DAB/peroxide

MATERIALS

DAB (3,3′-diaminobenzidine tetrahydrochloride)

Hydrogen peroxide (H₂O₂; 0.3%)

H₂O₂ is supplied as a 30% stock solution. It should be <6 mo old. To dilute to 0.3%, add 100 μL of H₂O₂ to 10 mL of H₂O.

Sodium phosphate (0.1 M, pH 7.4)

Aluminum foil

Hood with balance

Ice

Microcentrifuge tubes

Sonicator

Tubes with capacity of 10-15 mL

1. Weigh a 1.5-mL microcentrifuge tube. In a hood, add a very small quantity of DAB to the tube and weigh the tube again.
2. Calculate and add the amount of 0.1 M sodium phosphate (pH 7.4) needed to make a 0.5 mg/mL DAB solution (~10-15 mL).
3. Sonicate.
4. Add one drop (~100 μL) of 0.3% H₂O₂ to 10 mL of DAB solution.
5. Wrap the tube in aluminum foil (DAB/peroxide is light-sensitive), and place on ice until used.

DAB should be used within 1 h.