in situ hybridiation protocol.docx
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in situ hybridiation protocol.docx
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insituhybridiationprotocol
Insituhybridizationprotocol.
Figure1.Diagramshowingthetimelineofstepsintheinsituhybridizationprotocol.Tissueembeddingstepsareindicatedingreen,probe-preparationstepsinorange,andtheinsituhybridizationstepsinblue.ThistimelineconsidersthattheDNAtemplatefortheinvitrotranscriptionoftheprobeisavailable.Parafin-embeddedtissueblocksandDIG-labeledprobescanbepreparedbeforehandandstoredat4°Cand-80°C,respectively,untilthetimeofuse.Theinsituhybridizationprotocolcanthenbecompletedinaslittleasfourdays.
Note:
AllstepsuptoandincludingthehybridizationsteparesensitivetoRNAseactivity.Itisthereforeessentialtoworkincleanconditions.
Fresh4%paraformaldehyde(PFA)fixative
For500ml,warm400ml1xPBSto60°CanddissolvetwopelletsofNaOH.Inafumehood,add20gofparaformaldehydeandmixthoroughlyuntildissolved.PlacethesolutiononiceandwhencooledadjustthepHto7.2withH2SO4(1-2dropsfor100ml).Thenadjustthevolumeto500mLwith1xPBS.
Note:
DonotuseHCltoadjustthepHasthiswillreleasehighlytoxicfumes.Paraformaldehydeistoxic;thissolutionshouldbepreparedinafumehoodanddisposedofproperly.Also,Paraformaldehydeisstoredat4°Candshouldbeboughtnewevery6–9months.
Tissuefixation
1.Day1:
Harvesttissuesamplesandplacethemimmediatelyin15mLfreshPFAfixativeoniceinglassscintillationvials.Iftissuedissectionisrequired,thisisbestdoneoniceincoldfixative.(alargeexcessoffixative)
2.Applyavacuum(˜500mmHg)tothesampleswhileonice.Holdavacuumfor15-20minutes;smallbubblesshouldreleasefromthesamplesbutthefixativeshouldnotcometoaboil.Releasethevacuumslowly,andrenewthePFAfixativetoensurethefixativeremainsattherightconcentration.Repeatthisstepuntilthetissuessinkafterreleaseofvacuum.
3.ReplacethePFAfixativeoncemoreandkeepthevialsat4°Covernight.
Tissuedehyddration
4.Day2:
Pre-cool1×PBSandtheethanolseriesat4°C.
5.Rinsethesamplestwicefor30mineachin1×PBS,onice.(usealargeexcessofeachsolution)
6.Dehydratethesamplesbytakingthemthroughanethanolseries,onice,asfollow:
•10%ethanol30min,
•30%ethanol30min,
•50%ethanol1hour,
•70%ethanol1hour,
•85%ethanol1hour,
•95%ethanol1hour,
•3changesof100%ethanolfor1houreach.(Samplescanbestoredin70%ethanolat4°Cforseveralmonths.)
Solventinfiltration
7.Day3:
Thefollowingmorning,warmthesamplestoroomtemperatureforonehour.Thenreplacetheethanolgraduallywithhistoclearasfollows:
•ethanol/histoclear(3:
1v/v)1hour,
•ethanol/histoclear(1:
1v/v)1hour,
•ethanol/histoclear(1:
3v/v)1hour,
•histoclear1hour,
•histoclear1hour,
•histoclear1hour,
8.Attheendoftheday,add1/4volumeofParaplastPluschipsandplacethevialina60°Coven.AlsofillabeakerwithParaplastchipsandreplenishthisfrequentlyinordertoalwayshavefreshlymoltenwaxathand.(ThetemperatureoftheovenisimportantandprolongedheatingofParaplastabove60°Cshouldbeavoided.)
Waxinfiltration
9.Day4:
GraduallyreplacethehistoclearwithParaplastbyregularlyaddingmoreparaffinchips(everyhour).Attheendoftheday,carefullypouroffthehistoclear/waxmixtureandimmediatelyreplaceitwithpuremoltenwax.Leavethesamplesat60°Covernight.
10.Days5and6:
ChangetheParaplast3timesdaily,aboutevery4hours,withfreshmoltenwaxkeepingthesamplesat60°C.
Note:
Itispossibletochangethewaxonlyonceaday,butthetissuepreparationwouldtakeafewextradaystocomplete,asthewaxneedstobechangedatleast5or6times.Vacuuminfiltrationofthetissuesampleswhenthesearein100%EtOHand/orinwaxcanfurtherimprovetheinfiltrationandembeddingoftissues.Vacuuminfiltrationofsamplesinwaxshouldbedoneat60°C.
11.Day7:
Changethewaxoncemore.Thesmellofhistoclearshouldnowbegonecompletelyandthesamplescanbeembeddedlaterthatday.
12.Setupalargewarmingplateat60°Cprotectedbyaluminumfoil.
13.Themethodofembeddingwillvarydependingonthetypeoftissuetobeanalyzed.Themostimportantpointatthisstepistoensurethatthesamplesareorientedcorrectlyforsectioninglater.Onewayistouseplasticmoldsandrings.Warmupthemoldsonthewarmingplateandpourmoltenwaxintothebottompartofthemold.Transferatissuesampleintothemoldwithwarmedforcepsandorientitintothedesiredposition.Placethewhiteringontothemoldandaddadditionalmoltenwaxsuchthatthetissuesarecoveredcompletely.Carefullymovethemoldfromtheplatetothebench.Letthewaxcooldowngradually.
