20xSSC:3MNaCl0.3MNa-citrate;pH7.0HybridizationSol"n:5xSSC0.5%(w/v)BlockingReagent0.1%(w/v)N-lauroylsarcosine,Na-salt0.02%(w/v)SDS50%(w/v)formamide*Blockingreagentdoesnotdissolverapidly.Heatto~50-70°C;thesol"nremainsturbid.Make~1hourinadvance.Storehybridizationsolutionat4°Cinabottleorat-20°Cin50mlconicaltubes.Note:Prehybridizationsolutionisthehybridizationsolutionwithoutthelabelledprobe.Immunochemiluminescence:BlotWash#1:2xSSC;0.1%(w/v)SDS.BlotWash#2:0.1xSSC;0.1%(w/v)SDS.Buffer#1:100mMmaleicacid,pH7.5150mMNaClBuffer#2:2%BlockingReagentinBuffer#1Heatto50-70°C;prepare~60"inadvance;sol"nremainsturbidStoreat4°C.AddNaAzideforlongtermstorage.Buffer#3:100mMTris-Cl,pH9.5100mMNaCl50mMMgCl2Antibody:10µlanti-digoxigenin-APFabfragments(Boehringer-Manncat#1093274)40mlofBuffer#110mlofBuffer#2Thiswilllastseveralmonthsofreuseifstoredat4°C.CSPDReady-to-Use:Keepreusedportionsseparatefromused.Store@4°CPROTECTFROMLIGHT![ThisproductischeaperfromTropix,catalog#CD1000R-thisisa1Lbottleofthestuff...thatvolumeisnotcheap.Itcomesinsmallervolumes,dependingonyourusage.]NOTES:Allsolutionsshouldbeatroomtempwhenusedonblots.Wereuseprehybridizationsolution,probes,Buffer2,antibodyconjugate,andCSPDReady-to-Use.Asneeded,wereplacetheprehybridizationsolution,Buffer2andCSPDwithfreshsolutions.Theprobesandantibodysolutionsareusedforseveralmonthsandspikedormadefreshasneeded.Formostprobes,onlyhalfofthelabellingmixtureisneededforsufficientsignal.RecipesarefromtheBoehringerMannheimNonrADIoactiveDNALabelingandDetectionKitandprotocols.MiscellaneousLabLore:

1. Fragmentswithincorporatedalkali-lABIledignucleotidesmaybestrippedfromblots;thealkali-stableversionsdonotstripoffwell.
2. Theblockingreagent(Buffer2)seemstoworkbetterafteracoupleofuses.Inthefirstcoupleofuses,backgroundspotsmaybeabundant.
3. Depurinationisapparentlyunnecessaryforthetransferofsmall(<10kb)fragments.
4. Crosslinkingisunnecessaryiftheblotisnotgoingtobestrippedandre-probed.
5. Thereisapparentlyenoughblockingreagentintheantibodysolutionthatthepriorblockingstepcanbeskippedwithoutincreasingbackground.
6. Ifthethehalf-lifeofyourworkingCSPDsolutionbeginstodecreaserapidlyinstorage,tryusinganewstoragecontainer(e.g.,50-mlFalcontube);storagebottlesreusedmanytimesapparentlyaccumulatesomethingthataffectsCSPDstability.

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