Mapping Protein Distributions on Polytene Chromosomes by Immunostaining

INTRODUCTION

Theformidablesizeandstructureofpolytenechromosomesallowmappingofchromosomalproteindistributionsatveryhighresolution.ThisprotocoldescribesthepreparationofpolytenechromosomesfromDrosophilalarvae,immunostainingofthechromosomeswithaproteinofinterest,andcounterstainingwithGiemsaandHoechst.

RELATEDINFORMATION

TechnicalmodificationsincorporatedintothisprotocolthathaveenabledmorereproducIBLepatternsofbandinginimmunostainedchromosomeswereinitiallydescribedbySilverandElgin(1976).Forfurtherdetailsondissectinglarvalsalivaryglands,seeDissectionofLarvalSalivaryGlandsandPolyteneChromosomePreparation(Kennison2008).

MATERIALS

Reagents

Ammoniumsulfate(50%[w/v])(optional,seeStep23)

Antibodies,primary

Affinity-purifiedprimaryantibodiesaredilutedinPBScontaining1%BSA.Thedilutionsmustbeadjustedforeachindividualprimaryantibody.Typicaldilutionsforrabbitpolyclonalantibodiesrangebetween1:50and1:500.Fordouble-labelingexperiments,useprimaryantibodiesraisedintwodifferentspecies.

Antibodies,secondary

SecondaryantibodiesaredilutedinPBScontaining2%normalserum,obtainedfromthesamespeciesasthesecondaryantibody.Dependingonthemethodemployed,useanti-rabbitIgG(Fc)horserADIshperoxidase(HRP)conjugate(1:100dilution),abiotin-conjugatedsecondaryantibody,orafluorescence-conjugatedsecondaryantibody.Fordouble-labelingexperiments,usefluorescence-conjugatedsecondaryantibodiesraisedtothecorrespondingtypeofprimaryantibody.Thedilutionfactorforeachantibodyneedstobedeterminedexperimentally.

Blockingsolution(asmallspoonfulofnonfatdrymilkin40mLPBS)

Bovineserumalbumin(BSA)

3,3""-diaminobenzidinetetrahydrochloride(DAB;SigmaD5637)

Detergent(optional,seeStep2)

Drosophila(seeSteps7-9)

Entellan(EMD)(optional,seeStep33)

Ethanol(95%)

Fixingsolution

Giemsa(Merck109204)

Glycerol(99.5%)

H₂O₂(Merck107210)

Hoechststainingsolution

Liquidnitrogen

Methanol(optional,seeStep23)

Mowiol-DABCOstocksolution

Nutrient-richflymedium

Phosphate-bufferedsaline(PBS;pH7.5)

Poly-L-lysinesolution(0.1%[w/v]inH₂O;SigmaP8920)

Sodiumphosphatebuffer(10mM,pH6.8)

TritonX-100

VECTASTAINEliteABCKit(VectorLaboratoriesPK6100)(optional,seeStep30)

Washsolution1

Washsolution2

Equipment

Aluminumfoil

Diamondpen

Eyeprotection(seeStep21)

Filterpaper(e.g.,Whatman3MM)

Humidchamberforslideincubation

Latexgloves

Microscope,dissecting

Microscope,phase-contrast

Pencilwitheraserend

Rackforholdingslides

Razorblade

Shakingplatform

Siliconizedcoverslips(Corningorequivalent;22x22mm)

Slides

Slidejars

Squashingapparatus(optional,seeStep17)

Forextendedchromosome-spreadingsessions,usethecustom-madesquashingapparatus(Fig.1).

