Despite the health risks associated with diaminobenzidine (DAB), it remains the preferred substrate for demonstrating horseradish peroxidase (HRP) in brain tissue. Tetramethylbenzidine (TMB) is more sensitive and less hazardous, but it does not form an electron-dense substance with osmium and is inferior for immunohistochemistry. The DAB reaction product can be enhanced by adding cobalt chloride and nickel salts to the phosphate buffer. This method simplifies the procedure and produces a darker, electron-dense reaction product, making it useful for various applications, especially in electron microscopy and immunohistochemistry. The technique was developed while the author was a Staff Fellow at LNO, NINCDs, NIH.Despite the health risks associated with diaminobenzidine (DAB), it remains the preferred substrate for demonstrating horseradish peroxidase (HRP) in brain tissue. Tetramethylbenzidine (TMB) is more sensitive and less hazardous, but it does not form an electron-dense substance with osmium and is inferior for immunohistochemistry. The DAB reaction product can be enhanced by adding cobalt chloride and nickel salts to the phosphate buffer. This method simplifies the procedure and produces a darker, electron-dense reaction product, making it useful for various applications, especially in electron microscopy and immunohistochemistry. The technique was developed while the author was a Staff Fellow at LNO, NINCDs, NIH.