Immunofluorescence and Fluorescent In Situ Hybridization in Mouse Fibroblasts and Embryonic Stem Cells

TechniquesroutinelyusedinourlabforanalysingtheX-inactivationprocessindifferentiatingEScellsaredescribed.Wefocusparticularlyonchromatinchanges(histonemodifications,proteinassociation...)duringXinactivation,usingimmunofluorescence(IF)combinedwithRNAFISHorDNAFISHoninterphasenuclei.TheseshouldprovideatoolfordefiningpotentialcausalrelationshipsbetweendifferenteventsnotonlyduringXinactivationbutalsoduringtheestablishmentofotherpatternsofgeneactivity.

ThemainpurposeofacombinedIFandFISHanalysisis,ontheonehand,topreservenucleararchitectureandtheantibody"sepitopeasfaraspossIBLebut,ontheotherhand,toallowthepenetrationoftheFISHprobefordetectionofnucleartranscripts,genelocationorchromosometerritories.TheoptimalconditionsforIFareusuallypoorlycompatiblewiththoseforFISH.Wehavethereforetestedavarietyofmethodsandconditionsandwedescribeherethosethatwefindoptimalfortheimmuno-detectionofhistonemodificationscombinedwithRNAorDNAFISHonmousefibroblastsorembryonicstemcells.Formoredetails,thereaderisreferredtoChaumeiletal.,2002andChaumeiletal.,2004.

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Immunofluorescence

NumerousmethodsinvolvingavarietyoffixationandpermeABIlizationtechniquesareavailableforperformingIFandthechoicedependsoncelltype,epitopeandantibodybeingused(Spectoretal.,1998).

1. CultureundifferentiatedordifferentiatingEScellsongelatin-coatedcoverslipsforatleast24-48hours;
2. Washonceinfreshlyprepared1XPBS;
3. Fixinfreshlymade,filter-sterile3%paraformaldehyde/1XPBSfor10minutesatRTor4°C;
4. Wash3timesin1XPBSfor5minuteseach;
5. Permeabilizewithfreshlymade1XPBS/0.5%TritonX-100(containingtheRNAse-inhibitor,2mMVanadylRibonucleosideComplex,incaseofasubsequentRNA-FISH)onicefor3.5-5minutes(seenote1);
6. Wash3timesin1XPBSfor5minuteseach;
7. Blockin1XPBS/1%BSA(Gibco)for15minutesatRT;
8. Incubatewithprimaryantibodydilutedin1XPBS/1%BSA(containing0.4U/µlRNAGuardincaseofasubsequentRNA-FISH)for45minutesatRT(orotherconditions,seenote2)indark,humidchamber;
9. Washatleast3timesin1XPBSfor5minuteseach;
10. Incubatewithsecondaryantibody(dilutedinsamesolutionasaboved)for40minutesatRTindarkandhumidchamber(seenote3);
11. Washatleast3timesin1XPBSfor5minuteseach;
12. DNAcounterstaining(2minutesin1XPBScontaining0.2mg/mlDAPI);
13. Washtwicein1XPBS;
14. Mountcoversliponaslideinglycerolbasedmountingmedium.

RNAFISH

ForageneraldescriptionanddiscussionofRNAFISHprotocols,thereaderisreferredtoSpectoretal.,1998.ConditionsfordetectionofcytoplasmicversusnuclearRNAsaredifferent,andherewefocusonlyonthedetectionofnucleartranscripts.Todetecttheprimarytranscriptsofgenes,genomicprobesseveralkilobasepairslongshouldbeused.ProbesspanningintronsandexonswilldetectboththeprocessedmRNAandtheprimarytranscript.Oligonucleotideswithinintronicsequenceswillbespecificfortheprimarytranscript(seeRobertSinger"swebsiteformoredetailsonuseofoligosasprobes:http://singerlab.aecom.yu.edu/).ForthedetectionofXistRNAcoatingoftheXchromosomeincis,orprimarytranscriptsofX-linkedgenes,wehaveusedseveralgenomicDNAprobes,spanningaminimumof3kb,labelledbynicktranslationorrandompriming,withsuccess.AnexampleofRNAFISHisshowninFigure1.

PreparationofFISHprobes

1. DNAprobes,tobeusedforRNAorDNAFISH,arelabeledbynicktranslationusing1to2µgofDNAper50µlofreactionandfollowingmanufacturer"sinstructions(seenote4);
2. Approximately0.1µgofprobe(usually5µlofastandardnicktranslationreactionof50µl)isethanolprecipitatedtogetherwith10µgofsalmonspermper18x18mmcoverslip(seecomment1);
3. Performtwowashesofthepelletin70%ethanol(toremoveunincorporatednucleotides);
4. ThepelletisresUSPendedthoroughlyinformamide(5µlpercoverslip),bypipettingandincubatingat37°Cifnecessary;
5. Denaturetheprobefor7minutesat75°C;
6. Add5µlof2Xhybridizationbufferpercoverslip(seestocksolutionsection).Mixwellandkeeponice(probecanbekeptoniceforupto30minutes),whilecoverslipsarebeingpreparedfortheFISHstep(seecomment2).