Viewlargerversion(17K):[inthiswindow][inanewwindow]

Figure1.Schematicdiagramofthesquashingapparatus.Thisapparatuseasestheeffortwhensquashingchromosomesoveranextendedperiod,andproducesmorehomogeneouspressureovertheentirecoverslip.Asmallblockmadeofpolyvinylchloride(2.5x2.5x1.5cm)isattachedtoaflexibleTeflonribbonasshown.Theblockshouldhang~2-3mmabovetheplaneoftheholdingblock(alsomadeofpolyvinylchloride).Apieceoffilterpaper(i.e.,Whatman3MM)andtheslidewiththecoversliparethenpositionedontheholdingblockandheldinplacebythesUSPendedblock. Tweezers METHOD PreparationofPoly-L-Lysine-CoatedSlides 1.Place100-200slidesinracks. 2.(Optional)Washslidesfor2hinastrongdetergent.Ingeneral,high-qualityslidesdonotrequireanypretreatmentwithdetergent.However,insomecases,pretreatmentwithastrongdetergentmaybenecessarytoensureahomogeneouswettingofthesurfacebythepoly-L-lysinesolutioninStep5. 3.Washfor2hinrunningtapwater.RinsetwiceinH2O. 4.Dipslidestwicein95%ethanol.Air-dry. 5.Dipslidesintothepoly-L-lysinesolution.Thesolutionshouldwettheglasssurfaceuniformlyandstayontheslides. 6.Air-drytheslides,andstorethemat4°C. RaisingThird-InstarLarvae Thequalityofthechromosomesdependscriticallyonthestateofnutritionandwell-beingofthelarvae.Thisisparticularlyimportanttoconsiderincaseswherethegeneticbackground(e.g.,unhealthymutantsorcertaintransgeniclines)placesaheavytollonthedevelopmentofthelarvae. 7.Raisefliesin175-mLbottlescontainingnutrient-richflymedium. 8.Allowthefliestolayeggsjusttothepointwherelarvaewillhatchunderuncrowdedconditions(i.e.,fewerthan100larvaeina175-mLbottle).Letthelarvaedevelopat18°C. 9.Forsalivaryglandpreparations,usethird-instarlarvaethatarestillcrawlingandhavenotyetstartedtopupate. DissectingLarvalSalivaryGlandsandFixingChromosomes 10.Foreachslide,removetwolarvaefromthebottle,andwashtheminPBS. 11.KeepthelarvaeinPBS,anddissectoutthetwopairsofsalivaryglandsfromeachlarvausingtweezers.Carefullyremovemostoftheattachedwhitefat-bodycellswithoutdamagingorseparatingthesalivaryglands.Forfurtherdetailsondissectinglarvalsalivaryglands,seeDissectionofLarvalSalivaryGlandsandPolyteneChromosomePreparation(Kennison2008). 12.Holdthesalivaryglandsatthecommonductwithtweezers.Transfertheglandstoadropoffixingsolutiononasiliconizedcoverslip. 13.Incubatetheglandsfor10-20min,occasionallystirringwiththetipofthetweezerstoensurehomogeneousfixation.Thefixationtimeisanimportantparameterandneedstobeadjustedforeveryantigentested.Aimfortheshortesttimenecessarytovisualizethesignal,asextendedfixationtimeswillresultindifficultieswithspreadingthechromosomes.Unlikethesoletreatmentwithacids,e.g.,DissectionofLarvalSalivaryGlandsandPolyteneChromosomePreparation(Kennison2008),theinclusionofformaldehydeinthefixingsolutionkeepsthechromosomesticky,makingitmoredifficulttoobtainpreparationswithwell-spreadchromosomearms. PreparingChromosomeSquashes 14.Takeupthecoverslipwithapoly-L-lysine-treatedslide. 15.Tapthecoverslipwiththeeraserendofapenciluntilthecellsarebrokenup.Thiscanbemonitoredbestwhendoneagainstablackbackground. 