The article discusses the use of diaminobenzidine (DAB) as a substrate for horseradish peroxidase (HRP) in brain tissue, despite its health risks. While tetramethylbenzidine (TMB) is more sensitive and less hazardous, its reaction product does not complex with osmium to form an electron-dense substance. DAB-based products are preferred for immunohistochemistry, especially for delineating HRP-rich cells. A method to enhance DAB reaction product darkness was developed by using phosphate buffer with cobalt chloride. This method was later improved by adding nickel salts to the incubation medium. The new procedure simplifies the process by directly adding metal salts to the DAB solution in concentrations that barely saturate the medium. The steps include dissolving DAB in phosphate buffer, adding cobalt chloride and nickel ammonium sulfate, incubating the tissue, adding hydrogen peroxide, and rinsing. This method eliminates the need for a presoak in Tris-buffered cobalt chloride and offers increased sensitivity and a black reaction product suitable for immunohistochemistry. The technique was developed at the National Institute of Neurological Disorders and Stroke (NINCDS), NIH. The procedure is efficient and useful for various applications, including labeling anterogradely transported HRP. The article acknowledges the inherent risks of DAB but highlights the method's utility in electron microscopy and immunohistochemistry.The article discusses the use of diaminobenzidine (DAB) as a substrate for horseradish peroxidase (HRP) in brain tissue, despite its health risks. While tetramethylbenzidine (TMB) is more sensitive and less hazardous, its reaction product does not complex with osmium to form an electron-dense substance. DAB-based products are preferred for immunohistochemistry, especially for delineating HRP-rich cells. A method to enhance DAB reaction product darkness was developed by using phosphate buffer with cobalt chloride. This method was later improved by adding nickel salts to the incubation medium. The new procedure simplifies the process by directly adding metal salts to the DAB solution in concentrations that barely saturate the medium. The steps include dissolving DAB in phosphate buffer, adding cobalt chloride and nickel ammonium sulfate, incubating the tissue, adding hydrogen peroxide, and rinsing. This method eliminates the need for a presoak in Tris-buffered cobalt chloride and offers increased sensitivity and a black reaction product suitable for immunohistochemistry. The technique was developed at the National Institute of Neurological Disorders and Stroke (NINCDS), NIH. The procedure is efficient and useful for various applications, including labeling anterogradely transported HRP. The article acknowledges the inherent risks of DAB but highlights the method's utility in electron microscopy and immunohistochemistry.