Multiple Antigen Labeling - Vector Laboratories

7Fig. 5Fig. 6Fig.7Add Second PrimaryAntibodyAdd Second BiotinylatedSecondary AntibodyAdd ABCAdd Enzyme Substrate IIStaining for Second Antigen15. Avidin/biotin blocking step. PerformAvidin/Biotin blocking step if required. (This stepmay be necessary to prevent the interaction of thesecond set of labeling reagents with the first setof labeling reagents.)16. Protein blocking step. Incubate sectionsfor 20 minutes with buffer containing 5% NSprepared from the second VECTASTAIN ® kit, orincubate for 5-10 minutes in 10% NS.17. Primary antibody. Blot excess blockingsolution from sections and incubate with thesecond primary antibody diluted in 5% NS fromthe second VECTASTAIN ® kit using appropriateconcentration and length of incubation.18. Wash 2 x 3 minutes in buffer.19. Secondary antibody. Incubate sections for30 minutes with biotinylated secondary antibodyappropriate for labeling the second primaryantibody diluted in 5% NS. For a 5-10 minuteincubation, double the concentration of thebiotinylated antibody and normal serum.20. ABC. Incubate sections for 30 minutes withthe second VECTASTAIN ® ABC reagent preparedin advance as described in the kit instructions.For a 5-10 minute incubation, use the secondVECTASTAIN ® ABC reagent at twice the recommendedconcentration.21. Wash sections 2 x 3 minutes in buffer.22. Substrate. Incubate sections with theappropriate second, contrasting enzyme substrateuntil optimal color develops. Use therecommended times given in the substrate kitinstructions as a guideline.23. Wash sections in tap water for 5 minutes.24. Counterstain, clear, and mount in appropriatemounting medium.Fig. 8www.vectorlabs.com