Stabilizing and rapid thionin staining of TMB-based HRP reaction product

Tetramethyl benzidine (TMB) as a substrate for horseradish peroxidase (HRP) histochemistry offers a great advantage over other substrates in its sensitivity but the reaction product is unstable in alcohol. Immersing TMB-reacted sections in methyl salicylate renders the reaction product stable enough so that exposure to ethanol for 10 min causes no detectable loss of HRP labelled neurons. A rapid thionin stain is introduced which, after stabilizing the TMB-based reaction product in methyl salicylate, causes no detectable loss of HRP labelled neurons. These procedures should prove valuable to users of the popular HRP techniques for studying neural connections.