Protocol for Immunoperoxidase staining of tissue culture cells

1. Grow cells on sterile glass cover slips or slides overnight in 37^oC/5% CO₂ incubator
2. Rinse briefly with PBS
3. Fix cells by incubation with 1% formalin in PBS for 10 minutes
4. Rinse three times with PBS
5. Incubate for 10 minutes with 0.1% H₂O₂ in PBS to quench endogenous peroxidase activity
6. Rinse once with PBS
7. Incubate slides for 20 minutes in 10% goat serum in PBS (suppresses non-specific binding of IgG)
8. Wash with PBS
9. Incubate with primary antibody for 1 hour at room temperature or overnight at 4^oC. Optimal antibody concentration is usually from 1-10ug/ml in PBS-BSA
10. Wash three times with PBS
11. Incubate with biotin-conjugated secondary antibody for 45 minutes. Again, optimal antibody concentration is usually from 1-10ug/ml in PBS-BSA
12. Wash three times with PBS
13. Incubate with streptavadin-HRP for 15 minutes. This step should be titrated as excess streptavadin-HRP can lead to high background
14. Wash with 0.5% triton x-100 in PBS for 30 seconds
15. Incubate with fresh DAB solution for 5 minutes. This step can be lengthened if additional sensitivity is necessary. It is a good idea to do a quick spot check here
16. Rinse with distilled water
17. Counterstain with hematoxylin
18. Dehydrate with alcholos and xylene
19. Mount permanent coverslip

Note: Incubations are to be at room temperature or a 4^oC humidified chamber. Antibody concentrations here may not hold for all sources. These concentrations should be titrated for each source.