Hanker-Yates reagent

Common chromogen systems currently in use include diaminobenzidine (DAB), 3-amino-9-ethyl-carbazole (AEC), Hanker-Yates reagent, alpha-naphthol pyronin used with peroxidase as substrate fast blue, fast red, BCIP- (5-bromo-4-chloro-3-indolyl phosphate) NBT (nitroblue tetrazolium) used with alkaline phosphatase as substrate tetrazolium, tetranitroblue tetrazolium used with glucose oxidase as substrate, and immunogold with silver enhancement (Leong, 1993 Leong et al, 1997a). 

This gives a blue black reaction product which is insoluble in alcohol and other organic solvents. Like polymerized DAB, this reaction product can be osmicated. Varying results have been achieved with Hanker Yates reagent in immunoperoxidase techniques. 

HY, Hanker-Yates reagent CN, 4-chloro-l-naphthol AEC, 3-amino-9-ethylcarba-zole. 

Develop with Hanker-Yates reagent Development usually is complete by 15 min. However, intensity of development may vary depending on the primary antibody used and the amount of cannabinoid receptor present in tissue. A general approach is to place a negative control (e.g., cells on a cover slip treated with normal rabbit IgG as the primary antibody) side by side with a positive control (e.g., cells transfected with a cannabinoid receptor expression construct). The development reaction is terminated when the positive control qualitatively exhibits a twofold level of staining at the macroscopic level. Alternatively, the staining reaction can be monitored under an inverted cell culture microscope. 

As alternatives to DAB or for double immunoenzymatic staining. Hanker-Yates (HY) reagent, composed of p-phenylenediamine-HCl and two parts of pyrocatechol, 3-amino-9-ethylcarbazole (AEC), 3,3 -5,5 -tetramethylbenzidine (TMB), CN and a-naphthol/pyronin have received the most attention (Tubbs and Shebani, 1981 Sofroniew and Schrell, 1982). HY yields a purple-brown, almost black product, AEC a reddish product and TMB, CN a dark-blue product and naphthol/pyronin a red/purple product. 

---