A simple method for combining HRP-TMB histochemistry with tritiated thymidine autoradiography on the same tissue...

In order to determine the sequence of development of various types of spinal neurons defined by their projection, we have developed a method for combining tritiated thymidine autoradiography for birthdate determination, with the demonstration of retrogradely transported horseradish peroxidase (HRP) using tetramethylbenzidine (TMB) as the chromogen. Because of its greater sensitivity, TMB is the chromogen of choice for the demonstration of HRP. However, the HRP-TMB reaction product is unstable and completely destroyed when the tissue is processed for autoradiography. The present study describes the use of osmium tetroxide as a postreaction stabilizing agent which preserves the HRP-TMB reaction product in the form of a dark brown precipitate which is not destroyed when the tissue is subsequently processed for autoradiography. Background levels of autoradiographs stabilized with the osmium procedure are extremely low.