Multiple Antigen Labeling - Vector Laboratories

6Protocol: Multiple Antigen Labeling Using the VECTASTAIN ® SystemsStaining for First Antigen1. Preparation of tissue. Deparaffinize andrehydrate tissue sections following standardprotocols.Add First Primary Antibody2. Rinse in distilled water for 5 minutes.3. If endogenous enzyme activities are presentinactivate using appropriate methods (SeeAppendix 2).4. Wash sections 2 x 3 minutes in 10 mM sodiumphosphate buffer, pH 7.5, 150 mM NaCI (PBS).(Other buffers may be used).5. Avidin/biotin blocking step. PerformAvidin/Biotin blocking if required (Avidin/BiotinBlocking Kit, Cat. No. SP-2001). Incubate sectionswith Avidin Solution for 15 minutes. Rinsebriefly with buffer, then incubate in the BiotinSolution for 15 minutes. Wash sections 2 x 2minutes in buffer. This blocking step may beeliminated if suitable controls have determinedsuch background not to be a concern.6. Protein blocking step. Incubate sections for20 minutes with buffer containing 5% normalserum (NS) prepared from the first VECTASTAIN ®kit, or incubate for 5-10 minutes in 10% NS.7. Primary antibody. Blot excess blockingsolution from sections and incubate with thefirst primary antibody diluted in 5% NS fromthe first VECTASTAIN ® kit using appropriateconcentration and length of incubation.8. Wash 2 x 3 minutes in buffer.9. Secondary antibody. Incubate sections for30 minutes with biotinylated secondary antibodyfrom the first VECTASTAIN ® kit diluted in5% NS. For a 5-10 minute incubation, doublethe concentration of the biotinylated antibodyand normal serum.10. Wash sections 2 x 3 minutes in buffer.11. ABC. Incubate sections for 30 minutes withthe first VECTASTAIN ® ABC reagent prepared inadvance as described in the kit instructions. For a5-10 minute incubation, use the VECTASTAIN ® ABCreagent at twice the recommended concentration.12. Wash sections 2 x 3 minutes in buffer.13. Substrate. Incubate sections with theappropriate enzyme substrate until optimal colordevelops. Use the recommended times given inthe substrate kit instructions as a guideline.14. Wash sections 2 x 3 minutes in buffer.Fig. 1Add Biotinylated Secondary AntibodyFig. 2Add ABCFig. 3Add Enzyme Substrate IFig. 4www.vectorlabs.com