16.Movetheeraserendofthepenciloverthecoversliptospreadthechromosomes.Toavoidextensivemovementofthecoverslip,holddownthecoverslipwiththetipofafinger(uselatexglovestopreventacidburns).Donotcontinuetospreadthespecimenifthecoverslipstickstotheslide,asthiswillresultinshearingofthechromosomes.Thechromosomesareverybrittleatthispointoftheprocess.Theextentofspreadingdependsontheconstitutionandsizeofthechromosomes,aswellasonthetimeoffixation. 17.Inverttheslide,andplaceitoveronelayeroffilterpaper.Applyfirmpressurewiththethumb;thechromosomesbecomeflattenedandpressedtotheslide.Thesquashingapparatus(Fig.1)cansubstantiallyeasethisstepbecauseitallowsmorefirmandhomogeneouspressureonthechromosomeswithouttheriskofmovingthecoverslip. 18.Examinethepreparationbyphase-contrastmicroscopy.Useonlypreparationswithwell-spreadchromosomearmsshowinghigh-contrastbandingpatterns. 19.Markthepositionofthecoverslipwithadiamondpen. 20.Freezetheslideinliquidnitrogen. 21.Removecoverslipwitharazorblade(wearappropriateeyeprotection). 22.ImmediatelydiptheslideintoPBS,andwashitfor15minwithgentleshaking.RepeatthiswashonceinfreshPBS. 23.Proceedwiththeimmunostainingasdescribedbelow.Alternatively,keeptheslide(forupto1wk)in100%methanolorin50%(w/v)ammoniumsulfateat4°C.Certainantigensmightbeaffectedbyexposuretomethanol. Immunostaining 24.WashstoredslidestwiceinPBS(10mineachwash). 25.WashslidesonceinPBScontaining1%TritonX-100for10min. 26.Incubateslidesinaslidejarcontainingblockingsolutionfor1hatroomtemperatureorovernightat4°Cwithgentleshaking.Drainoffexcesssolution. 27.Add40µLofdilutedaffinity-purifiedprimaryantibodytoeachslide,andcoverwithacoverslip.Incubatetheslidesfor1hatroomtemperature. 28.Incubatetheslidesovernightat4°Cinahumidchamber. 29.RinsetheslidesinPBStoremovethecoverslips,andplacetheminarack.Washtheslidesthreetimesinblockingsolution(5mineachwash)withthoroughshaking. 30.Probetheslideswithasecondaryantibodydetectionsystemusingoneofthethreemethodsbelow.Ifonlyweaksignalsareobservedusingenzyme-coupledsecondaryantibodies(Steps30.i-30.vi),amplificationwiththebiotin-avidinsystemmightbehelpful(Steps30.vii-30.xiii).SecondaryantibodiestaggedwithaFluorochrome(Steps30.xiv-30.xvii)canbeusedtospeedupthedetectionprocedure,aswellasfordouble-labelingexperiments. Toimmunostainusingenzyme-coupledsecondaryantibodies: i.RinseslidesinPBSandremoveexcesssolution. ii.Add40µLofsecondaryantibody(e.g.,anti-rabbitIgG[Fc]HRPconjugate,1:100dilution)toeachslide,andcoverwithacoverslip.Incubatefor1hatroomtemperatureinahumidchamber.Often,commerciallyavailablesecondaryantibodypreparationsshowahighaffinityforDrosophilanuclearproteins,resultinginsomeexcellentsignalsonthechromosomes.Assuch,acorrespondingcontrolexperimentshouldalwaysbeincluded. iii.RinseslidesinPBSandplaceinrack.Washslidesinwashsolution1for15minandtheninwashsolution2for15min.Shakerackthoroughlyduringthewashingprocedure. iv.RinseslidesinPBS.Add100µLofasolutioncontaining0.5mg/mLDABand0.01%H2O2. v.Topreventoverstaining,followtheappearanceofthesignalsunderthemicroscopeusingbright-fieldoptics.StopthereactionbydippingtheslidesinPBS.WashinPBSfor10min. vi.CounterstainwithGiemsa(seeSteps30-33). Toimmunostainusingbiotin-conjugatedsecondaryantibodiesandenzymaticdetectionwiththeavidin/biotinsystem: