I. Required material
Reagents
- Primaries et secondaries antibodies
- Avidin/Biotin Blocking kits
- VECTASTAIN® kits
- VECTASTAIN® ABC kits
- Fluorescein Avidin DCS
Buffers
- PBS : 10mM sodium phosphate, 0.15M NaC1, pH 7.4-7.8
- others buffers
II. Experimentation length
- 30 mn or 1h of avidin/biotin blocking
- 20 mn or 40 mn of proteins blocking
- 20 mn ou 1h of sections incubation in secondary antibody
- 50 mn of rinsing steps
- TOTAL : 2h30 à 3h30
III. Protocol Multiple Labeling
Staining for First Antigen
1. Preparation of tissue. Deparaffinize and rehydrate tissue sections following standard protocols.
2. Rinse in distilled water for 5 minutes.
3. If endogenous enzyme activities are present inactivate using appropriate methods (See Appendix 2).
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).
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.
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.
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.
8. Wash 2 x 3 minutes in buffer.
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.
10. Wash sections 2 x 3 minutes in buffer.
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.
12. Wash sections 2 x 3 minutes in buffer.
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.
14. Wash sections 2 x 3 minutes in buffer.
Staining for Second Antigen
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.)
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.
17. Primary antibody. 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.
18. Wash 2 x 3 minutes in buffer.
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.
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.
21. Wash sections 2 x 3 minutes in buffer.
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.
23. Wash sections in tap water for 5 minutes.
24. Counterstain, clear, and mount in appropriate mounting medium.
IV.Search engine
- Primary antibody
- Secondary antibody
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