14.Anotherpossibilityistodistributeallthesamplesinapetridishcontainingmoltenwax(thesamplesshouldbecompletelycoveredbywax)whileworkingonthe60°Cwarmedplate.Orientatethesampleswithwarmforceps.Attheend,switchofftheplate.Whenthewaxissolidified,thepetridishcanbemovedtothebenchandthenstoreat4°C.Whenreadyforsectioning,smallblockscontainingatissuesamplecanbecutoutofthepetridishwithawarmedbladeandmountedontoamicrotomespecimenholderwithanextradropofmoltenwax.Letthesamplehardenforafewminutesbeforesectioning.
Note:
Blockscanbestoredat4°Cfor1yearorlonger.Foradditionalhelpfultipsontissuefixationandembedding,seeref.16
2.Probepreparation
Cloning
Probesareusuallygeneratedtooneormorespecificregionsofthegeneofinterest.Highlyrepetitivesequencesshouldbeexcludedfromtheprobe,butsequencescorrespondingtoconservedproteindomains,suchasDNA-bindingmotifsintranscriptionfactors.ThisbecausetheRNAseAtreatmentinthepost-hybridizationsteps(seebelow)reducesthelevelofprobecross-hybridizationbetweengenefamilymembers.RNAseAcleavesatmismatchesinRNAduplexes,fragmentingprobeshybridizedtotranscriptswithimperfectcomplementarity,whichwillbewashedoffinsubsequentsteps.
Ingeneral,severalprobesmayneedtobetestedforeachgeneofinterest.Probescanbeasshortas150basesinlength.Althoughthereisnolimittothelengthoftheprobe,fragmentsshorterthan1.5kbtypicallytranscribebetter.DNAfragmentstobetranscribedareclonedintoavectorcontainingT3,T7,orSP6promoters(e.g.pCRII-TOPO,Invitrogen).Alternatively,T3,T7orSP6promotersequencescanbeincludedasa5’-tailonthereverseprimerusedforamplifyingtheproberegion.Ineveryinsituhybridizationexperiment,weincludeapositivecontrolprobeforwhichthehybridizationpatternisknownandwhichworksconsistently,aswellasanegativecontrol.ThelattercouldbearandomRNAprobethatisknownnottohybridizetoanytranscriptsinthetissueofinterest.Althoughitiscommontousethesenseprobeofthegeneunderanalysis,thisisnotnecessaryandanynon-hybridizingRNAwillsuitthepurpose.Ifavailable,tissuesfromatranscriptionalnullalleleofthegeneofinterestwouldserveasanexcellentnegativecontrol.
2.Invitrotranscription
1.Linearizetheplasmidbydigestingapproximately5μgDNAwitharestrictionenzymethatdigestsattheendoftheinsertoppositethesiteofthepromoter.Donotuserestrictionenzymesthatleavea3'-overhang.Thiscanleadtotranscriptionartifactsbecausethepolymerasecanutilizethe3'overhangasasubstratetocontinuetranscription.Alternatively,theproberegionincludingtheT3,T7orSP6promotercanbeamplifiedbyPCRtogeneratetheDNAtemplatefortheinvitrotranscriptionreaction.
2.Checkanaliquotonageltoverifythatthereactionhasgonetocompletion.
3.ExtracttheDNAwithphenol/chloroform,precipitatewithethanolandresuspendtheDNApelletinmilliQH2Otoaconcentrationof1μg/μl.Alternatively,theDNAcanbecleanedusingstandardDNApurificationcolumns.
4.Setuptheinvitrotranscriptionreactionbymixing:
•1μlpurifiedDNA(1μg/μl)
•2μl10xtranscriptionbuffer
•2μL10xDIGRNAlabelingmix
•1μlRNAseOUT
•2μlT3,T7orSP6RNApolymerase(20U/μl)
•H2Oupto20μl.
Incubateat37°Cfor1to2hours.Keep1μltoanalyzeonagellater.
Note:
Fluorescence-labeledprobesarepreferredinanimalsystemsbutduetothehighauto-fluorescenceofmostplanttissues,Insituhybridizationprotocolsforplantsuseradiolabeledprobes17ormorecommonlyDIG-labeledprobesthataredetectedbyimmunohistochemistry.
5.RemovetheDNAtemplatebyadding75μLMilliQH2Oand5μLRQ1DNAse(1U/μl)tothetranscriptionreactionandincubatingthereactionat37°Cfor10min.
6.Purifytheinvitrotranscriptionreactiontoeliminatenon-incorporatednucleotidesandsmalltranscripts.OnepossibilityistouseasizeexclusionSephadex®column,suchastheminiQuickSpinRNAColumns(Roche).Alternatively,thetranscriptionreactioncanbepurifiedbyaconventionalLiCl/ethanolprecipitation(seeref.14).Keep1μltocheckonagellater.
7.Probesover250basesinlengthareusuallypartiallyhydrolizedtoobtainRNAmoleculesofapproximately150ntandthereforesmallenoughtoefficientlypenetratethetissuesections.Addanequalvolumeof2xcarbonatebuffer(80mMNaHCO3,120mMNa2CO3)tothepurifiedtranscriptionreactionandincubateat60°C.Theincubationtimeshouldbecalculatedbythefollowingformula:
time=(Li-Lf)/(KxLixLf)whereLi=initialprobelength(inkb);Lf=finalprobelength(0.150kb);K=0.11kb/minute.Keep2μLtocheckonagel.
8.Neutralizethehydrolysisreactionbyadding1/20volumeof10%aceticacid.
9.Theprobeisthenpurifiedbyadding1μloftRNA(100mg/
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