| ApplicationNotes | PROCEDUREFORBCIP/TNBT(ES007): 
 1)AfterthefinalbindingreactionwithanAPlabeledprobe,washtissuesectionsormembranethoroughlyinabuffersuchasTris/HClorTris-bufferedsalinecontaining0.1%Tween20.DONOTUSEPHOSPHATEBUFFERS.InorganicphosphateisapowerfulinhibitorofAP.
 
 2)AfterthefinalwashcompletelycovertissuesectionsormembranewithBCIP/TNBTSolutionandincubate5-15minutesprotectedfromlight.Longerincubationsmaybenecessarydependinguponenzymeactivity.Ifcolordevelopsalmostimmediately,theformazandepositsmayflakeoffofthetissuesectionsormembrane.Ifthisoccurs,furtherdilutionoftheAPprobeisnecessary.Afinelineofformazandepositcircumscribingthebandordot,withnodepositinthecenter,alsosuggestsfurtherdilutionoftheAPprobestobenecessary.
 
 3)Stoptheenzymereactionbythoroughlywashingtissuesectionsormembraneswithdeionizedwater.
 
 4)Ifdesired,counterstaintissuesectionsina0.5%NeutralRedsolutionpreparedinaMolL(-1)acetatebuffer.Hematoxylinsolutionswillnotprovideappropriatecontrast.Membranesshouldbedriedandstoredprotectedfromlight.Littleornobackgroundstainingshouldbeobservedonsectionsormembranes.Presenceofexcessivebackgroundstainingindicatesincompleteremovalofnon-boundAPfromthesectionsormembrane.Increasethenumberofwashesorthewashingtimetoremedythisproblem.
 
 Results:
 
 AfinedarkpurpleprecipitatewilllocalizeatsitesofAPactivityontissuesections.DarkpurplebandsordotswillbevisIBLeatthesitesofAPactivityonmembranes.
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