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)ConfocalimmunofluorescentanalysisofHeLacellsusingα-Tubulin(DM1A)MousemAb(green).Red=PropidiumIodide.P.marneffeiyeastcellswithpropidiumiodidelabellednuclei19當(dāng)前第19頁(yè)\共有24頁(yè)\編于星期三\4點(diǎn)九、DPH（

Diphenylhexatriene

）Diphenylhexatrieneisusedinthestudyofcellmembranes.Itisalmostnon-fluorescentinwater,butitexhi