Overview | SincefirstintroducedbyGrahamandKarnovsky(1966),numerousproceduresforitsuseinhistochemistry,immunohistochemistry,Westernanddotblotshavebeendescribed(Lewis&Knight,1977;Larsson,1988;Gallyasetal.,1982).InthepresenceofhorserADIshperoxidaseandhydrogenperoxide,DABoxidativelypolymerizestoaninsolublebrownpolymer.Thecolorcanbemodifiedandintensifiedbytreatmentwithmetalsaltsofsilver,copper,nickel,cobaltandosmium(Hsu&Soban,1982).DABisasUSPectedcarcinogenandmustbehandledwithcaution.CHEMICONsuppliesDABasastableliquidconcentrateeliminatingthenecessityofhandlingapotentiallyhazardousmaterial.ThestABIlizationsystemisuniqueandpreventsformationofpartiallyoxidizedDABexcludingnonspecificbindingtootherhemecontainingproteinssooftenobservedwithpowderedDABpreparations.TheconcentratecanbedilutedinappropriateperoxidecontainingbuffersprovidingtheresearcherwiththecapabilityofformulatinganyofthenumerouspublishedDABreactionsystems.ThemostcommonproceduresuggestsdilutingtheDAB(CatalogNumberES005)1:50ina100μmol/LTris-HClbuffer,pH7.6,andaddingH(2)O(2),toafinalconcentrationof0.01%.Incubationtimesrangefrom5-15minutes.Prolongedreactiontimewillresultinnon-specificstainingofotherhemeproteins. |