Immunoperoxidase staining of culture cells
Immunology
scientificprotocols authored almost 5 years ago
Method:
- Grow cells on sterile coverglasses or chamber slides overnight.
- Rinse briefly with PBS
- Fix cells by incubation with one of the following methods:
- 1% formalin in PBS for 10 minutes
- 80% methanol in PBS for 10 minutes
- Cold acetone for 5 minutes, air dry
- 4% paraformahyde
- Rinse three times with PBS
- Incubate with 1% H2O2 in PBS for 10 minutes to quench endogenous peroxidase activity
- Rinse with PBS twice
- Incubate slides for 30 minutes in 10% blocking serum in PBS (suppresses non-specific binding of IgG)
- Blot excess blocking serum
- Incubate with primary antibody for 1 hour at room temperature or overnight at 4°C. Optimal antibody concentration is usually from 0.5-10ug/ml in TBS.
- Wash three times with PBS
- Incubate with biotin-conjugated secondary antibody for 30-45 minutes at room temperature. Again, optimal antibody concentration is usually from 0.5-10ug/ml in TBS
- Wash three times with PBS
- Incubate with streptavadin-HRP for 15 minutes. This step should be titrated as excess streptavadin-HRP can lead to high background
- Wash with PBS three times
- Incubate with freshly mixed DAB solution for 3-10 minutes. We highly suggest that you should observe the color developing under microscopy.
- Rinse three times with distilled water
- Counterstain with hematoxylin
- Dehydrate with 70-100% alcohol and clear with xylene
- Mount with coverslip
