Diaminobenzidine (DAB) Staining

Abstract

This protocol is used to stain paraffin-embedded mouse brain tissue.

To paraffin-embedded tissue, deparaffinize in 100% xylenes for 3x 5 minutes.

Incubate in 100% ethanol for 2x 5 minutes.

Incubate in 70% ethanol for 5 minutes.

Incubate in 50% ethanol for 5 minutes.

Incubate in 1X PBS for 5 minutes.

Perform antigen retrieval by incubating tissue in 1X sodium citrate for 20 minutes at 95^oC.

Sodium citrate: 10mM sodium citrate + 0.05% Tween-20, pH6

Wash with 1X PBS for 5 minutes.

Incubate in 0.9% H₂O₂ for 10 minutes at room temperature.

Wash with 1X PBS for 5 minutes.

10.

Incubate in blocking buffer for 1 hour at room temperature.

Blocking buffer: 10% serum + 0.05% Triton X-100 in 1X PBS

11.

Wash with 1X PBS for 5 minutes.

12.

Incubate in primary antibody diluted in blocking buffer overnight at 4^oC.

13.

Wash with 1X PBS for 5x 5 minutes.

14.

Incubate in secondary antibody diluted in blocking buffer for 1 hour at room temperature.

15.

Wash with 1X PBS for 5x 5 minutes.

16.

Incubate in streptavidin horseradish peroxidase diluted in 1X PBS for 1 hour at room temperature.

17.

Wash with 1X PBS for 5x 5 minutes.

18.

Expose to DAB for 3-10 minutes then rinse in 1X PBS for 2x 2 minutes.

19.

Incubate in 50% ethanol for 2 minutes.

20.

Incubate in 70% ethanol for 2 minutes.

21.

Incubate in 100% ethanol for 2 minutes.

22.

Incubate in 100% xylenes for 5 minutes.

23.

Coverslip with Permount and a #1.5 coverslip.