I. Required material
Reagents
- Primary antibodies
- 2,5% Normal Horse Serum (NHS)
- BSA (Immunohistochemical grade)
- ImmPRESS™ Reagent
Buffers
- PBS : 10 mM sodium phosphate buffer, pH 7.5, 150 mM NaCI
- others buffers
II. Experimentation length
- 40mn of proteins blocking
- 1 h of sections incubation in ImmPRESS Reagent
- 40 mn of rinsing steps
- TOTAL : 2h21
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 peroxidase activity is present in the section, inactivate using an appropriate method.
4. Wash sections 2 x 3 minutes in PBS. (Other buffers may be used).
5. Protein blocking step.Incubate sections for 20 minutes with ready-to-use (2.5%) normal horse serum (NHS).
6. Primary antibody. Incubate sections with mouse or rabbit primary antibody diluted in appropriate antibody diluent (buffer containing diluted (2.5%) NHS or 0.1% immunohistochemical grade bovine serum albumin (BSA)).
7. Wash slides for 5 minutes in buffer.
8. ImmPRESS™ Reagent. Incubate sections for 30 minutes with appropriate ImmPRESS™ Reagent (anti-mouse Ig, anti-rabbit Ig ou universal).
9. Wash slides for 5 minutes in buffer.
10. Substrate. Incubate sections in peroxidase substrate solution until optimal color develops.
11. Rinse for 5 minutes in buffer.
Staining for Second Antigen
12. Protein blocking step Incubate sections for 20 minutes with ready-to-use NHS.
13. Primary antibody. Incubate sections with the second mouse or rabbit primary antibody diluted in appropriate antibody diluent (See step 6).
14. Wash for 5 minutes in buffer.
15. ImmPRESS™ Reagent. Incubate sections for 30 minutes with appropriate ImmPRESS™ Reagent (anti-mouse Ig, anti-rabbit Ig, or universal).
16. Wash for 5 minutes in buffer.
17. Substrate. Incubate sections in second, contrasting peroxidase substrate solution until optimal color develops.
18. Wash for 5 minutes in buffer.
19. Counterstain, clear, and mount in appropriate mounting medium.
IV.Search engine
- Primary antibody
- Secondary antibody
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