vii.Toeachslide,add40µLofsecondaryantibodyconjugatedtobiotin,andcoverwithacoverslip.Incubatefor1hatroomtemperatureinahumidchamber. viii.Duringsecondaryantibodyincubation,mixthebiotinsolutionandtheavidin-HRPsolutiontoallowtheformationofcomplexes.Mix40µLofsolutionAand40µLofsolutionB(fromtheVECTASTAINEliteABCkit)in1mLofPBScontaining0.1%BSAfor10minatroomtemperature. ix.Afterincubationwithsecondaryantibodies,rinseslidestwiceinPBScontaining0.1%BSA(10mineach).Removeexcessliquid. x.Place50µLofthebiotin-avidin-HRPmixonthepreparationandcoverwithacoverslip.Incubatetheslidesfor40minatroomtemperatureinahumidchamber. xi.Washslidesinwashsolution1for15minandtheninwashsolution2for15min. xii.RinseslidesinPBS.Add100µLofasolutioncontaining0.5mg/mLDABand0.01%H2O2. xiii.Topreventoverstaining,followtheappearanceofthesignalsunderthemicroscopeusingbright-fieldoptics.StopthereactionbydippingslidesinPBS.WashinPBSfor10min. Toimmunostainusingfluorescence-coupledsecondaryantibodies: xiv.RinseslidesinPBSandremoveexcesssolution.Afterthispoint,performallstepsunderdimmed-lightconditions.Duringincubations,wraptheslidejarsinaluminumfoiltominimizebleachingoffluorochromes. xv.Add40µLofdilutedfluorescence-conjugatedsecondaryantibodysolutiontotheslide,andcoverwithacoverslip.Incubatefor1hatroomtemperatureinahumidchamber.Fordouble-labelingexperiments,bothsecondaryantibodiesoftheappropriatetypecanbeappliedsimultaneouslyduringthisstep. xvi.RinseslidesinPBSandplaceinarack.Washslidesinwashsolution1for15minandtheninwashsolution2for15min. xvii.StoreslidesinPBSat4°CinthedarkorproceedtocounterstainwithHoechst33258(seeSteps35-37). GiemsaStaining ChromosomeswithHRPsignalsshouldbecounterstainedwithGiemsa. 31.Preparea1:130dilutionofGiemsain10mMsodiumphosphatebuffer(pH6.8). 32.Stainslidesfor30secto1min.RinseslidesbydippingseveraltimesintoH2O. 33.Mountin99.5%glycerolwithacoverslip.Immediatelyexaminetheslidesunderthemicroscope.Giemsastainwillfadewithinafewhours.However,chromosomescanbewashedinPBSandrestained. 34.Forstorage,slidescanbefrozenat–20°C.Entellan(EMD)canbeusedasapermanentmountingsolution. StainingwithHoechst33258 ChromosomeswithfluorescentsignalscanbecounterstainedwithHoechst33258.However,bandingpatternsproducedbythefluorescentDNAstainsareoftendifficulttocomparewiththepublishedpolytenechromosome-bandingpatterns.Itisoftenmoreconvenienttosimplyusephase-contrasttocorrelatethefluorescentsignalwiththecorrespondingchromosomebands. 35.StainslidesinHoechststainingsolutionfor5min. 36.MountpreparationswithfluorescentsignalsinMowiol-DABCOstocksolution. 