A sensitive method for histochemical demonstration of horseradish peroxidase in neurons following retrograde axonal transport.

A study was made on the effects of various fixatives and some other histochemical parameters used in the procedure for demonstrating labeled neurons following retrograde axonal transport of horseradish peroxidase (HRP). The enzyme was injected into the tongue of adult mice and the results were obtained by counting labeled hypoglossal neurons following certain variations in the procedure. Paraformaldehyde in the fixative should be avoided since it reduces the number of labeled neurons as compared to glutaraldehyde in a concentration of 1.5-2.5% Fixation for about 4 h is recommended followed by a wash in 5% sucrose buffer overnight. Variables in the histochemical procedure were systematically studied in order to determine optimal pH, buffer type, buffer concentration and substrate concentration. The effect of using a "preincubation" in buffer containing only diaminobenzidine tetrahydrochloride (DAB) was also examined. These results were used to develop a modified histochemical procedure which produced a substantial increase in the number of detectable HRP-labeled neurons as compared to equivalent sections that were reacted in the incubation medium described by Graham and Karnovsky. The modified histochemical procedure involves incubation (no preincubation with DAB only) of sections in the dark for 30 min in a solution consisting of 10 ml cacodylate buffer (pH 5.1;0.1 M), 20 mg DAB and 0.1 ml of 1% hydrogen peroxide. The Kodak Wratten no. 46 filter is recommended for light-microscopical identification of labeled neurons since it is closely matched to the absorption spectrum of the DAB reaction product and consequently greatly increases the contrast of HRP-labeled neurons.