RNAFISH

1. CultureEScellsongelatin-coatedglasscoverslipsorslides;
2. Washinfreshlymade,RNAse-free1XPBS;
3. PermeabilizeinfreshlymadeCSK(Cytoskeletal)buffer/0.5%TritonX-100containinganRNAseinhibitor(2mMVanadylRibonucleosideComplex)onicefor5-7minutes(seenote1andnote5andcomment3andcomment4);
4. Fixinsterileandfreshlymade3%Paraformaldehyde/1XPBSfor10minutesatRT;
5. Washtwicein70%ethanolfor5minuteseach(seenote6);
6. Dehydratein80%,95%,100%ethanol,for3minuteseach.Removecoverslipfrom100%ethanolandallowtoairdry(seecomment5);
7. Depositthedenaturedprobe(avoidingairbubbles)ontoRNAse-freeglassslideandthenplacecoverslipontothedrop,cell-sidedown;
8. Hybridizationovernightat37°Cinadarkandhumidchamber(madeusingpapertissuessoakedin50%formamide/2xSSC)(seenote7);
9. Washthreetimesin50%formamide/2XSSC(adjustedtopH7.2)for5minuteseachat42°C(seecomment6);
10. Washthreetimesin2XSSCfor5minuteseachat42°C;
11. DNAcounterstaining(2minutesin2XSSCcontaining0.2mg/mlDAPI);
12. Washtwicein2XSSCfor5minuteseach;
13. Mountthecoverslipsonaslideinglycerol-basedmountingmedium.

RNAFISHfollowingimmunofluorescence

WhenIFandFISHaretobecombined,weprefertoperformIF(underRNAse-freeconditions)priortotheFISH,astheformamidetreatmentduringtheFISHprocedureissometimesincompatiblewithpreservationoftheepitopesdetectedbysomeantibodies.AnexampleofXistRNAFISHcombinedwithIFisshowninFigure2.

PreparationofFISHprobes(seeRNAFISHsectionabove)

IF-RNAFISH

1. FollowIFprotocoluptoPBSwashesaftersecondaryantibody(noDAPIcoloration);
2. Post-fixationfor10minutesatRT,usingfilter-sterile,freshlymade3%PFA/1XPBS;
3. Washtwicein2XSSC(freshlymadefromasterile20Xstock)for5minutes;
4. Hybridizationandwashes:seeRNAFISHsectionabove.

DNAFISH

ForageneraldescriptionanddiscussionofDNAFISHprotocols,thereaderisreferredtoSpectoretal.,1998.

PreparationofFISHprobes

• Preparationofthenicktranslationprobe:
  1. FISHprobeislabeledovernight(seeRNAFISHsection);
  2. Precipitationof0.1µgofprobewith10µgofsalmonspermand5µgofCot-1DNAper18x18mmcoverslip;
  3. Twowashesofthepelletin70%ethanol;
  4. Resuspensionofthepelletin5µlofformamidepercoverslipat37°C;
  5. Denaturationfor7minutesat75°C;
  6. Competitionfor30minutesto1hourat37°C;
  7. Additionof5µlofhybridationbufferpercoverslip(seeRNAFISHsection).
• Forchromosomepaintprobes,wefollowthesupplier"srecommendations(Cambio).

DNAFISH

1. CultureEScellsongelatin-coatedglasscoverslipsorslides;
2. Washonceinfreshlymade1XPBS;
3. Fixinfilter-sterile,freshlymade3%Paraformaldehyde/1XPBSfor10minutesatRT(seecomment7);
4. Washtwicein1XPBSfor5minuteseach;
5. Permeabilizeinfreshlymade1XPBS/0.5%TritonX-100onicefor5-7minutes(seenote1);
6. Dehydratetheslidesin80%,95%,100%ethanol,for3minuteseach;
7. Airdry;
8. Denaturein50%formamide/2XSSC(adjustedtopH7.2)for30minutesat80°C(seecomment8);
9. Washtwiceinice-cold2XSSC;
10. Depositdenaturedprobesolutionontocellsonslide(orplacecoverslipcell-sidedownontoprobeonslide);
11. Hybridizewiththeprobeovernightat42°Cinadarkandhumidchamber(papertissuessoakedin50%formamide/2xSSC)(seenote7);
12. Wash3timesin50%formamide/2XSSC(adjustedtopH7.2)for5minuteseachat42°C;
13. Wash3timesin2XSSCfor5minuteseachat42°C;
14. Ifabiotin-labelledprobe(e.g.chromosomepaint)isused,adetectionstephastobeincluded;
15. Blockin4XSSC/0.1%Tween/5%BSA(Gibco)for15minutesatRT;
16. Incubateinfluorescentlylabelledstreptavidinoravidindilutedinblockingbufferfor40minutesatRTinhumidchamber;
17. Wash3timesin2XSSC;
18. DNAcounterstaining(2minutesin2XSSCcontaining0.2mg/mlDAPI);
19. Washtwicein2XSSCfor5minuteseach;
20. Mountcoversliponaslideinglycerolbasedmountingmedium.