37.Examinetheslidesusingafluorescencemicroscope. TROUBLESHOOTING Problem:Backgroundstainingishigh. [Steps30,33,or37] Solution:TheconcentrationofNaClinwashsolution2canberaisedto500mM. Problem:HRPstainingisweak. [Step30] Solution:SilvertreatmentofthepreparationturnsthebrowncolorofaDABsignaltoblack,thussubstantiallyimprovingthecontrast,whichisveryconvenientwhenblack-and-whitephotographyisusedfordocumentation.UsetheSigmaFASTDABwithmetalenhancer(SigmaD0426),followingthesupplier’sinstructions,butshortenthemetalamplificationstepto~1min. Problem:InGiemsa-stainedslides,stainingofchromosomebandsappearstooweakunderbright-fieldoptics. [Step33] Solution:RepeatGiemsastainingstartingwithStep32. DISCUSSION Immunostainingofpolytenechromosomesismostoftenusedformappingdistributionsofchromosome-associatedproteins,identifyingandcharacterizingcis-regulatoryDNAelementsboundbyparticularproteins,andmappingfunctionalproteindomainsnecessaryforchromosomalbindingorotheractivities,suchastheinteractionwithotherpartnerproteins.Forexample,atransgeneconstructcontainingacis-regulatoryDNAelementmaycreateanewprotein-bindingsiteattheintegrationsiteonachromosome(Fig.2). Viewlargerversion(95K):[inthiswindow][inanewwindow] Figure2.DistributionofthePolycombproteinatsection43-49ofthesecondchromosomevisualizedwithenzyme-conjugated(HRP)secondaryantibodies.(Top)ChromosomewithatransgeneconstructcontainingaPolycomb-bindingsite(PRE)at44E(arrowhead);(bottom)wild-typechromosome. 您的称呼： 单位名称： 您的邮箱： 您的电话： 品牌与型号： 其他要求： 新闻动态 行业前沿 技术文章 最新产品 中国官网，价格更新 中国试剂购买网站 2018-08-18 萌新求助本人注册了AOCS并获得了虎之穴id,paypal也申请过了。现在卡... 2021-03-25 AOCS脂肪酸检测方法_图文_百度文库 2021-03-25 AOCS是什么意思? - AOCS的全称 | 在线英文缩略词查询 2021-03-25 一般aocs的标准哪里找呀? - 食品标准化 - 食品论坛 - Powered by... 2021-03-25 AOCS-2021 2021-03-25 进口品牌清关代理 AOCS公司 2017-03-30 aocs标准-分析测试百科网 2021-03-25 Inverse PCR for PACend sequencing 反式PCR 生物在线 Labon... 2017-08-15 小心被忽悠!AOC显示器遭遇假李逵_AOC 176S_液晶显示器导购中关村... 2019-06-29 IsolationofPBMCsfrompatientbloodandbuffycoats1 ... 2018-05-12 Inverse PCR for PACend sequencing 反式PCR 生物在线 Labon... 2021-09-15 AOCSCe1b89脂肪酸检测方法 2021-09-16 【Protocol】 for competitive RTPCR PCR技术讨论版 专业... 2021-09-18 ImmunohistochemistyEnzymatic Protocol 2021-09-17 Isolation of PBMCs from patient blood and buffy coats 实验方... 2021-09-21 IsolationofPBMCsfrompatientbloodandbuffycoats1 ... 2021-09-06 Monoclonal Antibody Production 实验方法 2021-09-23 ImmunohistochemistyEnzymatic Protocol 免疫组织化学 资讯... 2021-09-11 AOCS/AOCS Official Method Ba 4f-00/Individual Methods/111450 2026-07-07 AOCS/AOCS官方方法Ba 8-78/个别方法/111458 2026-07-07 AOCS/Soybean BASF Plant Science LP BPS-CV127-9 Powder (10g) 0911-C/Certified Reference Materials/112626 2026-07-07 AOCS/AOCS官方方法Cd 11b-91/个别方法/111521 2026-07-07 AOCS/Marine Oil/Laboratory Proficiency Program/118335 2026-07-07 AOCS/AOCS官方方法Ch 3-91/个别方法/112529 2026-07-07 AOCS/AOCS Official Method Ba 4e-93/Individual Methods/111449 2026-07-07 AOCS/AOCS官方方法Ac 2-41/个别方法/111426 2026-07-07 Copyright@苏州蚂蚁淘生物科技有限公司 2003-2021  188进口试剂采购网版权所有     苏ICP备17049038号-9 友情链接：  其他 188进口试剂采购网 www.188bio.cn -中国试剂网,试剂网,化学试剂网,国药试剂,抗体公司,试剂公司,试剂盒公司,苏州试剂公司,北京化学试剂公司,天津化学试剂,试剂商城,试剂代理,流式抗体 细胞库查询 sitemap