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Staining for First Antigen
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1. Preparation of tissue. Deparaffinize and rehydrate tissue sections following standard protocols.
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2. Rinse in distilled water for 5 minutes.
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3. If endogenous enzyme activities are present inactivate using appropriate methods.
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4. Wash sections 2 x 3 minutes in 10 mM sodium phosphate buffer, pH 7.5, 150 mM NaCI (PBS). (Other buffers may be used).
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5. Avidin/biotin blocking step. Perform Avidin/Biotin blocking if required (Avidin/Biotin Blocking Kit, Cat. No. SP-2001). Incubate sections with Avidin Solution for 15 minutes. Rinse briefly with buffer, then incubate in the Biotin Solution for 15 minutes. Wash sections 2 x 2 minutes in buffer. This blocking step may be eliminated if suitable controls have determined such background not to be a concern.
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6. Protein blocking step. Incubate sections for 20 minutes with buffer containing 5% normal serum (NS) prepared from the first VECTASTAIN® kit, or incubate for 5-10 minutes in 10% NS.
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7. Primary antibody. Blot excess blocking solution from sections and incubate with the first primary antibody diluted in 5% NS from the first VECTASTAIN® kit using appropriate concentration and length of incubation.
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8. Wash 2 x 3 minutes in buffer.
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9. Secondary antibody. Incubate sections for 30 minutes with biotinylated secondary antibody from the first VECTASTAIN® kit diluted in 5% NS. For a 5-10 minute incubation, double the concentration of the biotinylated antibody and normal serum.
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10. Wash sections 2 x 3 minutes in buffer.
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11. ABC. Incubate sections for 30 minutes with the first VECTASTAIN® ABC reagent prepared in advance as described in the kit instructions. For a 5-10 minute incubation, use the VECTASTAIN® ABC reagent at twice the recommended concentration.
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12. Wash sections 2 x 3 minutes in buffer.
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13. Substrate. Incubate sections with the appropriate enzyme substrate until optimal color develops. Use the recommended times given in the substrate kit instructions as a guideline.
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14. Wash sections 2 x 3 minutes in buffer.
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Staining for Second Antigen
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15. Avidin/biotin blocking step. Perform Avidin/Biotin blocking step if required. (This step may be necessary to prevent the interaction of the second set of labeling reagents with the first set of labeling reagents.)
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16. Protein blocking step. Incubate sections for 20 minutes with buffer containing 5% NS prepared from the second VECTASTAIN® kit, or incubate for 5-10 minutes in 10% NS.
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17. Primary antibody. Blot excess blocking solution from sections and incubate with the second primary antibody diluted in 5% NS from the second VECTASTAIN® kit using appropriate concentration and length of incubation.
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18. Wash 2 x 3 minutes in buffer.
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19. Secondary antibody. Incubate sections for 30 minutes with biotinylated secondary antibody appropriate for labeling the second primary antibody diluted in 5% NS. For a 5-10 minute incubation, double the concentration of the biotinylated antibody and normal serum.
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20. ABC. Incubate sections for 30 minutes with the second VECTASTAIN® ABC reagent prepared in advance as described in the kit instructions. For a 5-10 minute incubation, use the second VECTASTAIN® ABC reagent at twice the recommended concentration.
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21. Wash sections 2 x 3 minutes in buffer.
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22. Substrate. Incubate sections with the appropriate second, contrasting enzyme substrate until optimal color develops. Use the recommended times given in the substrate kit instructions as a guideline.
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23. Wash sections in tap water for 5 minutes.
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24. Counterstain, clear, and mount in appropriate mounting medium.
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