Nickel DAB (N-DAB) Labeling

Contributed by Luke Hammond, QBI, The University of Queensland, Australia

Nickel DAB (N-DAB) Labeling for sections


Triton-X 100 Buffer 0.2% in 2%normal serum/1x PBS buffer Primary antibody diluted as per manufacturers recommendation (titration may be necessary) Appropriate secondary antibody diluted in Triton buffer AB complex Sodium acetate 0.175M. Nickel sulphate 1.25g in 50ml Sodium acetate buffer 0.175M (Make up fresh) DAB solution 10mg DAB in 50ml Nickel Sulphate solution. Dissolve and filter before use. 1% bleach solution for DAB deactivation.


  • Wash section with 0.2% Triton-X 100 buffer
  • Block section with Triton-X 100 0.2% normal serum -1hour
  • Remove blocking solution and incubate the section in primary antibody overnight at 40C
  • Wash in 1x PBS (3x 10minutes)
  • Incubate sections with secondary antibody 1hour
  • Wash in 1x PBS (3x 10minutes)
  • Incubate in AB complex as per manufacturer’s instructions
  • Wash in 1x PBS (3x 10minutes)
  • Incubate in DAB solution for 5minutes
  • Pour off the DAB solution and add 5ul of 30% Hydrogen Peroxide to this solution, mix and re incubate the sections. Check the sections microscopically and wash in PBS to stop the reaction once the desired staining intensity is reached.
  • Counterstain if necessary using a contrasting histochemical stain such as Nuclear Fast Red(note: Haematoxylin counterstain is effective only with a brown DAB reaction product)
  • Prepare sections for the microscope by dehydrating clearing and mounting in DPX
  • Decontaminate any DAB affected surfaces and glassware with bleach and dispose of waste as per local regulations.

This method is based, with permission, on an original protocol available here.