1. Fix and stain cells as described in Staining for immunohistochemistry or FACS , except that the secondary/last antibody is coupled with horseradish peroxide.

2. Make A + B solution: 10 ml A + 10 ml + 980 ml PBS. Mix and let stand at room temperature.

ABC staining is brown. The color changes to blue if the staining buffer contains 1% nickel chloride or dark if 0.5% cobalt chloride.

3. Add A + B solution to the cells, incubate at room temperature for 30 minutes.

4. Wash three times with PBS over a peroid of 30 minutes with gentle rock.

5. Remove PBS, add DAB solution (0.5 mg/ml diaminobenzidine in PBS). Add 30 ml of 0.3% H₂O₂ per ml of DAB solution. DAB should be protected from light and stock solution stored at -20 °C.

6. Observe the extent of staining under a microscope and, when the color is appropriately developed, stop the reaction by washing with several changes of PBS.

DAB is mutagenic, deactivated with bleach.

7. Dehydrate and mount.