Virtualab Protocols (Chang Bioscience)
The following protocol is for the VECTASTAIN ABC KIT (Vector Lab).
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.