DNA-FISHfollowingimmunofluorescence

ThedetectionofDNArequiresaDNAdenaturationstepwhichcandestroytheimmunofluorescencesignalinsomecases.Therefore,ifthecombinationdoesn"tworkproperly,imagesshouldberecordedpriortotheDNAFISHexperimentusingsquaredcoverslips.

AnexampleofIFcombinedwithDNAFISHisshowninFigure3.

PreparationofFISHprobes-seeDNAFISHsectionabove

IF-DNAFISH

1. FollowIFprotocoluptoPBSwashesfollowingsecondaryantibodydetection(noDAPIcoloration);
2. Postfixinfreshlymade,sterile3%PFA/1XPBSfor10minutesatRT;
3. Washtwicein2XSSC(freshlymadefroma20Xsterilestock)for5minutes;
4. Permeabilizeinfreshlymade0.1MHCl/0.7%Tritonfor10minutesonice;
5. Washtwicein2XSSCfor5minuteseach;
6. Denaturein50%formamide/2XSSC(adjustedtopH7.2)for30minutesat80°C(seenote8);
7. Washseveraltimesinice-cold2XSSC;
8. HybridizewithdenaturedprobeO/Netc:seeDNAFISHsection.

DNAFISHfollowingRNAFISH

ThedetectionofDNArequiresaDNAdenaturationstepwhichcandestroytheRNAFISHsignalinsomecases,renderingthesimultaneouscombination(oncoverslips)impossible.Post-fixation(3%PFA/1XPBSfor10minatRT)oftheRNAsignalpriortotheDNA-FISHcanbeperformed;however,thispost-fixationstepcandramaticallyaffecttheefficiencyofDNAdenaturation.Inthesecases,itispossibletoperformtheRNA-FISHprocedurefirstandtorecordtheRNAFISHimagespriortoperformingDNAFISH(onslides),usingamicroscopecapableoftrackingnucleuscoordinates.

SimultaneousRNA-DNAFISH(oncoverslips)

1. FollowRNAFISHandDNAFISHsectionsforthepreparationoftheFISHprobes(seenote9).
2. FollowDNAFISHsectionforpreparationofcoverslips,denaturationandhybridization(seenote10).

SequentialRNA-DNAFISH(onslides)

1. FollowRNAFISHsectionforthepreparationoftheFISHprobeandpreparationofslides(ascoverslips).
2. Recordimagesandcoordinatesofnucleionanappropriatemicroscope.
3. Scratchoffthenailpolish.
4. Washoffthemountingmediumin4XSSC/0.2%Tween,3timesat42°C.
5. FollowtheDNAFISHsectionforpreparationofFISHprobes.
6. RNasetreatthesamplesat37°Cforonehour(1U/mlRNaseA(fermentas)+10U/ml(NEB)in2XSSC).
7. Denaturein70%formamide/2XSSC(adjustedtopH7.2)for2-4minat75°C.
8. FollowtheendoftheDNAFISHsection(steps9to20).

Backtotop

2Xhybridizationbuffer	(Storageat-20°C)4XSSC20%dextransulfate2mg/mlBSA(Biolabs)40mMVanadylRibonucleosideComplex(VRC)
mountingmedium	SeeSpectoretal(1998).Storeat-20°C,alwayskeepinthedarkandonicewhenaliquoting.90%glycerol0.1XPBS0.1%p-phenylenediamine,pH9.(Shouldbe"straw"colored-ifitveerstopurpleoryellow,discard.)
CSK(Cytoskeletal)buffer	MakeupinsterileH2O.PIPEScanbeaddedaspowderandpHadjustedwithNaOH.Filter,steriliseandstoreinaliquotsat-20°C.100mMNaCl300mMsucrose3mMMgCl210mMPIPESpH6.8

20XSSC-Sigma®S6639Alexasecondaryantibodies(includinghighlycross-adsorbed2aryAbs)-Molecularprobes™BSA7.5%-Invitrogen™(Gibco®)15260-037BSA100X-N.E.Biolabs®B9001SCot-1mouseDNA-Gibco®/Invitrogen™18440-016Coverslips18x18mm(100)-ESCO(VWR9611301)Dextransulfate-Sigma®31403Formamide-Sigma®F5786Gelatin250g-Merck®(VWR24360233)Glycerol-Sigma®G5516MouseX-chromosomepaint-Cambio1187-XMB-02Paraformaldehyde-RE/PURO387507PBS10X500ml-Sigma®D1408P-phenylenediamine(1g)-Sigma®27-515-8RNAGuard(PHARMACIA)-Amersham™270815-01SalmonspermDNA(10mg/ml)-Boehringer™(Mol.Biol.grade)SpectrumGreendUTP-Vysis®6J9410SpectumReddUTP-Vysis®6J9420SlidesSuperfrost(50)-MenzelGläser(VWR8032-E01)Triton®-X-100-ICN807423Tween20-Sigma®P9416VanadylRibonucleosideComplex(VRC)-N.E.Biolabs®S1402SNickTranslationKit-VYSIS®(Abbott32-801300)Water500ml-Sigma®W3500

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